Giovanna Suzzi, Università di Teramo, Italy

#### Reviewed by:

Laurent Guillier, Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (ANSES), France Giorgia Perpetuini, Università di Teramo, Italy Arun K. Bhunia, Purdue University, United States

> \*Correspondence: Edward M. Fox<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 22 December 2017 Accepted: 15 March 2018 Published: 03 April 2018

#### Citation:

Gray JA, Chandry PS, Kaur M, Kocharunchitt C, Bowman JP and Fox EM (2018) Novel Biocontrol Methods for Listeria monocytogenes Biofilms in Food Production Facilities. Front. Microbiol. 9:605. doi: 10.3389/fmicb.2018.00605 High mortality and hospitalization rates have seen Listeria monocytogenes as a foodborne pathogen of public health importance for many years and of particular concern for high-risk population groups. Food manufactures face an ongoing challenge in preventing the entry of L. monocytogenes into food production environments (FPEs) due to its ubiquitous nature. In addition to this, the capacity of L. monocytogenes strains to colonize FPEs can lead to repeated identification of L. monocytogenes in FPE surveillance. The contamination of food products requiring product recall presents large economic burden to industry and is further exacerbated by damage to the brand. Poor equipment design, facility layout, and worn or damaged equipment can result in Listeria hotspots and biofilms where traditional cleaning and disinfecting procedures may be inadequate. Novel biocontrol methods may offer FPEs effective means to help improve control of L. monocytogenes and decrease cross contamination of food. Bacteriophages have been used as a medical treatment for many years for their ability to infect and lyse specific bacteria. Endolysins, the hydrolytic enzymes of bacteriophages responsible for breaking the cell wall of Gram-positive bacteria, are being explored as a biocontrol method for food preservation and in nanotechnology and medical applications. Antibacterial proteins known as bacteriocins have been used as alternatives to antibiotics for biopreservation and food product shelf life extension. Essential oils are natural antimicrobials formed by plants and have been used as food additives and preservatives for many years and more recently as a method to prevent food spoilage by microorganisms. Competitive exclusion occurs naturally among bacteria in the environment. However, intentionally selecting and applying bacteria to effect competitive exclusion of food borne pathogens has potential as a biocontrol application. This review discusses these novel biocontrol methods and their use in food safety and prevention of spoilage, and examines their potential to control L. monocytogenes within biofilms in food production facilities.

Keywords: Listeria monocytogenes, biofilms, biocontrol, bacteriophages, bacteriocins, endolysins, competitive exclusion, essential oils

## INTRODUCTION

fmicb-09-00605 March 28, 2018 Time: 17:7 # 2

Listeria monocytogenes is a Gram-positive, rod shaped, facultative anaerobe capable of causing food borne illnesses particularly in high-risk population groups including the elderly, immune compromised, pregnant women, and neonates (Farber and Peterkin, 1991). While L. monocytogenes associated illness is not as common as that of other food borne pathogens like Salmonella, Campylobacter, or Escherichia coli, its mortality rate can be considered the highest. Approximately 30 % of invasive listeriosis cases lead to mortalities with most requiring hospitalization, and therefore demanding L. monocytogenes can be considered as a food borne pathogen of public health importance (Lomonaco et al., 2015; Véghová et al., 2016). Due to its ubiquitous nature, L. monocytogenes poses a food safety risk as it is frequently introduced into the processing environment through raw ingredients. L. monocytogenes can adhere to a variety of abiotic surfaces with some strains persisting for numerous years and acting as a source of continuous cross contamination (Fox E. et al., 2011; Coughlan et al., 2016; Colagiorgi et al., 2017).

Due to significant food safety risks, the control of L. monocytogenes has become a regulatory requirement that food business operators must adhere to. Regular cleaning, disinfecting, and sanitizing of food contact and non-food contact surfaces are required as part of a sanitation plan that also incorporates maintenance of equipment and buildings, pest control, and general hygiene. In addition, the implementation of good manufacturing practices and effective hazard analysis critical control point plan aids in reducing the risk of food borne illness (Drew and Clydesdale, 2015). However, L. monocytogenes is a difficult organism to eradicate and its presence still occurs even with the best management plans (Tompkin, 2002; Drew and Clydesdale, 2015).

While the exact mechanisms can be unclear for how L. monocytogenes is able to persist in food production environments (FPEs) so successfully, researchers have proposed that there are numerous factors at play. Poorly maintained equipment, surfaces, and unhygienic factory design can result in niches containing adequate nutrients, water, and protection from cleaning allowing bacteria to survive and grow while also introducing bacteria to subinhibitory levels of disinfectants (Carpentier and Cerf, 2011; Fox E.M. et al., 2011; Ibba et al., 2013; Møretrø et al., 2017). Typically disinfectants, when applied correctly, can sufficiently inhibit the colonization of introduced planktonic cells; however, dosing failures and applying disinfectants to wet surfaces can result in equipment being inadequately disinfected and bacteria being exposed to subinhibitory chemical levels (Martinez-Suarez et al., 2016; Møretrø et al., 2017). Incorporating desiccation processes has been shown to increase the effectiveness of disinfections procedures (Overney et al., 2017); however, an ample amount of drying time is difficult when continuous or even daily production runs are required. It is also important to note the difference between resistance, an increase in concentration or time required to exert the same reduction, and tolerance, an adaptation in a microbe's susceptibility potentially the result of exposure to subinhibitory levels

(Cerf et al., 2010; Ortega Morente et al., 2013). For example, some L. monocytogenes strains are known to carry genes for disinfectant chemical efflux pumps, such as qacH and bcrABC. The distribution of these genes tends to vary on a strain by strain basis instead of being unique to a specific lineage or subtype (Dutta et al., 2013; Ortiz et al., 2015; Møretrø et al., 2017). Although it has been reported that these genes only result in tolerance to quaternary ammonium compounds at levels far below the concentrations actually used in the food industry (Tezel and Pavlostathis, 2015), the ability to form biofilms is also a crucial factor in the survival of L. monocytogenes. Biofilms are composed of numerous cells attached to each other or an abiotic surface surrounded by an extracellular matrix containing a mixture of polysaccharides, proteins, and extracellular DNA (da Silva Fernandes et al., 2015; Fagerlund et al., 2017). This extracellular matrix provides a protective barrier around the internalized microbial cells from desiccation and heat, contributes to increased adhesion, and is a reservoir of nutrients (Colagiorgi et al., 2016). In addition, biofilms can impede the activity of antimicrobial agents as the matrix limits their diffusion potential and contains cells with differing susceptibility while also allowing for the acquisition of new genetic traits like those mentioned above through horizontal gene transfer. Further, biofilms typically consist of multiple species that can allow for the colonization of transient strains or provide increased attachment and survival to strains not typically good biofilm formers (Coughlan et al., 2016).

### THE BIOCONTROL METHODS MOVEMENT

While tolerance to disinfectants and sanitizers is not considered as significant an issue as antibiotic resistance, their continued use and potential ineffectiveness against biofilms warrant new strategies for the control of L. monocytogenes. As consumers become more conscious of food safety significance, the use of novel biocontrol methods is gaining further interest. This return to biocontrol methods of microbes and plants has the potential to relieve some of the tolerance to disinfectants and decrease some of the selective pressures that their overuse has on maintaining resistance markers (Coughlan et al., 2016). Biocontrol methods with potential to act against listerial biofilms include bacteriophages, their endolysins, competitive bacterial species and their antimicrobial products, bacteriocins, and plant-derived products and will be discussed in this review.

### BACTERIOPHAGES

The most abundant microorganism on earth, bacteriophages (phages) are viruses that infect bacteria for propagation, live naturally in the environment, and anywhere host bacteria are found (Bai et al., 2016; Pérez Pulido et al., 2016). Phages are classified based upon their morphology (head and

tail, either contractile or non-contractile, or no tail), nucleic acid (double stranded or single stranded; deoxyribonucleic or ribonucleic acid), and life cycle, which is of most relevance for biocontrol. There are two types of life cycles phages can undergo after entering the bacterial cell: the lysogenic cycle (temperate phages) or the lytic cycle (**Figure 1**). Phages may be capable of a lysogenic cycle that converts to the lytic cycle in unfavorable conditions, or undergo a solely lytic life cycle. Temperate phages are not suitable as a biocontrol agent as integration into the host genome may result in increased pathogenicity through horizontal gene transfer (Hagens and Loessner, 2007; Salmond and Fineran, 2015). In comparison, lytic phages are ideal as a biocontrol agent due to their fast-lytic action.

Although identified over a hundred years ago, interest in phages has only recently been reignited with the rise of antibiotic resistance among bacteria (Hagens and Loessner, 2007). The utility of phages has included the treatment of diseases in humans and animals, typing of bacterial strains, decontaminating meat carcasses after slaughter, and targeted inactivation of pathogenic and spoilage bacteria on food contact and noncontact surfaces as well as surfaces of ready to eat products and during packaging and storage (Hagens and Loessner, 2007; Strauch et al., 2007). The application of phages as an innovative approach to control biofilms in the FPE is also beginning to be explored. While there has been great achievement in the use of phages from a therapeutic perspective, their success in the FPE is not as simple. Factors like the composition and structure of the biofilm, temperature, the metabolic state of the bacteria in the biofilm, the extracellular matrix in general, food components, and nutrients all provide additional challenges to the effectiveness of phage application (Parasion et al., 2014). While there have been some reports of phage resistance (Fister et al., 2016), it occurs more gradually than the development of antibiotic resistance as phages are able to mutate continuously, like bacteria, and resistance is further slowed by using a combination of phages active against the one bacterial species (Hagens and Loessner, 2007; Sadekuzzaman et al., 2017). There is a substantial amount of research conducted on phages' ability to protect food from Listeria, with two commercial Listeria phage products, ListShieldTM and ListexTM P100 approved as food preservatives with the generally recognized as safe status since 2006. However, studies investigating the efficacy of these products and other Listeria phages against biofilms are few, with most having focused on ListexTM P100.

Biofilm maturity has the potential to reduce the efficacy of phage treatment, as well as any control method. Various

studies have examined this concept utilizing preformed biofilms at various maturity levels, ranging from 24 h to 2 weeks, with most studies reporting a minimum 1-log reduction. Most studies to date have utilized stainless steel as the surface to form L. monocytogenes biofilms and examine the efficacy of bacteriophage treatments. This reflects the widespread presence of these surfaces, both food contact and non-contact in food processing environments. The success of bacteriophage treatments at inactivating L. monocytogenes biofilms on these surfaces, however, has shown mixed results. A number of studies demonstrated promising results when ListexTM P100 was applied to L. monocytogenes biofilms on stainless steel, with reductions in the order of 5-log being achieved (Soni and Nannapaneni, 2010; Montañez-Izquierdo et al., 2012). Both of these studies used an application treatment of 24 h at ambient room temperature. Iacumin et al. (2016) also applied ListexTM P100 for 24 h at 20◦C onto stainless steel wafers and report the complete elimination of L. monocytogenes biofilm. This prolonged treatment application, however, in many cases is not practical in an FPE. In addition, Iacumin et al. (2016) pressed the stainless steel wafer onto an agar plate to replicate the process of cross-contamination in the FPE; however, it did not take into consideration the phage products ability or inability to act on biofilms in the crevices or corners where these might be thicker than a flat surface.

A shorter treatment time of 2 h was applied by Sadekuzzaman et al. (2017) when running a similar inactivation test with ListShieldTM; however, this was associated with a much lower inactivation of just 2-log when applied to L. monocytogenes biofilm on stainless steel. This was even less effective on a rubber surface, achieving a 1-log reduction in L. monocytogenes cell numbers. The results of Sadekuzzaman et al. (2017) also reflect those observed by Gutiérrez et al. (2017) who saw a similarly low inactivation achieved by a 4 h ListShieldTM treatment, typically 1-log or less. Although the latter study did show greater inactivation with ListexTM P100 under the same treatment conditions, the ListexTM P100 commercial phage preparation showed a reduced activity range, only capable of infecting 7 of the 11 strains tested. An important aspect in phage application is the ratio of phage to bacteria known as the multiplicity of infectivity. To increase the likelihood the phage will infect the bacterium, the phage needs to be at a higher ratio than the number of target bacterial cells (Montañez-Izquierdo et al., 2012). High multiplicity of infectivity has been reported to result in efficient phage treatment with one study recommending a multiplicity of infectivity around five was required for adequate reductions by ListexTM P100 (Montañez-Izquierdo et al., 2012).

Apart from temperature, multiplicity of infectivity, and treatment time, other factors may influence efficacy of biocontrol treatments, notably the presence of organic matter such as the food matrix. A further parameter which must be considered when examining efficacy of treatment on surfaces is the surface architecture itself, which may range from a smooth rendered surface to a scored surface with associated crevices which may be colonized by bacteria and their biofilms. Chaitiemwong et al. (2014) considered both surface crevices and food matrices (diluted food residues of ham, salmon, endive, or milk) when measuring the efficacy of ListexTM P100 treatment. Results suggested deeper crevice features on the surface decreased the treatment efficacy, with inactivation in the magnitude of > 3-log achieved on 0.2 mm crevices compared to the max 1.4-log CFU/mL observed in crevice depth of 5 mm. Of particular note was the difference seen when comparing the food matrix, with lower inactivation observed for milk and vegetable when compared with meat or fish. Ganegama Arachchi et al. (2013) mimicked conditions in fish processing and demonstrated the presence of fish protein led to a lower associated biofilm density compared to control stainless steel experiments when a fish protein matrix was added to the cultivation of L. monocytogenes biofilm on stainless steel. This highlights the complex role the food matrix may play in both biofilm formation and subsequent efficacy of bacteriophage treatment, demonstrating the need for further studies to understand the significance of food matrix on bacteriophage treatment efficacy.

Taken together, current literature detailing phage biocontrol studies directed at L. monocytogenes, such as those detailed above, shows differing success in their ability to decrease established biofilms. The often low reductions achieved demonstrate the challenges biofilms pose for not only bacteriophages but all control methods, but this is not to say that there is no place for phages as a potential biocontrol method. As with many disinfection regimes, additional interventions such as steps to loosen biofilm or remove organic matter can increase the success of phage treatments (Ganegama Arachchi et al., 2013). Further research considering multi-species biofilms and infacility application will help determine the true potential of this biocontrol approach.

### ENDOLYSINS

Endolysins (lysins) are hydrolytic enzymes required for bacteriophage dissemination from the host bacterial cell. They occur at the end of the lytic cycle to release the phage virions by breaking down peptidoglycan in the bacterial cell wall in what is termed lysis from within (Chan and Abedon, 2015; Schmelcher and Loessner, 2016). Researchers have harnessed lysins through protein expression production systems, generally in E. coli. Following purification of the lysin, it can be applied externally to the cell wall, thus not requiring phage infection, for biopreservation and biocontrol application (García et al., 2010). Lysins are grouped based upon the cell wall component they attack with the five main classes being N-acetylglucosaminidases, endo-β-N-acetlyglucosaminidases, lytic transglycosylases, endopeptidases, and N-acetylmuramoyl-L-alanine amidases (García et al., 2010; Schmelcher and Loessner, 2016). Lysins are highly specific with a narrow spectrum of activity making them host specific with some lysins only being active on the bacterial strain the phage was isolated from (Oliveira et al., 2012). In addition, they are fast acting and no development of resistance has been reported to date (Schmelcher and Loessner, 2016). Most

research has occurred on Gram-positive bacteria using the lysis from without approach as the peptidoglycan layer is exposed. Although limited, antimicrobial activity of lysins on Gram-negative bacteria has been reported when used in conjunction with EDTA, a membrane permeablizer (Chan and Abedon, 2015).

The antimicrobial activity of lysins has mostly focused on infection control of staphylococcal bacteria. Other applications that have been considered include use in agriculture to prevent plant disease by either intense application of cell lysates expressing a chosen lysin or development of transgenic plants by incorporation of the lysin gene into the plant genome (Düring et al., 1993; Kim et al., 2004); as a rapid detection and imaging method of pathogenic bacteria (Schmelcher et al., 2010; Bai et al., 2016); and transformation of listerial bacteriophage endolysin encoding genes into dairy starter cultures as a biopreservation method (Gaeng et al., 2000). Antilisterial lysins isolated to date have predominately focused on the control of planktonic cells in vitro with promising results although further validation is required (**Table 1**). Only a few antilisterial lysins have been assessed in food products and the food matrix and environment have been found to affect the antimicrobial activity (Oliveira et al., 2012).

To date there is only one lysin, PlyLM, which has been tested against L. monocytogenes biofilms after 100 % susceptibility on planktonic L. monocytogenes and Listeria innocua cells was achieved (Simmons et al., 2012). PlyLM reduced the monolayer biofilm to the same level as the application of lysozyme and proteinase K. When used in combination with proteinase K, or both proteinase K and lysozyme, synergistic effects were observed, and the biofilm was effectively digested. However, biofilms were only grown for 24 h at 37◦C, and therefore the efficacy of these enzymes under other conditions merits further investigation, for example, performance at lower temperatures which are more reflective of those of most FPEs. More research has been undertaken on staphylococcal biofilms, predominantly monospecies biofilms, which have achieved reductions in biofilm mass. Of interest is their efficacy against persister cells. Persister cells are metabolically inactive subpopulations of cells, which are "super-resistant" to antimicrobial agents such as antibiotics (Brooun et al., 2000; Wood, 2017). Studies have shown these persister cells occur as a subpopulation of bacterial biofilms, and as such can present a significant obstacle to biofilm inactivation by antimicrobials (Brooun et al., 2000; Singh et al., 2009). Several studies have shown a promising role for lysins to inactivate persister cells in biofilms (Gutiérrez et al., 2014; Schuch et al., 2017). The success being reported against staphylococcal biofilms suggests that the potential lysins may have against biofilms in a food production context, particularly in targeting Listeria biofilms, which are a significant problem in FPEs. Another phage enzyme, extracellular polysaccharide depolymerase, has also be shown to degrade biofilm EPS; however, they are highly specific to the strains the phage infects (Chan and Abedon, 2015). A similar approach targeting L. monocytogenes in biofilms could also present an alternative control measure.

## COMPETITIVE BACTERIAL SPECIES

Competitive exclusion is where one bacterial species competes with another species over resources and/or space in a habitat, successfully reducing the number of cells or excluding that species (Hibbing et al., 2010). This competitive exclusion can be the result of the production of antimicrobials such as bacteriocins, organic acids either acting directly against the species it is competing with or acting on the environment altering the pH, or alternatively physically outcompeting other bacterial species for nutrients and/or space and limiting normal survival or proliferation of those competitive species. This strategy is typically categorized into three components: competition, where planktonic cells of both species are co-cultured for a period of time; exclusion, where the antagonistic species are grown to a biofilm cell density prior to the addition of planktonic cells of the target species; or displacement, in which the target species are grown to biofilm cell density prior to addition of planktonic antagonists (Woo and Ahn, 2013; Pérez-Ibarreche et al., 2016). As biofilms protect microorganisms from chemical cleaners and disinfectants, the use of non-pathogenic microorganisms may assist sanitation approaches in controlling, preventing, or eradicating unwanted species like food borne pathogens.

Competitive exclusion studies typically pit planktonic cells of the antagonist species (i.e., the species which will exert a competitive exclusion effect) against planktonic cells of the target species in a competition assay, grown together for a period of time facilitating biofilm formation. Daneshvar Alavi and Truelstrup Hansen (2013) used a short incubation time of 72 h which resulted in a 1-log decrease in L. monocytogenes cell density after application of Serratia proteamaculans. A similar reduction was also reported by Fox et al. (2014) of L. monocytogenes biofilm cell density after 96 h when grown in co-culture with Janthinobacterium lividum. However, greater reductions have been reported when cells were incubated for longer periods with results around log 4.5 and 5.5 on stainless steel coupons and polytetrafluoroethylene, respectively (Pérez-Ibarreche et al., 2016). Zhao et al. (2004) also reported higher magnitude reductions of 7.8-log reduction over 28 days at 15◦C by two bacterial isolates, Lactococcus lactis (Lc. lactis) and Enterococcus durans. In another experiment performed at 8◦C for 28 days, four isolates, including the previous two isolates were also capable of reductions around 7-log units. However, the higher reductions reported by Zhao et al. (2004) and Pérez-Ibarreche et al. (2016) were produced by lactic acid bacteria (LAB) whose inhibitory activity has been studied extensively for many years, particularly as probiotics (Jeong and Frank, 1994).

The inhibitory effect of LAB was further explored by Guerrieri et al. (2009) and Gómez et al. (2016) as a preformed biofilm preventing L. monocytogenes biofilm formation as part of the exclusion strategy. Gómez et al. (2016) tested a variety of LAB strains and found reductions ranged from 4- to 7-log units over 24 and 48 h; however, by 72 h, L. monocytogenes growth had increased by almost half fold of the control indicating that these strains were only capable of exclusion

TABLE 1 | Antilisterial lysins reported in literature, key summary, and application.

BC, biocontrol; IC, infection control; RMD, rapid multiplex detection; BP, biopreservation.

within the first 24–48 h. However, Lc. lactis 368 strain was able to completely exclude the growth of L. monocytogenes for the entire period, although it should be noted that all experiments were performed at a relatively elevated temperature and as such lower temperatures reflective of many FPEs require further consideration. In comparison, Guerrieri et al. (2009) showed the potential of LAB bacteria at refrigeration temperatures with a Lactobacillus plantarum (Lb. plantarum) strain capable of a 4-log reduction over a 10-day period. Mariani et al. (2011) used the native biofilm microflora of wooden cheese ripening shelves to achieve a 1- to 2-log reduction over a 12-day period, although this reduction was less than that observed in Guerrieri et al. (2009) and Gómez et al. (2016).

The third strategy displacement, as reviewed by Woo and Ahn (2013), demonstrated that the use of planktonic antagonist LAB strains as a post-treatment control method targeting L. monocytogenes was less effective compared to pre-treatment, although two strains (Lactobacillus paracasei and Lactobacillus rhamnosus) were capable of a 3-log reduction in L. monocytogenes biofilm cell density over 24 h when incubated at 37◦C.

While most studies are performed in laboratories, Zhao et al. (2006, 2013) took the concept of competitive exclusion a step further and looked at its applicability in poultry processing facilities. In a fresh poultry facility, two LAB strains (Lc. lactis and E. durans) were added to two enzyme-based cleaners and applied as a foam to selected drains four times in the first week and then two times for the following 3 weeks. Sampling continued for 18 weeks after the last treatment. Most drains experienced significant reductions within the first week after only four applications and all drains maintained lower levels of Listeria throughout the sampling period (Zhao et al., 2006). Importantly, two drains reported significant reductions 16 weeks after treatments finished. Similar parameters were applied to the application of the same strains at a ready to eat poultry processing facility. By the end of the first week of application, Listeria was not detected in five of the six drains with all drains reporting negative results between weeks 8 and 13 (Zhao et al., 2013). It should also be noted that the strains utilized were known to either possess nisin or other forms of antimicrobials; however, it was not elucidated if the inhibition was the result of the production of antimicrobials.

There have been some encouraging results in the use of LAB against L. monocytogenes biofilm cells in laboratory-based experiments (**Table 2**); however, very few have been trialed in actual FPEs, apart from Zhao et al. (2006, 2013). The results from their two studies have shown promising results as an alternative control method utilizing E. durans and Lc. lactis; however, further longitudinal research surrounding the in-facility application is required. In addition, the application of other bacterial species identified in some of the studies mentioned above, for example, J. lividum and S. proteamaculans, warrants in-facility testing. However, it should be noted that the LAB strains utilized for in-facility application studies were isolated from the production environment indicating that specific strains may work best in the environment they were isolated from and these strains may vary depending on the food industry.

Houry et al. (2012) reported the use of bacterial species in a novel biocontrol approach. In the study, they identified a subpopulation of bacilli known as bacterial swimmers which were capable of creating transient pores within the biofilm structure. By pre-treating Staphylococcus aureus biofilms with bacterial swimmers, which also produced an anti-stapylococcal bactericide, they achieved a greater inactivation of S. aureus in biofilm by facilitating access of toxic substances in the environment into the biofilm.

### BACTERIOCINS

An important component of the competitive survival strategy of bacteria is the production of antimicrobial products. One group of ribosomally synthesized antimicrobials are the heat stable peptides known as bacteriocins (Cotter et al., 2005; Gálvez et al., 2008; Winkelströter et al., 2015). It has been suggested that most bacteria produce at least one bacteriocin and LAB are known to be prolific producers (Cotter et al., 2005). Most

TABLE 2 | Bacterial species active against L. monocytogenes and purported mode of action.

bacteriocins have a narrow spectrum of activity, that is, they are active against the same species that produces them but the producer is immune to them, while some have a broad spectrum of activity acting on members within the same genus as well as other genera and species (Cotter et al., 2005). The mode of activity varies depending on the particular class of bacteriocin and can include pore formation, or inhibition of key cellular processes such as peptidoglycan production, DNA replication, mRNA, or protein synthesis, to name a few (Cotter et al., 2005). There are two main groups: Class I (also known as lantibiotics), peptides that undergo post-translational changes, and Class II, which do not (Cotter et al., 2013). Among the most well-characterized and successful bacteriocins to date is nisin, a Class I bacteriocin from Lc. lactis which has been approved for use in food as a preservative/additive by the World Health Organization, European Union, and the United States Food and Drug Authority (Cotter et al., 2005). A great deal of research has gone into identifying more bacteriocins active against L. monocytogenes planktonic cells and biofilms, an important arena as nisin resistance is slowly being reported.

Most studies can be classified into two groups based upon how the bacteriocin is applied: either as whole bacterial cells known or suspected of bacteriocin production, or alternatively the bacteriocin extract itself, applied either as a crude or semi-purified product. Their utility against preformed L. monocytogenes biofilms of varying times has been the subject of numerous studies, with some reporting promising results. For example, Gómez et al. (2016) assessed Lc. lactis, Lactobacillus sakei, and Lactobacillus curvatus, all known to produce nisin Z, sakacine A, and sakacine P, respectively, against 48 h preformed biofilms. Lb. sakei and Lb. curvatus were capable of complete inactivation over 72 h whereas the two Lc. lactis strains provided a 6-log reduction by the end of the test period. Winkelströter et al. (2015), however, were unable to produce results of a similar magnitude when L. monocytogenes was co-cultured with Lb. paraplantarum, only achieving 2-log inactivation at 24 and 48 h before decreasing by 72 h. Guerrieri et al. (2009) took an alternative approach and reported that Lb. plantarum and Enterococcus casseliflavus were able to inactivate L. monocytogenes 7-day preformed biofilms by 3.9- and 3.7-logs over a 10 dayperiod. Importantly, the results could be associated with bacteriocin production, as no changes to the pH were observed.

Another technique is extracting the bacteriocin in the form of cell-free supernatant (CFS), as a crude bacteriocin fermentate or semi-purifying the product. The antimicrobial activity of CFS has shown mixed success in co-inoculation studies to prevent the formation of biofilms by L. monocytogenes, with Camargo et al. (2016) reporting significant reductions after 24 h, whereas Bolocan et al. (2017) only observed between 1.6- and 3.6-log CFU/cm<sup>2</sup> reduction after 72 h depending on the media used. In the latter study, however, the CFS extract which produced the highest reduction was from an isolate known to also produce an organic acid which was not

removed, and therefore this result may not be associated solely to the antimicrobial activity of the bacteriocin. When Camargo et al. (2016) applied the CFS to 24 h preformed biofilms for 2.5 h, they found biofilm formation continued in some isolates.

Other researchers have compared the two methods, bacterial cells and extracts again with varying results. García-Almendárez et al.'s (2008) analysis on 4-day preformed biofilms demonstrated a crude bacteriocin fermentate from Lc. lactis known to produce nisin A was capable of a 2.7-log reduction over 24 h. However, a greater reduction over 5-logs was achieved when the Lc. lactis was applied for 6 h, then rinsed, and placed in a desiccator for five days. Whereas, Winkelströter et al. (2011) co-inoculated L. monocytogenes with Lb. sakei or its CFS and found that any decreases observed in the first 24 h were diminished with time, as results at 48 h were comparable to the pure culture levels. A promising approach by Pérez-Ibarreche et al. (2016) involved the supplementation of Lb. sakei cells with a semi-purified bacteriocin for 6 h, which resulted in a twofold reduction in L. monocytogenes numbers on the stainless steel surface, or an additional 1-log reduction on polytetrafluoroethylene.

As mentioned previously, the bacteriocin nisin has been approved for commercial purposes and has paved the way as an alternative biocontrol method. Research into bacteriocins has been performed with comparable results to the other noncommercial bacteriocins discussed above. Minei et al. (2008) found that nisin was capable of inhibiting L. monocytogenes biofilm formation for 9 h on stainless steel coupons, and although cell growth did recommence after this time, a 3.5-log inactivation was still maintained by 48 h. On the other hand, Henriques and Fraqueza (2017) shortened the treatment time to 5 min and even at the highest concentration, no activity was recorded, although activity was defined as a ≥ 5-log decrease.

From the above, it is obvious that results vary significantly depending on if bacteriocin producing bacterial cells or the bacteriocin extracts is used. Results from bacteriocin extracts can be correlated to the antimicrobial action of the bacteriocin with greater certainty; however, additional analysis is required particularly when whole cells are used to help ensure that the measured inhibition is not the result of competitive exclusion or the production of other antimicrobials such as organic acids. The co-inoculation and preformed biofilm studies reflect the ability of the bacteriocin to either prevent the formation or affect the removal of established biofilms in the FPE; however, the length of time the biofilms are grown for prior to the bacteriocin being applied also affects the antimicrobial activity as mature biofilms may provide better resistance. Although several studies show that promising results most require additional analysis at temperatures and other environmental conditions mirroring the FPE to identify potential candidates suitable for further testing. With the potential resistance to nisin arising, the identification of other bacteriocins is essential. In addition, the application of synergistic antimicrobials to further combat the development of resistance should be considered.

### PLANT-DERIVED ANTIMICROBIAL PRODUCTS – ESSENTIAL OILS

An alternative to the use of chemicals, microorganisms, or their derivatives is the use of plant-derived antimicrobial products such as essential oils (EOs). Herbs and spices are commonly known to exhibit antimicrobial activity and have been used by various cultures for flavoring, as a food preservative or for medicinal purposes. EOs play a key role in protecting plants from bacteria, fungi, viruses, insects, and animals (Perricone et al., 2015). Traditional distillation, cold press/expressing, solvent extractions, and enfleurage methods have been used to extract EOs from plant-derived materials; more recently, modern techniques including microwave or ultra sound assisted extraction, pressurized extractions, and super critical fluid extraction have been used to obtain EOs from a variety of plant sources (including roots, wood, bark, twigs, leaves, seeds, buds, flowers, and fruits). However, the constituents and compositions of EOs vary significantly from high concentrations to trace amounts based upon the plant part, plant age, and extraction method used, in turn influencing their antimicrobial activity (Lemberkovics et al., 2004; Reyes-Jurado et al., 2014; Perricone et al., 2015; Xia et al., 2017). Key molecules in EOs with the most effective antibacterial activity are typically from aldehyde and phenol chemical classes which include compounds such as cinnamaldehyde, carvacrol, eugenol, or thymol (Bakkali et al., 2008; Perricone et al., 2015). EOs are able to permeabilize the cell membrane resulting in the leakage of ions or other cell content, and may also disrupt key genetic functions and/or cellular components like proteins, polysaccharides, phospholipids, fatty acids, and essential enzymes due to the lipophilic nature of EOs (Bakkali et al., 2008; de Oliveira et al., 2010, 2012a; Perricone et al., 2015).

While there are thousands of EOs described, it is reported around 300 of these have generally recognized as safe approval and are used commercially for flavoring or fragrance; however, more detailed information is required for their use as a biocontrol agent (Burt, 2004; Reyes-Jurado et al., 2014). Most research surrounding the antimicrobial activity of EOs focuses on their effects on planktonic cells of food spoilage and pathogenic bacteria either in standard laboratory conditions or in their application on food items. This application on food as a biocide has major limitations as higher concentrations are required potentially interfering with the sensory attributes of the food (Burt, 2004; Chorianopoulos et al., 2008). In addition, some components of food items, mainly fats, proteins, carbohydrates, water, salt, antioxidants, pH, and other preservatives or additives used may impact upon the activity of the EOs (Perricone et al., 2015). Further research is required to understand the impact EOs have on bacterial pathogens and in particular their ability to prevent or eradicate biofilms in FPEs. Some research is occurring within this space; however, there is limited research against L. monocytogenes biofilms with a few studies looking at the extracted EOs, the active components of specific EOs, or altering the EO chemical composition.

de Oliveira et al. (2010) assessed the EOs from fresh citronella (Cymbopogon nardus) and lemongrass (Cymbopogon citratus) leaves applied alone or in combination; however, it was the Citronella EO which demonstrated the highest reductions against both the 3 and 240 h preformed biofilms with complete reduction after 60 min of application. Similar results reported in another study by de Oliveira et al. (2012b) found 2% (vol/vol) Chinese cinnamon extract (Cinnamomum cassia) was capable of reducing a 48 h preformed biofilm to below the detection limit (2.84-log CFU/cm<sup>2</sup> ) after 20 min; however, both of these studies applied the EOs at temperatures above 20◦C.

Essential oils contain a mixture of major and minor molecules responsible for their antimicrobial activity with some of the major components being explored further. The active components of clove (eugenol) and spearmint (carvone) EOs were tested on a 6 h preformed L. monocytogenes biofilm but were found to increase biofilm mass by Leonard et al. (2010). Citral and nerol, in contrast, both major components from lemongrass (C. citratus) and Lippia rehmannii (nerol only), displayed a similar reduction as the positive control ciprofloxacin.

Additional microbial species can also impact upon the activity of the EO or active component. For example, Leonard et al.'s (2010) study as mentioned above was on L. monocytogenes monospecies biofilms and reported a mixture of results among the EO and the various active components tested, whereas de Oliveira et al. (2012a) looked at the activity of Chinese cinnamon and its active component, Cinnamaldehyde, on a mixed biofilm of L. monocytogenes and enteropathogenic E. coli on stainless steel coupons dipped in reconstituted whole milk. The EO and cinnamaldehyde were both capable of reducing the mixed biofilm to below the detection limit of 2.84 log CFU/cm<sup>2</sup> whereas the EO and active components only provided reductions just over 2-logs on the L. monocytogenes biofilm. Chorianopoulos et al. (2008) examined the EO and hydrosol (by-product of the steam distillation) of Satureja thymbra (Pink Savory) and showed similar results when grown in a mixed biofilm with a food borne pathogen (L. monocytogenes and Salmonella enterica) and a spoilage bacterium (Pseudomonas putida). It was noted that the optimized application time was 60 min and any increase in time provided no additional reduction. The impact other microbial players may have on the activity of EOs requires further exploration in order to gain insights into the various relationships at play.

A common problem for the use of EOs as a biocontrol method on food products is the associated impacts on taste at concentrations required for appropriate antimicrobial effect. A process to concentrate the EOs for application at a lower volume with the same potentially high antimicrobial activity may be required in the case of some EOs. Krogsgård Nielsen et al. (2017) looked at emulsifying and encapsulating isoeugenol oil to increase the antimicrobial effectiveness at a smaller volume with the addition of electrostatic forces to attract negatively charged bacteria to positively charged EOs. Although the concept of emulsification and encapsulation sounds promising, the minimal biofilm eradication concentrations (MBECs) for the coated and uncoated emulsified products were only half a log lower than the pure isoeugenol when tested in standard laboratory medium at three temperatures (6, 12, and 25◦C) and no difference was observed in carrot juice. This observation requires further exploration as the reductions in the MBEC did not correlate to observations under confocal microscopy. Of note was the morphological changes observed in the mixed biofilms of Pseudomonas fluorescens and S. aureus from uniform layers to clusters of numerous cells, which requires further research to determine if there are any implications.

As mentioned previously, the use of EOs at concentrations to exhibit sufficient antimicrobial activity has the potential to impart undesirable flavor and when applied in an FPE may also result in an excessive sensorial impact. In addition, the interactions of EOs with components of the food matrix from food debris may also impact on the applicability of EOs in food environments. Only a few studies have investigated the application of EOs to disrupt or prevent the formation of biofilms. Further research on parameters specific to industry will allow a better decision on the application of EOs as an alternative or supplementary biocontrol method.

### CONCLUDING REMARKS

While current sanitation processes are effective against planktonic cells, the potential for tolerant strains to increase due to interactions at subinhibitory levels and the potential reliance on them as antimicrobials, as the case in the health industry, is a cause for concern. The ability to eradicate established biofilms and prevent new biofilms from being formed is a challenging task which food production managers are charged with, as biofilms can present increased food safety risks. A useful tool in understanding the microbial community is metagenomics analysis of the FPE. By understanding the FPE microbiome, valuable information can be gained regarding persistence or transience of strains. This facilitates source tracking of persistent strains, can identify other microbial species that may provide either a positive or negative effect on the target strain, and can identify strains surviving the disinfection processes (Dass and Anandappa, 2017; Doyle et al., 2017). From this information, the appropriate biocontrol method can then be determined. There have been some significant advances in the development of biocontrol methods, particularly bacteriophages that have progressed to commercial products with the results of some studies validating their progression to commercialization. The use of competitive bacterial species has also showed some promising results with the concept of utilizing antagonist strains isolated from the production environment providing individualized treatment options. Bacteriocins and endolysins have also shown their ability to significantly reduce established biofilms; however, they typically require some form of purification process to achieve these results. The sensory implications of EOs at concentrations required to exert antimicrobial effects are a limiting factor

in their use as a sole biocontrol method, and therefore they may find more appropriate utility as a supplementary method targeting non-food contact surfaces. However, like all biocontrol methods, efficacy can be impacted by a variety of factors including temperature or time the control method was applied for, the use of one species or multiple species biofilms, biofilm growth method, or surface matrix composition. Standardization in the assessment of novel biocontrol methods against biofilms is required, in addition to assessment under conditions reflective of FPEs before appropriate comparisons can be made.

### REFERENCES

### AUTHOR CONTRIBUTIONS

JG and EF conceived the study and drafted the manuscript. All authors corrected and approved the manuscript.

### FUNDING

This work was supported by University of Tasmania and CSIRO joint funding. JG was a recipient of University of Tasmania and CSIRO Ph.D. Scholarship.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer GP and handling Editor declared their shared affiliation.

Copyright © 2018 Gray, Chandry, Kaur, Kocharunchitt, Bowman and Fox. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Complete Genomic Analysis of a Salmonella enterica Serovar Typhimurium Isolate Cultured From Ready-to-Eat Pork in China Carrying One Large Plasmid Containing mcr-1

### Edited by:

Giovanna Suzzi, Università di Teramo, Italy

#### Reviewed by:

Haijian Zhou, Chinese Center for Disease Control and Prevention, China Rong Zhang, Second Affiliated Hospital of Zhejiang University School of Medicine, China Maria Schirone, Università di Teramo, Italy

#### \*Correspondence:

Fengqin Li<EMAIL_ADDRESS>Séamus Fanning<EMAIL_ADDRESS>
†These authors have contributed equally to this work as co-first authors.

#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 12 December 2017 Accepted: 16 March 2018 Published: 27 April 2018

#### Citation:

Wang W, Baloch Z, Zou M, Dong Y, Peng Z, Hu Y, Xu J, Yasmeen N, Li F and Fanning S (2018) Complete Genomic Analysis of a Salmonella enterica Serovar Typhimurium Isolate Cultured From Ready-to-Eat Pork in China Carrying One Large Plasmid Containing mcr-1. Front. Microbiol. 9:616. doi: 10.3389/fmicb.2018.00616 Wei Wang<sup>1</sup>† , Zulqarnain Baloch<sup>2</sup>† , Mingyuan Zou<sup>3</sup>† , Yinping Dong<sup>1</sup> , Zixin Peng<sup>1</sup> , Yujie Hu<sup>1</sup> , Jin Xu<sup>1</sup> , Nafeesa Yasmeen<sup>4</sup> , Fengqin Li<sup>1</sup> \* and Séamus Fanning1,5,6 \*

<sup>1</sup> Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing, China, <sup>2</sup> College of Veterinary Medicine, South China Agricultural University, Guangzhou, China, <sup>3</sup> Heilongjiang Provincial Center for Disease Control and Prevention, Harbin, China, <sup>4</sup> Institute of Microbiology, University of Agriculture Faisalabad, Faisalabad, Pakistan, <sup>5</sup> UCD-Centre for Food Safety, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Dublin, Ireland, <sup>6</sup> Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Belfast, Ireland

One mcr-1-carrying ST34-type Salmonella Typhimurium WW012 was cultured from 3,200 ready-to-eat (RTE) pork samples in 2014 in China. Broth dilution method was applied to obtain the antimicrobial susceptibility of Salmonella Typhimurium WW012. Broth matting assays were carried out to detect transferability of this phenotype and whole-genome sequencing was performed to analyze its genomic characteristic. Thirty out of 3,200 RTE samples were positive for Salmonella and the three most frequent serotypes were identified as S. Derby (n = 8), S. Typhimurium (n = 6), and S. Enteritidis (n = 6). One S. Typhimurium isolate (S. Typhimurium WW012) cultured from RTE prepared pork was found to contain the mcr-1 gene. S. Typhimurium WW012 expressed a level of high resistance to seven different antimicrobial compounds in addition to colistin (MIC = 8 mg/L). A single plasmid, pWW012 (151,609-bp) was identified and found to be of an IncHI2/HI2A type that encoded a mcr-1 gene along with six additional antimicrobial resistance genes. Plasmid pWW012 contained an IS30-mcr-1-orf-orf-IS30 composite transposon that can be successfully transferred to Escherichia coli J53. When assessed further, the latter demonstrated considerable similarity to three plasmids pHYEC7-mcr-1, pSCC4, and pHNSHP45-2, respectively. Furthermore, plasmid pWW012 also contained a multidrug resistance (MDR) genetic structure IS26-aadA2-cmlA2-aadA1-IS406-sul3-IS26-dfrA12-aadA2-IS26, which showed high similarity to two plasmids, pHNLDF400 and pHNSHP45-2, respectively. Moreover, genes mapping to the chromosome (4,991,167-bp) were found to carry 28 mutations, related to two component regulatory systems (pmrAB, phoPQ) leading to modifications of lipid A component of the lipopolysaccharide structure. Additionally, one mutation (D87N) in the quinolone resistance determining region (QRDR) gene of gyrA was identified in this mcr-1 harboring S. Typhimurium. In addition, various virulence factors and heavy metal resistance-encoding genes were also identified on the genome of S. Typhimurium WW012. This is the first report of the complete nucleotide sequence of mcr-1-carrying MDR S. Typhimurium strain from RTE pork in China.

Keywords: MDR Salmonella enteric serovar Typhimurium, conjugation, mcr-1, phoP/Q, pmrA/B, plasmids, readyto-eat pork

### INTRODUCTION

fmicb-09-00616 April 25, 2018 Time: 15:27 # 2

Salmonella is often acquired from contaminated food, and is an important zoonotic bacterium linked to cases of gastroenteritis and bacteremia. This bacterium is now the leading cause of global bacterial food poisoning out breaks and is associated with increased morbidity and mortality (Kirk et al., 2015). There are estimated to be 94 million cases of gastroenteritis globally per year, with 155,000 deaths attributed to this bacterium (Majowicz et al., 2010; Gallati et al., 2013). It was reported that between 1994 and 2005, approximately 22% of food borne infections in China were caused by Salmonella (Wang et al., 2007). Of note, Salmonella Typhimurium is one of the most prevalent serovars identified among these food borne illnesses in China (Zhang et al., 2014). Salmonella Typhimurium strains are most commonly isolated from retail meat particularly pork in China, and both sporadic and outbreakassociated cases of human salmonellosis caused by this bacterium often need clinical therapy (Zhang et al., 2016). Moreover, this bacterium has the potential to act as a reservoir for different antimicrobial resistance-encoding genes (Zhang et al., 2016).

The relevance of S. Typhimurium is also marked by its capability to acquire resistance determinants, to various drug classes, especially those that are third-generation cephalosporins, tetracyclines, (fluoro) quinolones, folate pathway inhibitors, phenicols, penicillines, aminoglycosides and macrolides (Wong et al., 2014; Yi et al., 2017). Resistance to polymyxin in bacteria including Salmonella is commonly due to chromosomal mutations in two component regulatory systems (e.g., pmrAB and phoPQ) resulting in structural modification of lipid A. Therefore, bacterial resistant to polymyxin was historically thought to be rare (Lee et al., 2014). However, recently, a new plasmid-encoding polymyxin resistance gene, mcr-1, was discovered from bacterial isolated from the environment, animals, and humans in China (Liu et al., 2016). Subsequently, occurrence of the mcr-1 gene has been reported worldwide, indicating a newly recognized global distribution. Furthermore, the mcr-1-bearing plasmids were found to possess the ability to disseminate between food-producing animal and human bacteria and were also highly stable in these backgrounds, even in the absence of polymyxin selection (Anjum et al., 2016; Kempf et al., 2016; Liu et al., 2016; El Garch et al., 2017).

The mcr-1 gene in Salmonella was originally detected from human and food in England and Wales, including 8 S. Typhimurium, 1 S. Paratyphi B var Java and 1 S. Virchow strains (Doumith et al., 2016). Since then, this gene was reported from Salmonella isolates in Europe, the United States, and China, from humans, food-producing animals, and their surrounding environment, and retail meat (Anjum et al., 2016; Campos et al., 2016; Figueiredo et al., 2016; Quesada et al., 2016; Vinueza-Burgos et al., 2016; Yang et al., 2016). Nonetheless, to date, few reports of the detection of mcr-1 have been cited in Salmonella from ready-to-eat (RTE) pork meat in China. In this study, we report for the first time, the prevalence of Salmonella species among RTE pork in China, and while one S. Typhimurium isolate was then found to be positive for the mcr-1 gene, which was located on a large transferable plasmid, along with multiple antimicrobial resistance genes. The complete genome sequence of both the plasmid and chromosome was determined to shed insight into the diversity and complexity of this interesting strain.

### MATERIALS AND METHODS

### Strains Isolation

A total of 3,200 RTE pork samples were collected from 32 retail outlets and 32 commercial hypermarkets, in 32 provincial capitals of China in 2014 (**Figure 1**). Fifty RTE pork samples were collected at each sampling site and all were stored inside tightly sealed aseptic bags, surrounded by a biological ice bag, and then placed in a box maintained at a temperature lower than 4◦C. Samples were immediately transported to the laboratory and subjected to microbiological analysis within 2 h. All samples were subjected to qualitative analysis for Salmonella using an enrichment method described by the National Food Safety Standard of China-Food microbiological examination, Salmonella (GB 4789.4-2010). Finally, presumptive Salmonella were selected for biochemical confirmation using API 20E test identification test strips (bioMérieux, Marcy l<sup>0</sup> Etoile, France), as well as for molecular identification using PCR assay targeting the invA gene (Malorny et al., 2003). For all of the confirmed Salmonella isolates, serotypes were determined by the slide agglutination test, using Salmonella antisera (Statens Serum Institute, Denmark) according to the Kauffmann–White scheme. All confirmed Salmonella isolates were stored in brain heart infusion broth with 40% [v/v] glycerol (Land Bridge, Beijing, China) at −80◦C. Each sample retained was represented by at least one bacterial isolate.

### Detection of mcr-1 Gene

The frozen strains were cultured overnight at 37◦C in brain heart infusion broth. A TIANamp Bacterial DNA

extraction kit (TianGen DNA Kit DP302, Beijing, China) was used to extract the genomic DNA from the culture according to the manufacturer's instructions. A NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, NH, United States) was then used to evaluate the quality of DNA. PCR was performed to confirm the presence of the mcr-1 gene among the studied strains using primers targeting mcr-1 as reported previously (Liu et al., 2016). The genetic identity of all amplicons was subsequently confirmed by nucleotide sequencing (Liu et al., 2016). Finally, one S. Typhimurium isolate designated as S. Typhimurium WW012 was determined to harbor the mcr-1 gene and this isolate was selected to perform the follow-up experiments.

### Antimicrobial Susceptibility Tests (AST)

Antimicrobial susceptibility testing (AST) of the S. Typhimurium WW012 isolate was performed using broth dilution method, using the Biofosun <sup>R</sup> Gram-negative panel (Fosun Diagnostics, Shanghai, China). Data obtained was interpreted according to standards and guidelines described by Clinical and Laboratory Standards Institute [CLSI] (2016). Additionally, we also used the National Antimicrobial Resistance Monitoring System (NARMS) protocol when CLSI standards were not available. The panel of 21 antimicrobial compounds as shown in **Table 1** was tested Reference Escherichia coli strain ATCCTM25922 was used as the quality control.

### Conjugal Plasmid Transfer and Characterization

The mcr-1 harboring S. Typhimurium WW012 isolate was analyzed for its ability to transfer the colistin resistance phenotype using broth matting to the plasmid-free recipients, E. coli J53. Transfer of mcr-1 to transconjugants was confirmed by PCR (Cui et al., 2017). Plasmid DNA profiles of both the donor and transconjugants were carried out using the S1-nuclease digestion pulsed-field gel electrophoresis (S1-PFGE) molecular sub-typing method (Liu et al., 2016).

### Whole Genome Sequencing and Annotation

To assess the genomic background of S. Typhimurium WW012, whole-genome sequencing (WGS) was performed using the SMRT <sup>R</sup> Pacific Biosciences RS II platform (Pacific Biosciences, Menlo Park, CA, United States). Annotation of the genomes was performed using RAST<sup>1</sup> , BLASTn and BLASTp<sup>2</sup> programs. The open reading frame (ORF) Finder program<sup>3</sup> was used to identify features of public health interest. PlasmidFinder-1.3<sup>4</sup>

<sup>1</sup>http://rast.nmpdr.org

<sup>2</sup>http://blast.ncbi.nlm.nih.gov/Blast.cgi

<sup>3</sup>http://www.ncbi.nlm.nih.gov/orffinder

<sup>4</sup>https://cge.cbs.dtu.dk/services/PlasmidFinder/

<sup>∗</sup>Data was interpreted according to (a) CLSI; (b) NARMS; €Used as a feed additive in animal production.

was used to identify plasmid replicon types. The comprehensive antibiotic resistance database (CARD)<sup>5</sup> was used to identify antimicrobial resistance genes. Virulence factor database (VFDB)<sup>6</sup> was queried to predict the presence of virulence factors.

Three complete genomes, Salmonella Typhimurium LT2 (Accession number AE006468.1), Salmonella Typhi CT18 (AL513382.1), and Salmonella Indiana C629 (Accession number CP015724) were available from GenBank and used to compare the Salmonella pathogenicity islands (SPIs) with S. Typhimurium WW012 in this study by SPIFinder<sup>7</sup> . Finally, the antibacterial biocide and metal resistance genes database (BacMet)<sup>8</sup> was used to predict the presence of any metal-resistance genes.

### Nucleotide Accession Numbers

The complete genome of S. Typhimurium WW012 was deposited at GenBank under the Accession number CP022168 (chromosome) and CP022169 (plasmid p WW012).

### RESULTS

### Prevalence of mcr-1-Positive Salmonella

In this study, we collected and tested a total of 3,200 RTE pork samples from 32 provincial capitals in China in 2014. Thirty (30/3,200, 0.94%) samples were positive for Salmonella; one isolate from each sample was selected for subsequent analyses. In total, thirty isolates were recovered (comprising 10 serotypes), and the three most frequent serotypes among RTE pork samples covering 66.7% of the total, were identified as S. Derby (n = 8), S. Typhimurium (n = 6), and S. Enteritidis (n = 6). One (1/30, 3.3%) S. Typhimurium isolate (designated as S. Typhimurium WW012) was cultured from RTE prepared pork collected from a convenience market in Nanning city, Guangxi Province, and found to contain the mcr-1 gene. The sampling details of isolates are shown in **Supplementary Table S1**.

### Susceptibility to Antimicrobial Compounds

In this study, S. Typhimurium WW012 expressed a level of high resistance to seven different antimicrobial compounds in addition to colistin (MIC = 8 mg/L) (**Table 1**). The isolate was susceptible to all tested penicillins, cephalosporins, carbapenems, and macrolides (**Table 1**). Moreover, S. Typhimurium WW012 was also found to be susceptible to enrofloxacin, whilst expressing an intermediate resistant phenotype to ciprofloxacin (**Table 1**).

### Horizontal Transfer of mcr-1 Genes and Associated Determinants

One plasmid was identified from S. Typhimurium WW012 by S1-PFGE and the conjugation assay showed that it could be transferred to a plasmid-free recipient E. coli J53 with a conjugation frequency of 1.2 × 10−<sup>6</sup> as determined under laboratory conditions (**Table 2** and **Supplementary Figure S1**). MIC value of the transconjugant were recorded at 8mg/L using broth dilution test, which showed 64-fold increase in when compared with the recipient E. coli J53 (0.125 mg/L). This transconjugant expressed a MDR phenotype and was found to be resistance to five antimicrobial compounds including of trimethoprim, sulfamethoxazole, streptomycin, tetracycline, chloramphenicol, and florfenicol, in addition to colistin.

### Genome Sequence Information

The S. Typhimurium WW012 genome consisted of a single circular chromosome and a circular plasmid (**Table 2** and **Supplementary Figure S2**). The chromosome consisted of 4,991,167 bp with 4,684 predicted ORFs along with 110 RNAs. Meanwhile, a single circular extra-chromosomal element was identified and denoted as plasmid pWW012 of IncHI2 and IncHI2A replicon types. The total size of plasmid pWW012 was 151,609 bp including 168 predicted ORFs with the average GC content of 45.0% (**Table 2**). Moreover, the multilocus sequence type (MLST) of S. Typhimurium WW012 was identified as ST34 (**Table 2**).

<sup>5</sup>https://card.mcmaster.ca/analyze

<sup>6</sup>http://www.mgc.ac.cn/VFs/main.htm

<sup>7</sup>http://cge.cbs.dtu.dk/services/SPIFinder

<sup>8</sup>http://bacmet.biomedicine.gu.se/

.

TABLE 2 | The summary of the features associated with the genome and plasmid identified in S. Typhimurium WW012<sup>∗</sup>

<sup>∗</sup>No features were identified.

### Acquired Antimicrobial Resistance-Encoding Genes Identified in the Bacterial Genome

The CARD database was queried to identify resistance related genotypes on the genome of S. Typhimurium WW012. A total of 21 antimicrobial resistance genes (two genes were identified as aadA2) were identified, which encoded resistance to 11 antimicrobial agents of 10 different classes, including 14 genes on the chromosome and 7 on the plasmid (**Table 2** and **Supplementary Figure S2**). In detail, when compared with S. Typhimurium LT2, one missense mutation in gyrA (D87N) and a total of 24 missense mutations in the PmrAB and PhoPQ systems were found within the chromosome of S. Typhimurium WW012. Moreover, four and five additional antimicrobial resistance genes of strA/B, sul2, tet(B) were also found within the chromosome and plasmid, respectively, (**Table 2** and **Supplementary Figure S2**).

### Virulence Factors Annotated in the Bacterial Genome

To identify potential virulence-encoding genes in S. Typhimurium WW012, the genome was used to query those factors listed in the VFDB. These were aligned to the ORF protein sequences using BLASTP and filtered with 90% identity and match length. Using this approach, 201 virulence factors were identified on the chromosome, among which 66 genes belonged to type-three secretion system (T3SS) encoding genes (**Supplementary Figure S2** and **Supplementary Table S2**). The genomes of S. Typhimurium WW012 were also compared with those of S. Typhimurium LT2, S. Typhi CT18, and S. Indiana C629 in NCBI to identify the presence of SPIs. It was found that S. Typhimurium WW012 possessed SPI-3, SPI-4, SPI-5, SPI-13, SPI-14, and C63PI (**Table 3**) in addition to SPI-1 and SPI-2. Moreover, 63 fimbrial adherence-encoding genes and other virulence-encoding genes were identified on the chromosome of S. Typhimurium WW012 (**Supplementary Figure S2** and **Supplementary Table S2**).

### Metal Resistance Genes Annotated in the Bacterial Genome

The antibacterial biocide and metal resistance-encoding gene database (BacMet) was used to predict the presence of metalresistance genes encoded on the genome of S. Typhimurium WW012. Twenty-eight resistance genes related to arsenic, copper, iron, zinc, magnesium, cobalt, mercury, molybdenum, nickel, and silver, were located on the chromosome, whilst 7 tellurium resistance encoding genes were identified as terA/B/C/D/E/W/Z (**Supplementary Figure S2** and **Supplementary Table S3**) on plasmid pWW012.

### Comparative Analysis of Plasmid pWW012

The mcr-1 gene located on plasmid pWW012 was bracketed by two IS30 elements that were located in the same orientation. Similar structures were found in plasmids pHYEC7-mcr-1

<sup>∗</sup>Not identified.

(KX518745), pHNSHP45-2 (KU341381), and pSCC4 (CP021078) recovered previously from E. coli and Citrobacter braakii, respectively, (**Supplementary Figure S3A**). The insertion element IS30 showed 100% (95% query coverage) of homologous sequence with ISApl1 in plasmid pHNSHP45-2, where in 17 amino acids were missing at the 5<sup>0</sup> -end comparing with the latter (**Supplementary Figure S3A**). In addition to the mcr-1 gene locus, plasmid pWW012 also contained an additional MDR gene cassette embedded in a complex class 1 integron of 15.2-kbp (**Supplementary Figure S3B**) bracketed by two IS26 elements in but found in inverted orientations directions, at a distance from the mcr-1 gene locus (**Supplementary Figure S2**). The class 1 integron was preceded by a copy of insertion element IS26, and two transposase-encoding genes tnpR and tnpM, followed by the aadA2, cmlA1, and aadA1 genes. Downstream of these resistance genes, an interesting resistance gene locus consisting of IS406–sul3-orf-orf-orf-IS26 was identified. This locus was followed by one integrase, two resistance genes of dfrA12 and aadA2, and the IS26 element. This structure has also been identified in two E. coli originated plasmids designated as pHNLDF400 (KV019258.1) and pHNSHP45-2 (KU341381) (**Supplementary Figure S3B**).

### DISCUSSION

In the past, Salmonella has attracted much attention owing to its important role as a food-borne pathogen. Recent studies reported on the prevalence of Salmonella and plasmid-encoded polymyxin resistance genes, containing mcr-1, among humans, foodproducing animals and food samples in China and elsewhere (Anjum et al., 2016; Campos et al., 2016; Doumith et al., 2016; El Garch et al., 2016; Figueiredo et al., 2016; Kempf et al., 2016; Liu et al., 2016; Quesada et al., 2016; Yang et al., 2016). However, limited comprehensive epidemiological data are available describing the prevalence of Salmonella and mcr-1 gene among RTE pork samples in China, although pork and pork-related products are the main source of animal protein for Chinese consumers. To the best of our knowledge, the current study was the first report on the epidemiological prevalence and detection of Salmonella and mcr-1 gene among RTE pork samples in China. Generally, the prevalence rate of Salmonella and the mcr-1 gene among RTE pork samples was relatively low in this study, while similar results were observed from clinical, pigs and retail pork in previous studies (Figueiredo et al., 2016; Quesada et al., 2016; Cui et al., 2017). However, considering the fact that these RTE pork samples are positive for Salmonella and therefore would not be heated or cooked the infectious risk would increase. In China, only prepackaged RTE foodstuffs have a pathogen limiting standard applied at National level. In this study all RTE pork samples were collected from marketing sites and nearly 87% of the samples that were positive for Salmonella were unpackaged. Although these pork samples were permitted for sale by the food hygiene bureau at the beginning, unpacked treatment may later increase the probability of the contamination of Salmonella during their shelf-life. Therefore, limited standard and effective legislation in RTE pork to prevent and control the contamination of Salmonella should now be considered.

In this study, we successfully recovered and characterized one Salmonella isolate (S. Typhimurium WW012) that harbored a mcr-1-bearing plasmid from a RTE prepared pork sample in China. The genus Salmonella consists of a number of serovars, where in it is reported that the most often detected serovar harboring the mcr-1-was Typhimurium (Doumith et al., 2016; Cui et al., 2017), suggesting that the acquisition and prevalence of mcr-1-bearing mobile elements may require a specific genetic background. The relationship between S. Typhimurium and mcr-1-bearing plasmids warrants further investigation (Doumith et al., 2016). This RTE pork isolate expressed a MDR phenotype in addition to colistin. Notably, all of the resistant antimicrobial agents tested in this study are widely used at human and veterinary clinics. Carnevali et al. (2016) screened for the mcr-1 gene in 4,473 Salmonella isolates from human, foodproducing animal, food and environment samples and identified 25 positive isolates of human and veterinary origin, all of which were susceptible to broad-spectrum cephalosporins. The S. Typhimurium WW012 isolate in this study was also susceptible to all tested cephalosporin antimicrobials. Similar results were reported from human, food-producing animals in China (Li et al., 2016; Yi et al., 2017).

A laboratory-based conjugation assay revealed that the mcr-1 gene was transferrable to the plasmid-free recipient E. coli J53, with a frequency of 1.2 × 10−<sup>6</sup> . In contrast, the transfer frequency of the IncI2-type plasmid carrying the mcr-1 gene reported in the original study was surprisingly higher, ranging from 10−<sup>1</sup> to 10−<sup>3</sup> between E. coli strains (Liu et al., 2016). In this study these data showed that the transfer of the mcr-1 gene from S. Typhimurium WW012 to E. coli had a frequency that was lower. Nonetheless, it was confirmed that the mcr-1-carrying IncHI2-type plasmid in this study can be transferred between different genera of Enterobacteriaceae.

Of note, according to previous reports, the IncHI2 replicon was one of the most common found in Salmonella (Campos et al., 2016; Doumith et al., 2016).  Notably, the current data showed that this transconjugant showed resistance to five antimicrobial agents in addition to colistin, indicating that S. Typhimurium WW012 could not only transfer the mcr-1 gene but also other MDRencoding genes into other Enterobacteriaceae strains. Therefore, once this clone carrying mcr-1 gene disseminates from RTE pork to humans via the food chain, it could be expected to promote the forward dissemination of the latter marker and other members of the Enterobacteriaceae. In this study S. Typhimurium WW012 belonged to ST34, which is the predominant sequence type in southern China (Sun et al., 2014). Recently, the mcr-1 gene was also detected in MDR S. Typhimurium (ST34) in China and Europe (Doumith et al., 2016; Li et al., 2016).

In this study, the plasmid mediated colistin resistance gene mcr-1 was found to be located in a composite transposon with the structure IS30-mcr-1-orf-orf-IS30, which was similar to those found previously on two E. coli plasmids pHYEC7 mcr-1 (KX518745) and pHNSHP45-2 (KU341381), and one C. braakii plasmid pSCC4 (CP021078, unpublished) (Liu et al., 2016; Wang et al., 2018). Unlike plasmid pHNSHP45-2, the original IncHI2-type mcr-1-carrying episome, the mcr-1 gene located on plasmids pWW012, pHYEC7-mcr-1 and pSCC4 was bracketed by IS30 elements. According to a latest report, ISApl1 is a member of the IS30 family of insertion sequences, which utilize a 'copy-out, paste-in' mechanism with a targeted transposition pathway requiring the formation of a synaptic complex between an inverted repeat (IR) in the transposon circle and an IR-like sequence in the target (Wang et al., 2018). IS30 is related to site-specific recombination, which could mediate the transmission of mcr-1 among different bacterial species. Moreover, it was found that several resistance genes were also found to be present and located between two IS26 elements on plasmid pWW012, in which a class 1 integron similar to that found in plasmids pHNSHP45-2 and pHNLDF400 (KY019258.1) also existed (Liu et al., 2016; Wang et al., 2017). The recovery of plasmid pWW012 that harbored structurally similar mobile elements to these contained in these reported plasmids implied that genetic exchange between such plasmids was common, and that such plasmids could readily be transferred to other bacteria, constituting one major evolution and resistance development route for bacterial pathogens.

In addition to the plasmid-mediated colistin resistance gene mcr-1, genes found on chromosome containing mutations, encoding two component regulatory systems (pmrAB, phoPQ), were also found in this study. These chromosomal mutations mediated mechanisms were thought to lead to the modification of lipid A, which anchors the lipopolysaccharide molecule to the outer membrane, resulting in reduction in the affinity to colistin (Lee et al., 2014). Moreover, a target gene mutation in the quinolone resistance determining region (QRDR) on the chromosome were identified in gyrA mutation (giving rise to D87N amino acid substitution) in this mcr-1 harboring S. Typhimurium. The latter is frequently reported in other MDR Salmonella species in China (Wong et al., 2014). In addition, strA/B encoding streptomycin resistance and sul2 encoding sulfamethoxazole resistances were also detected to locate on the chromosome of S. Typhimurium WW012.

In this study, 201 virulence genes were found on the chromosome of S. Typhimurium WW012 when queried through VFDB. Type-three secretion systems (T3SS) encoding genes related to SPI-1 and SPI-2 were commonly detected. Both SPI-1 and SPI-2 contained two independent type three secretion systems (denoted as TTSS SPI-1 and TTSS SPI-2, respectively) that can inject effect or proteins into host cells, which are crucial for various stages of infection (Weening et al., 2005). Comparison of the genomes S. Typhimurium WW012 with the reference or complete sequences in NCBI for the presence of SPIs showed that SPIs in S. Typhimurium WW012 were closely related to their equivalents in S. Typhimurium LT2 (McClelland et al., 2001), and shared some of those in S. Typhi CT18 and S. Indiana C629 (Parkhill et al., 2001; Wang et al., 2016). Additionally, 63 fimbrial adherence encoding genes on the chromosome of S. Typhimurium WW012 were also identified in this study. Several in vitro studies have suggested that fimbrial-encoding genes found in Salmonella could mediate the attachment of this bacterium to epithelial cells in the host (Leclerc et al., 2016; Peters et al., 2017). The latter phenotype may support long-term intestinal carriage of this bacterium.

Heavy metal compounds as well as antimicrobial agents are widely used as feed additives in food-producing animals for therapy, and growth purposes in China (Hu et al., 2017). Because of their stable and persistent characteristics, when heavy metals accumulate to specific concentrations, they potentially result in resistance among bacteria cultured from food-producing animals. Heavy metal resistance-encoding genes have also been identified in different environments (Deng et al., 2017). Recent research data showed that a relationship between the acquisition of heavy metal resistance genes and antimicrobial resistance genes, and antimicrobial resistance may arise through coresistance or cross-resistance to metals or co-regulation of resistance pathways (Argudín et al., 2016; Deng et al., 2017). In this study, 28 and 7 heavy metal resistance genes were found on the chromosome and plasmid, respectively. These heavy metal resistance genes might increase antimicrobial resistance capacity within this bacterium, through co-selection of determinants.

### CONCLUSION

This study firstly reported the epidemiological prevalence and detection of Salmonella and mcr-1 gene among RTE pork samples in China. These data highlight the importance of role played by S. Typhimurium in the dissemination of MDR genes. Although the mechanisms remain to be further described, the emergence of MDR genes particularly mcr-1, along with various virulence factors and heavy metal resistance genes, on the chromosome and

plasmid from S. Typhimurium, will challenge therapeutic options for clinicians and others. Moreover, successful transmission of the mcr-1 gene poses a challenge to those with an interest in the protection of public health.

### AVAILABILITY OF DATA AND MATERIALS

The aggregate data supporting findings contained within this manuscript will be shared upon request submitted to the corresponding author.

### AUTHOR CONTRIBUTIONS

WW, ZB, SF, and FL designed the experiments and wrote the manuscript. MZ, YD, YH, ZP, and JX carried out the experiments. ZB, WW, and NY analyzed the experimental results.

### FUNDING

This study was funded by the National Key R&D Program of China (2016YFD0401102) and China Food Safety Talent Competency Development Initiative: CFSA 523 Program.

### ACKNOWLEDGMENTS

We sincerely thank all the participants who took part in this study.

### REFERENCES

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00616/full#supplementary-material

FIGURE S1 | S1-PFGE indicating the transfer of single plasmid harboring mcr-1. Lane 1, Escherichia coli J53 (recipient); Lane 2, S. Typhimurium WW012 (donor); Lane 3, CT-E. coli J53(transconjugant); Lane 4, H-9812(PFGE marker strain, S.BraenderupH9812).

FIGURE S2 | A schematic representation of the complete chromosome (A) and plasmid (B) of S. Typhimurium WW012. Each ring, beginning from the inside to out represents the following features: GC content (black); GC skew (green and purple); CDS (blue); Antimicrobial resistance genes (shown in blue colored font), metal resistance genes (shown in green colored font), and virulence factor genes (shown in black colored font).

FIGURE S3 | Major structural features of plasmid pWW012 compared with several other plasmids identified from NCBI. (A) Major structural features of plasmid pWW012 compared with plasmids pHYEC7-mcr-1, pSCC4, and pHNSHP45-2 identified in E. coli and Citrobacter braakii, respectively. (B) Major structural features of plasmid pWW012 compared with plasmids pHNLDF400 and pHNSHP45-2 both identified in E. coli. ORFs are shown with arrows (blue arrows, transposase; red arrows, antibiotic resistance genes; green arrows, integrase; gray arrows, member protein; white arrows, hypothetical protein–see the key to the right hand side of the panel). The missing amino acids in IS30 comparing with ISApl1 were showed in red box.

TABLE S1 | Sampling information of Salmonella isolates in this study.

TABLE S2 | Virulence resistance-encoding genes identified in S. Typhimurium WW012 when comparing the genome of the latter against the current version of the VFDB database.

TABLE S3 | Metal resistance genes of S. Typhimurium WW012 with the current version of the BacMet database.

with disinfectant and heavy metal resistance. Microb. Drug Resist. doi: 10.1089/ mdr.2017.0127 [Epub ahead of print].

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer MS and handling Editor declared their shared affiliation.

Copyright © 2018 Wang, Baloch, Zou, Dong, Peng, Hu, Xu, Yasmeen, Li and Fanning. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Virulence Gene Sequencing Highlights Similarities and Differences in Sequences in *Listeria monocytogenes* Serotype 1/2a and 4b Strains of Clinical and Food Origin From 3 Different Geographic Locations

### *Edited by:*

Maria Schirone, Università di Teramo, Italy

#### *Reviewed by:*

Arun K. Bhunia, Purdue University, United States Valentina Bernini, Università degli Studi di Parma, Italy Hongxia Wang, University of Alabama at Birmingham, United States

> *\*Correspondence:* Kieran Jordan<EMAIL_ADDRESS>
#### *†Present Address:*

Marion Dalmasso, Normandie Univ, UNICAEN, ABTE, Caen, France

#### *Specialty section:*

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

*Received:* 06 September 2017 *Accepted:* 08 May 2018 *Published:* 05 June 2018

#### *Citation:*

Poimenidou SV, Dalmasso M, Papadimitriou K, Fox EM, Skandamis PN and Jordan K (2018) Virulence Gene Sequencing Highlights Similarities and Differences in Sequences in Listeria monocytogenes Serotype 1/2a and 4b Strains of Clinical and Food Origin From 3 Different Geographic Locations. Front. Microbiol. 9:1103. doi: 10.3389/fmicb.2018.01103 Sofia V. Poimenidou<sup>1</sup> , Marion Dalmasso2†, Konstantinos Papadimitriou<sup>3</sup> , Edward M. Fox <sup>4</sup> , Panagiotis N. Skandamis <sup>1</sup> and Kieran Jordan<sup>2</sup> \*

<sup>1</sup> Laboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, Greece, <sup>2</sup> Teagasc Food Research Centre, Moorepark, Fermoy, Co., Cork, Ireland, <sup>3</sup> Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, Greece, <sup>4</sup> CSIRO Agriculture and Food, Werribee, VIC, Australia

The prfA-virulence gene cluster (pVGC) is the main pathogenicity island in Listeria monocytogenes, comprising the prfA, plcA, hly, mpl, actA, and plcB genes. In this study, the pVGC of 36 L. monocytogenes isolates with respect to different serotypes (1/2a or 4b), geographical origin (Australia, Greece or Ireland) and isolation source (food-associated or clinical) was characterized. The most conserved genes were prfA and hly, with the lowest nucleotide diversity (π) among all genes (P < 0.05), and the lowest number of alleles, substitutions and non-synonymous substitutions for prfA. Conversely, the most diverse gene was actA, which presented the highest number of alleles (n = 20) and showed the highest nucleotide diversity. Grouping by serotype had a significantly lower π value (P < 0.0001) compared to isolation source or geographical origin, suggesting a distinct and well-defined unit compared to other groupings. Among all tested genes, only hly and mpl were those with lower nucleotide diversity in 1/2a serotype than 4b serotype, reflecting a high within-1/2a serotype divergence compared to 4b serotype. Geographical divergence was noted with respect to the hly gene, where serotype 4b Irish strains were distinct from Greek and Australian strains. Australian strains showed less diversity in plcB and mpl relative to Irish or Greek strains. Notable differences regarding sequence mutations were identified between food-associated and clinical isolates in prfA, actA, and plcB sequences. Overall, these results indicate that virulence genes follow different evolutionary pathways, which are affected by a strain's origin and serotype and may influence virulence and/or epidemiological dominance of certain subgroups.

Keywords: *Listeria monocytogenes*, virulence, gene sequencing, diversity, *prfA*, *hly*, *actA*

## INTRODUCTION

Listeria monocytogenes is a facultative intracellular foodborne pathogen, with pregnant women and neonates, immunocompromised individuals, and the elderly representing high risk groups for infection (Farber and Peterkin, 1991; EFSA ECDC, 2015). It is equally capable of both a saprophytic lifecycle in the environment and human infection causing the severe disease of listeriosis (Gray et al., 2006). Due to its wide variety of reservoirs (Farber and Peterkin, 1991; Lianou and Sofos, 2007), its ability to colonize abiotic surfaces (Møretrø and Langsrud, 2004; Poimenidou et al., 2016b) and to withstand environmental stresses (Hill et al., 2002; Poimenidou et al., 2016a), it is frequently implicated in food processing plant contamination, where it is able to persist for several months or years (Halberg Larsen et al., 2014), thus raising the risk to food safety. After transmission via contaminated food to humans, L. monocytogenes cells may cause illnesses such as gastroenteritis or invasive listeriosis following intestinal translocation. It may then be carried by blood or lymph fluid and reach the mesenteric lymph nodes, spleen and/or the liver, leading to subclinical pyogranulomatous hepatitis, meningoencephalitis, septicemia, placentitis, abortion, or neonatal septicemia (Vázquez-Boland et al., 2001b). Within the host, L. monocytogenes parasitizes macrophages and invades non-phagocytic cells, utilizing its virulence factors to mediate cell-to-cell spread (de las Heras et al., 2011).

The virulence potential of L. monocytogenes relies on several molecular determinants (Camejo et al., 2011), which play key roles at different stages of the infection process. Among the early stages of the infection process, genes including the internalins (inlA, inlB, inlF, inlJ) play key roles in adhesion and invasion. Intracellular pathogenesis heavily relies on factors transcribed by genes located in the major prfA-regulated virulence gene cluster (pVGC), also referred to as Listeria pathogenicity island 1 or LIPI-1 (Vázquez-Boland et al., 2001a; Ward et al., 2004). pVGC genes facilitate the intracellular growth and spread of the bacterium in the host and consist of a monocistron hly, which occupies the central position in the locus, a lecithinase operon comprising mpl, actA, and plcB genes, which is located downstream from hly and transcribed in the same orientation, and the plcA-prfA operon located upstream from hly and transcribed in the reverse direction (Portnoy et al., 1992; Vázquez-Boland et al., 2001b; Roberts and Wiedmann, 2003). The prfA gene encodes the PrfA protein, which is required for the transcription of pVGC, and prfA itself. Listeriolysin O (LLO) encoded by the hly gene is a pore-forming toxin that mediates lysis of bacterium-containing phagocytic vacuole, resulting in the release of bacterial cells into the host cytoplasm. plcA and plcB encode the phosphatidylinositol-specific phospholipase C (PI-PLC) and zinc-dependent broad-spectrum phospholipase C (PC-PLC), respectively, which synergistically with LLO mediate the escape of the pathogen from the single- and double-membranebound vacuoles. After lysis, the intracellular motility and cell-tocell spread are mediated by the surface protein actin A (ActA) through actin polymerization, for which additional functions (i.e., role in invasion, aggregation, colonization and persistence in the gut lumen) have been reported (Suárez et al., 2001; Travier et al., 2013). mpl encodes a zinc metalloproteinase needed to activate PC-PLC in order to initiate a new infection cycle.

Listeria monocytogenes is a genetically diverse species; its isolates form a structured population and are differentiated into four distinct lineages and 13 serotypes (Orsi et al., 2011), with the majority of isolates clustering into lineage I (serotypes 1/2b, 3b, 3c, 4b) and lineage II (serotypes 1/2a, 1/2c, 3a).  Additionally, various L. monocytogenes strains have presented diversity in virulence potential (Brosch et al., 1993; Chakraborty et al., 1994; Jaradat and Bhunia, 2003; Roche et al., 2003; Neves et al., 2008). Defective forms of virulence determinants were identified as the source of such virulence attenuation (Olier et al., 2002, 2003; Roberts et al., 2005; Roche et al., 2005; Témoin et al., 2008; Van Stelten et al., 2011).

The reasons that 1/2a serotype strains predominate among food environment isolates and 4b serotype strains among human listeriosis isolates are under investigation, with no clear inference made so far (Jaradat et al., 2002; Larsen et al., 2002; Gray et al., 2004; Jensen et al., 2007, 2008; Neves et al., 2008; Houhoula et al., 2012). On the other hand, there are indications of selective pressure for maintenance or specific adaptation of the pVGC genes in particular environments (Roberts et al., 2005; Orsi et al., 2008; Travier et al., 2013). Comparative genotyping could contribute to identifying unique genetic determinants toward the intraspecific pathogenic characteristics of L. monocytogenes isolates. Considering the above, the objective of this study was to examine the nucleotide diversity of the pVGC genes of L. monocytogenes strains isolated from human clinical cases and food or food-related environments, which belonged to the serotypes 4b and 1/2a and originated from three distinct geographical locations (i.e., Australia, Greece, and Ireland). Studying these variations may provide valuable information toward understanding the significance of virulence gene variation and the influence of environmental pressures acting on the genes.

### MATERIALS AND METHODS

### Bacterial Strains

A total of 36 Listeria monocytogenes strains (**Table 1**) were analyzed in this study. The strains represented three distinct geographically dispersed regions (Australia, Greece, Ireland), two serotypes (serotype 4b and 1/2a) and two isolation sources (clinical and food–related isolates). The clinical strains were kindly provided by Dr. Josheph Papaparaskevas (Houhoula et al., 2012) and Prof. Martin Cormican (University College Hospital, Galway, Ireland). The food-associated isolates were obtained from food and the food-processing environment. The strains were serotyped using a combination of antisera specific to the L. monocytogenes somatic O-antigen (Denka Seiken Co., Ltd., Tokyo, Japan), in tandem with a PCR-based serovar

TABLE 1 | Origins and characteristics of 36 L. monocytogenes strains used in the study.

determination assay (Doumith et al., 2004), as described by Fox et al. (2009). Bacterial strains were stored at −80◦C in Tryptic Soy broth (TSB) containing 20% glycerol and were cultured in TSB supplemented with 0.6% yeast extract (YE) at 37◦C overnight, prior to pulsed-field gel electrophoresis (PFGE) and DNA extraction.

### PFGE of *L. monocytogenes* Isolates

PFGE was carried out using the International Standard PulseNet protocol (Pulsenet USA, 2009). Two restriction enzymes, AscI and ApaI, were used and band patterns were analyzed using Bionumerics version 5.10 software (Applied Maths, Belgium), as previously described (Fox et al., 2012). Briefly, band matching was performed using the DICE coefficient, with both optimization and tolerance settings of 1%. Dendrograms were created using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Strains were considered to be indistinguishable when their pulsotypes displayed 100% similarity on the dendrogram and after confirmation by visual examination of the bands. To help support population diversity, all isolates were confirmed as having a unique pulsotype relative to any other isolate included in this study.

### DNA Extraction

Following overnight culture of each strain, DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen, UK) for strains from both Greece and Australia, or the QIAmp DNA mini kits (Qiagen) for strains from Ireland. A cell lysis step preceded DNA extraction and consisted in incubation of the cells in lysis buffer (20 mM TrisHCl, pH 8; 2 mM EDTA, pH 8; 1.2% Triton <sup>R</sup> −100; 20 mg/ml lysozyme) for 1 h at 37◦C. DNA was stored at −20◦C before use.

### Nucleotide Sequencing of *actA, hly, mpl, plcA, plcB, prfA*

PCR amplification of the targeted genes was performed using genomic DNA extracted as described above. Primer design was based on available sequences of the targeted genes in public databases using Primer3Plus software version 2.3.5 (Untergasser et al., 2012). The primers and PCR conditions, all including 35 cycles, are described in **Table 2**. Phusion <sup>R</sup> High-Fidelity DNA polymerase (New England Biolabs <sup>R</sup> Inc, USA) and AccuTaqTM LA DNA polymerase (Sigma, USA) were used for PCR reactions on 50 ng DNA for strains from Greece and Ireland, respectively. Following amplification, PCR products were purified using MinElute Gel Extraction kit (Qiagen). DNA sequencing was performed using external forward and reverse PCR primers at CEMIA SA (Larisa, Greece) and Source Biosciences (Dublin, Ireland) for Greece and Ireland PCR products, respectively. In the case of Australian isolates, sequences were extracted in silico from draft genomes using the same primer sets (**Table 2**) with Geneious <sup>R</sup> software version 9 (Kearse et al., 2012). DNA sequencing chromatograms were saved as ABI files for analysis.

### Data Analysis

Sequence assembly was performed using SeqMan Pro application in Lasergene <sup>R</sup> Genomics suite (DNASTAR, USA). Geneious <sup>R</sup> software version 9 (Kearse et al., 2012) was used to construct translation alignments for each gene separately and the pVGC (a concatenated sequence comprising the prfA, plcA, hly, mpl, actA, plcB sequences).

### Descriptive Analysis

Number of polymorphic sites (S), nucleotide diversity (π; average pairwise nucleotide differences per site), number of segregating sites (θ), and Tajima's D for neutrality were calculated using DnaSP software version 5 (Librado and Rozas, 2009). Number of polymorphic sites, number of substitutions, number of synonymous substitutions (SS) and non-synonymous substitutions (NSS), and the G + C content

<sup>a</sup>External primers located in upstream and downstream regions surrounding the targeted gene. <sup>b</sup> Internal primers. <sup>c</sup>Roche et al., 2005.

(%) were defined using Geneious software version 9 (Kearse et al., 2012). The dN/d<sup>S</sup> ratios or ω [number of non-synonymous substitutions/nonsynonymous sites (dN) to the number of synonymous substitutions/synonymous sites (dS)] were calculated using the Datamonkey online platform (Kosakovsky Pond and Frost, 2005). 3D scatterplots were created using 'Excel 3D Scatter Plot' version 2.1 (available at: http://www.doka.ch/ Excel3Dscatterplot.htm).

### Phylogenetic Analysis

Phylogenetic trees were generated using the NeighborNet algorithm (Bryant and Moulton, 2004) as adopted in SplitsTree software (Huson, 1998).

### Statistical Analysis

Descriptive analysis data calculated for individual genes were used in order to compare π, θ, and ω parameters for the pVGC with regard to different serotypes, geographical origin or isolation source using Student's t test (JMP version 9.0); significance level was set at α = 0.05.

### RESULTS

Among the 36 strain sequences analyzed, representing distinct PFGE profiles (Supplementary Material), 26 unique alleles were identified for pVGC (**Table 3**, Supplementary Dataset S1). Twenty-three isolates harbored a full length cluster of 7,503 nucleotides; 12 isolates had a 105 bp deletion in their actA sequence and as such had a 7,398 bp pVGC; one isolate had a single nucleotide deletion in its actA gene sequence and thus a 7,502 bp pVGC.

The pVGC contained 439 polymorphic sites, with 281 synonymous and 182 non-synonymous substitutions. The G + C% content was 37.2%. The overall nucleotide diversity was π = 0.02427 and θ = 0.01601. Although π and θ values for serotype 1/2a strains were higher than for 4b strains, the difference was not significant (P > 0.05). No significant π difference was observed among strains of different geographical origin, or between food environment and clinical origin. Comparing groupings by serotype, geographical origin or isolation source, grouping by serotype had a significantly lower π value (P < 0.0001). Serotype groups also exhibited distinct clustering on the 3D-scatter plot (**Figure 1A**), showing divergence from the other groupings. Divergence between the two serotypes in dN/d<sup>S</sup> ratio was also observed, suggesting different selective pressure acting on the two serotypes, with higher values among the serotype 1/2a group. The pVGC phylogenetic tree (**Figure 2**) showed two major distinct clusters representing the two serotypes, 1/2a and 4b. No specific pattern of origin-based classification was observed, with strains isolated in different countries or from different sources (i.e., foodassociated or clinical) sharing an identical nucleotide sequence. In each serotype group, stains were clustered in short distances to each other, with only strain GR\_PL50 distant to the others.

Eight haplotypes among the 36 strains were recovered for the prfA gene (5 for 1/2a serotype and 3 for 4b serotype). This gene possessed the lowest number of polymorphic sites (n = 24) with the lowest number of substitutions (n = 24) and non-synonymous substitutions (n = 4) compared to all fragments tested (**Table 3**). The overall nucleotide diversity was π = 0.01551 and θ = 0.01296. Groups containing strains of different geographical origin were clustered closely to each other (**Figure 1B**), while food isolates were distinct from the clinical isolates with respect to π values. Divergence in dN/dS, π and θ parameters resulted in distinct clustering of serotype groups compared to other groupings. The phylogenetic tree of prfA gene (**Figure 3**) showed the lowest degree of divergence among all tested genes, with longer branch lengths observed for 1/2a serotype isolates than for 4b isolates, which is in accordance with the higher nucleotide diversity within 1/2a serotype than 4b serotype (**Table 3**). Among the 19 substitutions observed for clinical isolates, none of them were non-synonymous.

The nucleotide sequence of the plcA gene (13 haplotypes; π = 0.02215) was diversified into 10 unique alleles of 1/2a serotype strains (π = 0.01624) and 3 alleles of 4b serotype strains (π = 0.0419). Serotype 4b strains had the lowest number of substitutions (n = 6) compared to the other subgroups (n = 39– 57), which resulted in the lowest nucleotide diversity. Serotype 1/2a strains differed from the other groups in dN/d<sup>S</sup> ratio values and serotype 4b strains in θ values, resulting in distinct clustering on the 3D-scatter plot (**Figure 1C**). The phylogenetic tree of the plcA gene (**Figure 4**) showed that isolates of the 1/2a serotype were highly divergent with more distant branches compared to 4b serotype strains. Unique sequence types in the group of 1/2a serotype belonged to Australian or Irish origin strains.

Analysis of the hly gene showed 19 haplotypes among the 36 strains with overall nucleotide diversity π = 0.01409 and θ = 0.01044. Higher diversity was observed among 1/2a serotype than 4b serotype strains (11 and 8 unique alleles, respectively). Groups of different geographical origin or groups of different isolation source (i.e., food environment or clinical) were clustered closely to each other (**Figure 1D**), in contrast to different serotypes, where the two groups (i.e., 1/2a and 4b serotypes) clustered apart along the dN/d<sup>S</sup> ratio axis showing a diverse selective pressure acting on the gene within each serotype. As illustrated in **Figure 5**, a high divergence in hly gene sequences among strains of 4b serotype was observed; two subpopulations were identified, one of which only included Irish isolates. The second subpopulation contained two sets of strains with shared sequences between Australian and Greek strains, and three unique alleles (i.e., one Greek strain and two Irish).

The mpl gene was represented by 14 unique alleles, 10 for 1/2a serotype and 4 for 4b serotype, with π = 0.02413 and θ = 0.01873. Grouping according to serotypes resulted in distinct clusters compared to the other groupings (**Figure 1E**), due to lower π values, while additionally the two serotype groups (i.e., 1/2a and 4b) differed in their dN/d<sup>S</sup> ratio demonstrating diverse selective pressure acting on the strains of each serotype within this gene. The phylogenetic tree for the mpl gene (**Figure 6**)

TABLE 3 |

Continued

from 0 are indicated with \*(0.05 < P < 0.1) or with \*\*(P < 0.05).

 The value is not available, as it could be not evaluated due to the low number of alleles (4 or more sequences needed).

showed a similar clustering of the strains between the two serotypes with respect to branch lengths, and higher divergence within the 1/2a serotype compared to 4b serotype, in terms of unique alleles.

The actA gene was represented by 20 unique alleles, the highest number among any of the pVGC genes, with overall nucleotide diversity π = 0.03782 and θ = 0.029. Groups containing strains of various origins or serotypes were highly variant, as illustrated in **Figure 1F**, confirming the diversity of this particular gene. Strains of serotype 1/2a were more diverse (π = 0.01819, θ = 0.01594) than serotype 4b strains (π = 0.0055, θ = 0.00572). This was also evident from the phylogenetic tree (**Figure 7**), where 13 different nucleotide sequences were found among 18 isolates of 1/2a serotype, with longer branch lengths compared 4b serotype strains. Food isolates had the highest number of non-synonymous substitutions (n = 103) among all subgroups within this gene and clinical isolates the lowest (n = 12). A large variation between the dN/d<sup>S</sup> ratio values was observed for food and clinical isolates, suggesting a different selective pressure acting on these two groups. Divergence in dN/d<sup>S</sup> was also observed between Australian and Greek or Irish isolates. Twelve isolates, representing 5 unique alleles, had a 105-bp deletion in their sequences; 8 of these isolates were of food environment origin and 4 of clinical origin. The isolate (AU\_Lm14-002) that had a single nucleotide deletion was of food origin.

For the plcB gene, 12 haplotypes were observed among the 36 strains, with nucleotide diversity π = 0.02254 and θ = 0.01751. Serotype 1/2a strains were more diverse than 4b strains, represented by higher numbers of unique alleles (8 and 4, respectively), and higher π and θ values. Food-related strains differed from clinical strains, and Australian strains clustered apart from Greek and Irish strains (**Figure 1G**), showing lower nucleotide diversity and thus, a higher genetic uniformity within the former groups (i.e., food or Australian) compared to the latter (i.e., clinical, Greek, or Irish). In the phylogenetic tree (**Figure 8**), the short length of the branches indicated the small divergence level among strains within each serotype.

Comparing all genes, the most diverse gene was actA (π = 0.03782) and the most conserved hly (π = 0.01409) and prfA (π = 0.01551); the π value of actA was significantly higher compared to hly (P = 0.0095) or prfA (P = 0.0088). Additionally, for pVGC no significant difference in nucleotide diversity was observed between the two serotype groupings, the two isolation sources or the three geographical origin groups. Higher nucleotide diversity in serotype 4b vs. serotype 1/2a was only observed for mpl and hly genes. Regarding the selective pressure acting on the genes, the highest values of the dN/d<sup>S</sup> ratio were observed for actA and the lowest on prfA and hly genes (P < 0.05).

Tajima's D-test for neutrality (Tajima, 1989; Simonsen et al., 1995), which examines whether the occurring mutations are

a result of selection or random (neutral) evolution, showed a significantly positive value for the test for the pVGC (**Table 3**). This suggests that the gene evolution deviates significantly from the standard neutral model and is under balancing selection, decrease in population size or a subdivision of the population structure. High Tajima's D-values (0.1 > P > 0.05) were also observed for food and clinical isolates in the pVGC, for Irish isolates in the mpl gene and for clinical isolates in the plcB gene. Negative values were observed for serotype 1/2a strains in the pVGC, prfA, and plcB genes, and for 4b serotype in plcB; however, these were not statistically significant and therefore are unlikely to represent a population bottleneck, a selective sweep or purifying selection.

### DISCUSSION

In the present study, the intraspecies variations in the prfA virulence gene cluster among 36 L. monocytogenes strains, with respect to different serotype (i.e., 1/2a and 4b), geographical origin (Australian, Greek, and Irish isolates), or isolation source (i.e., food environment or clinical isolates) was investigated. Consistent with previous classification studies (Ward et al., 2004; Orsi et al., 2008), within all six virulence genes analyzed and the pVGC, strains were divided into two major clusters, each representing one serotype, i.e., 4b and 1/2a serotype, which belong to lineage I and II, respectively. L. monocytogenes is a highly diverse species and lineages I and II are considered to be deeply separated evolutionary lineages (Nightingale et al., 2005). Significant association between lineage and the origin

of the strains has been reported (Wiedmann et al., 1997), while additionally, molecular types of the strains were shown to be associated with specific food types (Gray et al., 2004). Strains of different lineages are also divergent in terms of their virulence potential. While higher virulence associated with the lineage I population relative to that of lineage II has been reported, (Wiedmann et al., 1997; Norton et al., 2001; Gray et al., 2004; Jensen et al., 2007), others found no statistical correlation between virulence of the strains and their serotypes (Conter et al., 2009). Therefore, molecular typing and a better understanding of virulence stratification among serotypes and lineages are essential in epidemiological surveys and risk estimation procedures. The analysis in this study also showed that 4b serotype strains exhibited lower diversity than the 1/2a strains. This is consistent with previous findings where lineage II strains were genetically more diverse compared to lineage I, based on molecular typing of seven genetic loci including four housekeeping genes, two virulence genes and stress response sigB gene (den Bakker et al., 2008), ribotyping and random multiprimer PCR analysis (Mereghetti et al., 2002), or analysis of the prfA virulence gene cluster (Orsi et al., 2008). In addition to these reports, it was shown here that ω values were similar between the serotype groups for prfA and plcB, while varied largely for the pVGC, plcA, hly, mpl, and actA, indicating a different selective pressure

acting on these genes within each serotype. Furthermore, the opposite (i.e., negative vs. positive) Tajima's D values for the serotype groups within pVGC, hly, mpl, and actA suggest that these genes follow a different evolutionary pathway across serotypes.

Results of this study showed that among the six genes examined, only the hly gene of 4b serotype strains was partially correlated with geographical origin, with strains separating into two distinct subpopulations: one containing only Irish strains, the other containing Greek and Australian strains and two Irish strains. Since serotype 4b strains have been found as the etiological agent of the majority of epidemic or sporadic human listeriosis cases in many countries, including Ireland (Schuchat et al., 1991; Swaminathan and Gerner-Smidt, 2007; Fox et al., 2012), and hly is a key gene for the virulence potential of L. monocytogenes (Gaillard et al., 1986; Roberts et al., 2005), the correlation between Irish strains and hly could imply a possible impact of geographical-specific in LLO protein among 150 strains of food and human origin, while slight changes in the hly gene did not imply alterations on LLO molecular weight (Jacquet et al., 2002). Furthermore, no significant differences in the LLO protein among different serotypes 4b and 1/2a were reported (Matar et al., 1992; Jacquet et al., 2002). Nonetheless, Gray et al. (2004) reported a significant correlation between hly allelic types and origin of the strains (i.e., food vs. human isolates); hly type 1 was significantly more common among human isolates and was associated with larger plaque forming, indicative of in vitro cytopathogenicity, compared with other hly types (Gray et al., 2004). Therefore, such correlation of origin and hly types might be important in epidemiological studies. Additionally, studies based on ribotype analysis showed no specific clustering among L. monocytogenes strains distributed across different geographical locations, and therefore no significant effect of geographical distribution on their genetic diversity (Gendel and

Ulaszek, 2000; Jaradat et al., 2002; Mereghetti et al., 2002). The sequence diversity analysis in the current study showed that the groups of Greek, Australian and Irish isolates within the pVGC form distinct clusters based on parameters π, θ, and ω, which may underlie diverse evolutionary pathways for each group; this was also observed for all individual genes except the prfA gene. Origin-based pattern in nucleotide diversity was observed for Australian strains, which showed less diversity in plcB and mpl sequences relative to their Irish or Greek counterparts. The Tajima's D-values for Australian isolates were close to 0 contrary to Greek and Irish isolates with increased Tajima's D-values. This indicates a differentiation in the evolutionary pathway of Australian compared to Greek and Irish isolates within these genes.

Although serotype 4b strains predominate among human clinical isolates and serotype 1/2a strains among food isolates, gene-specific pattern between clinical isolates and 4b serotype strains or between food isolates and 1/2a serotype strains were not observed; food and clinical isolates could share alleles for all genes tested. However, descriptive analysis revealed that food and clinical isolates formed distinct clusters regarding their π and ω parameters for all the genes tested, with larger variations within prfA, actA, and plcB genes. This divergence might indicate that these genes were adapted differentially within each group, and this adaptation correlated with their prevalence in food or virulence phenotype, respectively. Previous studies investigating the correlation of isolation source and virulence of strains yielded differing conclusions. Some showed lower virulence potential for strains isolated from food environments compared to human clinical isolates (Norton et al., 2001; Jensen et al., 2008). Conversely, Larsen et al. (2002) reported no significant correlation between food or human origin of strains and invasiveness in the Caco-2 cell infection model, while all strains managed equally to multiply once inside the host cells when an in vivo test was used. Similarly others found no systematic differences in virulence between food or clinical isolates (Brosch et al., 1993; Gray et al., 2004; Neves et al., 2008; Bueno et al., 2010).

The results of this study also showed that the most conserved genes were prfA and hly and the most diverse was actA. Proteins PrfA, LLO and ActA are considered essential virulence factors (Gaillard et al., 1986; Nishibori et al., 1995; Vázquez-Boland et al., 2001a; Travier and Lecuit, 2014). It seems that there is a selective pressure on L. monocytogenes to maintain the former genes, while the increased diversity of actA compared to the other genes is consistent with previous findings (Orsi et al., 2008) and is attributed to increased recombination events occurring in actA, and to evolution by positive selection in both lineages I and II. Rapid PCR-based methods utilize species-specific genes to detect L. monocytogenes in food samples, aiming at preventing the unnecessary recalls of food products. It is of great importance to use target sequences of highly conserved regions rather than genes prone to genetic variability (Rodríguez-Lázaro et al., 2004). Virulence associated genes (e.g. actA, hly, inlA, inlB, prfA, plcA, plcB) and 16S/23S rRNA genes have been studied toward the development of such methods (Liu, 2006). The results indicated that due to the diversity seen, PCR assays based on prfA or hly as opposed to actA would be more reliable, covering isolates of different origin, serotype or isolation source.

In the current study, actA showed the highest number of alleles among all genes tested; 13 alleles were observed for serotype 1/2a strains and 7 alleles for serotype 4b strains. One food isolate (AU\_Lm14-002) had a single nucleotide deletion. Although this deletion would lead to a premature stop codon and a predicted truncated 487 amino acid protein, it was located immediately upstream of a poly(A) tract of 7 adenine residues. These mutations may have a role in influencing gene regulation, which allows phase switching and inactivation, and may be influencing actA transcription in this isolate, whereby a full length ActA may still be synthesized (Orsi et al., 2010). Twelve isolates representing 5 unique alleles had a 105 bp deletion, which comprises a 35 amino acid Proline-Rich Repeats (PRRs) fragment (Wiedmann et al., 1997; Jacquet et al., 2002; Orsi et al., 2008; Holen et al., 2010); the encoded proteins possess 3 instead of 4 PRRs. The number of PRRs contributes to bacterial movement (Lasa et al., 1995; Smith et al., 1996), however no significant effect on virulence potential of the strains has been shown (Roberts and Wiedmann, 2006; Holen et al., 2010). Among the isolates tested in this study, the 105 bp deletion was observed for 4 out of 18 isolates of 1/2a serotype and 8 out of 18 isolates of 4b serotype. Of these, 8 strains (which includes 3 alleles) were isolated from the food environment and 4 strains (2 alleles) were clinical isolates. Similar results were demonstrated by Wiedmann et al. (1997), who observed a predominance of 3-PRRs actA sequence among lineage I isolates compared to isolates of lineage II. This could indicate that this deletion does not influence the pathogenic potential of L. monocytogenes. Jacquet et al. (2002) observed that polymorphism in ActA proteins was rather correlated with origin (human or food isolates) than with serotype of the strains, while Conter et al. (2009) could not correlate actA polymorphism to the virulence of the strains. Based on the sequence analysis in the current study, no clear driving factor appeared to influence the nucleotide sequence or mutations in this gene, as all of the groups were dispersed regarding the parameters π, θ, and ω, while phylogenetic trees showed no consistent pattern between origin or environment of the strains and their genetic polymorphisms. These findings, along with the adapting character to certain functions previously suggested for this gene, and the increased recombination events (Orsi et al., 2008) might imply its multi-functionality recently reported (Travier et al., 2013).

Overall, this study provides insights into the selective pressures acting on the main virulence gene cluster of L. monocytogenes, and suggests differences based on serotype, geographic location and source. The selective pressure to minimize diversification was noted with the key virulence regulatory gene prfA, therefore results of this study support the key role of the global regulator prfA in the lifecycle of L. monocytogenes. In contrast to this, conservation of the actA gene sequence was lowest, with a greater sequence variation and number of alleles. Broadly speaking, higher conservation was noted among isolates sharing a serotype when compared with other groupings such as geographical location or source. Food and clinical isolates largely varied with respect to nucleotide diversity within prfA, actA, and plcB genes, possibly suggesting that a particular adaptation correlated with their prevalence in food or virulence phenotype, respectively. Geographical divergence was noted with respect to the hly gene, with serotype 4b Irish strains distinct to Greek and Australian strains. Future studies will be needed in order to clarify the correlation of geographical distribution of strains and their hly sequence, as well as the impact of such correlation on LLO functionality. Additionally, actA polymorphism should be further evaluated for other phenotypes that might result from its increased diversity among strains and diverse origins. In the present study, strains were selected to represent the distribution of L. monocytogenes based on prevalent serotypes and clinical or food associated origin. Further, a larger data set comprising strains of more serotypes, geographical or isolation origin and year of isolation should be investigated in order to infer significant conclusions regarding the impact of these parameters on LIPI-1 evolution and its correlation to virulence potential of the pathogen.

### AUTHOR CONTRIBUTIONS

The study was conceived and designed by KJ, PS, and EF. All authors contributed to acquisition, analysis, and interpretation of the data. The work was drafted and revised by SP and EF. All authors approved and agreed in the final version of the manuscript.

### FUNDING

This study was supported by the 7th Framework Programme projects PROMISE, contract number 265877.

### REFERENCES

### ACKNOWLEDGMENTS

SP would like to acknowledge the Greek State Scholarships Foundation (IKY) for providing her a Ph.D. fellowship.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.01103/full#supplementary-material

carrying a premature stop codon mutation in inlA. Appl. Environ. Microbiol. 77, 2479–2487. doi: 10.1128/AEM.02626-10

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Poimenidou, Dalmasso, Papadimitriou, Fox, Skandamis and Jordan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Enhanced Biofilm Formation by Ferrous and Ferric Iron Through Oxidative Stress in Campylobacter jejuni

Euna Oh, Katelyn J. Andrews and Byeonghwa Jeon\*

School of Public Health, University of Alberta, Edmonton, AB, Canada

Campylobacter is a leading foodborne pathogen worldwide. Biofilm formation is an important survival mechanism that sustains the viability of Campylobacter under harsh stress conditions. Iron affects biofilm formation in some other bacteria; however, the effect of iron on biofilm formation has not been investigated in Campylobacter. In this study, we discovered that ferrous (Fe2+) and ferric (Fe3+) iron stimulated biofilm formation in Campylobacter jejuni. The sequestration of iron with an iron chelator prevented the iron-mediated biofilm stimulation. The level of total reactive oxygen species (ROS) in biofilms was increased by iron. However, the supplementation with an antioxidant prevented the total ROS level from being increased in biofilms by iron and also inhibited iron-mediated biofilm stimulation in C. jejuni. This suggests that iron promotes biofilm formation through oxidative stress. Based on the results of fluorescence microscopic analysis, Fe2<sup>+</sup> and Fe3<sup>+</sup> enhanced both microcolony formation and biofilm maturation. The levels of extracellular DNA and polysaccharides in biofilms were increased by iron supplementation. The effect of iron on biofilm formation was also investigated with 70 C. jejuni isolates from raw chicken. Regardless of the inherent levels of biofilm formation, iron stimulated biofilm formation in all tested strains; however, there were strain variations in iron concentrations affecting biofilm formation. The biofilm formation of 92.9% (65 of 70) strains was enhanced by either 40 µM Fe2<sup>+</sup> or 20 µM Fe3<sup>+</sup> or both (the iron concentrations that enhanced biofilm formation in C. jejuni NCTC 11168), whereas different iron concentrations were required to promote biofilms in the rest of the strains. The findings in this study showed that Fe2<sup>+</sup> and Fe3<sup>+</sup> contributed to the stimulation of biofilm formation in C. jejuni through oxidative stress.

Keywords: Campylobacter, biofilms, oxidative stress, iron, survival mechanisms

### INTRODUCTION

Campylobacter is a leading bacterial cause of gastroenteritis and is responsible for approximately 166 million diarrheal cases and 37,600 deaths worldwide per year (Kirk et al., 2015). In addition to gastrointestinal infections, in some cases, Campylobacter jejuni may result in the development of Guillain–Barré syndrome (GBS), an acute flaccid paralysis (Willison et al., 2016). Although C. jejuni is isolated from a wide range of domestic, companion, and wild

#### Edited by:

Giovanna Suzzi, Università di Teramo, Italy

#### Reviewed by:

Odile Tresse, INRA – Centre Angers-Nantes, France Giorgia Perpetuini, Università di Teramo, Italy

> \*Correspondence: Byeonghwa Jeon<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 15 January 2018 Accepted: 17 May 2018 Published: 06 June 2018

#### Citation:

Oh E, Andrews KJ and Jeon B (2018) Enhanced Biofilm Formation by Ferrous and Ferric Iron Through Oxidative Stress in Campylobacter jejuni. Front. Microbiol. 9:1204. doi: 10.3389/fmicb.2018.01204

animals (Huang et al., 2015), poultry is considered as the most important reservoir for foodborne transmission of C. jejuni to humans (Hermans et al., 2012). Compared to other foodborne pathogens, such as Salmonella and pathogenic Escherichia coli, C. jejuni is physiologically unique (e.g., microaerophilic and asaccharolytic) and fastidious to culture (Silva et al., 2011). Thus, specific culture conditions are required for the growth of C. jejuni. For example, low oxygen concentrations (e.g., 5% O2) and high growth temperatures (e.g., 37∼42◦C) are needed for the optimal growth of C. jejuni (Davis and DiRita, 2008). As a capnophile, additionally, C. jejuni requires CO2, and carbonic anhydrase that is encoded by canB contributes to C. jejuni growth under low (such as 1%) CO<sup>2</sup> conditions (Al-Haideri et al., 2016).

A biofilm is microbial communities that are encased in a matrix of self-produced extracellular polymeric substance (EPS), including extracellular DNA (eDNA), polysaccharides, and proteins (Flemming et al., 2016). C. jejuni is capable of forming biofilms on various abiotic surfaces and frequently isolated from environmental samples (Kemp et al., 2005; Jokinen et al., 2011). Particularly, biofilm formation is deemed as an important survival mechanism in C. jejuni (Buswell et al., 1998; Murphy et al., 2006). As bacteria are usually found in biofilms in natural settings (Branda et al., 2005), Campylobacter is also found in biofilms on the surface of river rock and wood in the environment (Maal-Bared et al., 2012).

Several environmental factors affecting biofilm formation in C. jejuni have been reported. The biofilm formation of this microaerophilic bacterium is enhanced under aerobic conditions (Reuter et al., 2010; Turonova et al., 2015). Oxygen-rich conditions enhance the expression of membrane proteins, such as Peb4 and CadF, which are involved in the adhesion of C. jejuni to abiotic surfaces (Asakura et al., 2007; Sulaeman et al., 2012). Increased oxidative stress under aerobic conditions is associated with biofilm stimulation in C. jejuni (Oh et al., 2016). In addition, biofilm formation is also affected by nutritional factors. For instance, nutrient-rich culture media and high salt concentrations reduce biofilm formation in C. jejuni (Reeser et al., 2007). Iron is an essential nutrient required for all organisms (Chandrangsu et al., 2017) and is associated with biofilm formation in some bacteria, such as Streptococcus mutans (Berlutti et al., 2004). Staphylococcus aureus (Lin et al., 2012), and Pseudomonas aeruginosa (Banin et al., 2005). Since iron affects various biological processes in C. jejuni, such as gene expression regulation (e.g., Fur regulon) and protein glycosylation (e.g., pglA, pglC, and pglH) (Palyada et al., 2004), we hypothesized that iron may be involved in biofilm formation in C. jejuni. To prove this hypothesis, in this study, we investigated the effect of ferrous (Fe2+) and ferric (Fe3+) iron on biofilm formation in C. jejuni NCTC 11168 and 70 C. jejuni strains isolated from raw chicken.

### RESULTS

### Stimulation of Biofilm Formation by Iron