Liu M, Kemper N, Volkmann N and Schulz J (2018) Resistance of Enterococcus spp. in Dust From Farm Animal Houses: A Retrospective Study. Front. Microbiol. 9:3074. doi: 10.3389/fmicb.2018.03074

**300**

resistance genes to pathogenic bacteria. This concern is one of the reasons why trends of resistances in animal isolates are monitored in member states of the European Union and other areas (EFSA, 2015).

Isolates for monitoring programs are commonly obtained from animals, meat, and fecal samples. Furthermore, enterococci are suggested as useful indicator organisms for fecal contaminations of the environment because of their relatively high tenacity outside their hosts (Lukasova and Sustackova, 2003). Since fecal particles are a component of dust in animal housing they could be a source of fecal bacteria (Schulz et al., 2016). This fact explains, for instance, the detection of vancomycin-resistant enterococci (VRE) in dust samples from turkey flocks (Sting et al., 2013). Evidence also suggests that dust from farm animal houses might be a reservoir for multidrugresistant fecal enterococci, as shown for fecal Enterobacteriaceae (Schulz et al., 2016).

Therefore, this retrospective study analyzed the occurrence of enterococci in 125 dust samples from cattle, pig, and poultry barns and the resistance profiles of these bacteria. The dust samples originated from different investigations and studies conducted between 1980 and 2015. During this time span, fluoroquinolones were introduced in the market for use in animal husbandry (Guardabassi et al., 2008). In the same time span, the use of antibiotics as growth promoters was forbidden (Wegener et al., 1999). These events might have had an impact on the resistances of isolates from animal husbandry. Antimicrobial susceptibility testing to determine resistance profiles could be a useful tool in assessing the impacts on the antimicrobial resistances of isolates from farm animal houses (Wiedemann and Heisig, 1999).

## MATERIALS AND METHODS

#### Origins of Dust Samples

From 1980 to 2009, 125 dust samples were collected by sedimentation in Northern Germany. The samples originate from five pig houses, eight poultry barns, and one cattle barn. The samples were taken as parts of various studies. The sedimentation dust samples were collected and stored as described by Schulz et al. (2016). Briefly, collected sedimentation dust samples (between 5 and 50 g) were stored in sterile glass cylinders subsequently sealed with sterile corks and stored in an airconditioned room at 4◦C in the dark.

Additionally, five pooled dust samples collected from a broiler barn in 2015 were included in the study. Dust was transferred by sterile brushes into sterile bags from different dusty surfaces in a barn. After transport to the laboratory, the dust samples were also transferred in sterile glass cylinders. However, a freezer was used to store the samples at 4◦C in the dark.

## Isolation and Identification of Enterococcus spp.

Dust suspensions were prepared as described by Schulz et al. (2016). Subsequently, aliquots (0.1 ml and 0.1 ml of a tenfold dilution and 0.1 ml of a hundred-fold dilution) were plated in triplicate on Bile Aesculin Agar (BAA) (Oxoid Deutschland GmbH, Wesel, Germany) and on BAA supplemented with ciprofloxacin at 4 mg/L (BAACIP) (CIP: Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The detection limit was 1,000 cfu/g of dust. The buffer used to prepare the dust suspensions was plated as a negative control. Enterococcus faecium (DSM 2918) and Enterococcus fecalis (DSM 20478) were streaked on BAA as growth controls. The plates were incubated at 37◦C for 48 h. Presumed enterococci colonies appear with diameters of 1– 2 mm and are usually larger than common streptococci, shiny in appearance, and brown with brown or black halos on BAA (Public Health England, 2014; Thermofisher.com, 2017).

At least two putative enterococci colonies of every cultivable sample were randomly selected, streaked on Columbia Agar with sheep blood (COLSB) (Oxoid Deutschland GmbH, Wesel, Germany), and identified as described in the thesis from Liu (2017). Briefly, presumed Enterococcus spp. isolates were incubated on API R 20 STREP biochemical test strips in accordance with the manufacturer's protocol (bioMérieux SA, Marcy-l'Étoile, France). After 24 h of incubation, results were analyzed using the apiwebTM–API 20 STREP V7.0 software (bioMérieux, Deutschland GmbH, Germany). When the probability of identification was more than 90%, the result was seen as confirmed. However, the differentiation of species from the E. faecium group by biochemical tests can fail (Devrise et al., 2002). Therefore, a molecular biological method was used to identify isolates to species level (Stepien-Py ´ ´sniak et al., 2017). Stored isolates (at minus 80◦C) were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Isolates were incubated on CLOSB at 37◦C overnight, afterwards being analyzed by Bruker MALDI Biotyper (Bruker Daltonics, Billerica, USA) in accordance with the manufacturer's protocol. Identified species are summarized in **Table 1**. More detailed results (log(score) values) are shown in the **Supplementary Table 1**.

#### Antimicrobial Susceptibility

An antimicrobial sensitivity test was performed by the microdilution method for all confirmed enterococci isolates. The code of the commercially prepared microdilution panels is CMV3AGPF (Thermo Fisher Scientific Inc., Waltham, USA). The 15 antibiotics tested were tigecycline (TGC), tetracycline (TET), chloramphenicol (CHL), daptomycin (DAP), streptomycin (STR), tylosin (TYLT), quinupristin/dalfopristin (synercid) (SYN), linezolid (LZD), penicillin (PEN), kanamycin (KAN), erythromycin (ERY), ciprofloxacin (CIP), vancomycin (VAN), lincomycin (LIN), and gentamicin (GEN). The antibiotic concentrations tested are shown in **Tables 2**, **3**.

Due to the absence of trimethoprim-sulfamethoxazole (TMP/SMX) in the prepared panel, sensitivity to these agents was measured separately. Trimethoprim and sulfamethoxazole (Sigma-Aldrich, co., St. Louis, USA) dissolved in methanol were mixed in sterile broth (ratio 1:19). After dilution, the trimethoprim-sulfamethoxazole suspension was added to blank panels. The concentration ranges are also shown in **Tables 2**, **3**.

Fresh Enterococci broth suspension was prepared, and all panels were incubated at 37◦C for 24 hours (CLSI, 2016; EUCAST, 2016). E. faecium (DSM 2918) was used as a quality

TABLE 1 | Origin and number of isolated species.

control. The results were read using the VIZION R system (TREK Diagnostik Systems Ltd., West Sussex, UK). According to guidelines of the Clinical and Laboratory Standards Institute (CLSI), tiny buttons of growth were ignored when reading the minimum inhibitory concentration (MIC) of CHL, ERY, LZD, and TET (CLSI, 2016).

Breakpoints were adopted from CLSI (2016) when available. Three aminoglycosides (gentamicin, kanamycin, and streptomycin) were only tested for high-level resistance, and their breakpoints were obtained from the National Antimicrobial Resistance Monitoring System Animal Isolates (NARMS) of the United States Department of Agriculture (NARMS, 2016). The breakpoints for LIN and TYLT were obtained from NARMS as well (NARMS, 2016). The breakpoints for tigecycline and trimethoprim-sulfamethoxazole were obtained from the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2016). These figures are also included in **Tables 2**, **3**.

#### Statistical Analyses

Statistical analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA). For each antibiotic, significant differences between the number of resistant isolates from pigs, fattening poultry (broilers, turkeys, ducks), laying hens, and cattle were analyzed by exact conditional logistic regression using the GENMOD procedure. Exact score tests and odds ratios were calculated to estimate significant differences between the animal groups (Stokes et al., 2012). P-values ≤ 0.05 were interpreted as statistically significant. Isolates were collected from samples between 1980 and 2015. The CORR procedure was used to test if the number of resistances is associated with the age of isolated Enterococcus spp. (**Supplementary Table 1**). Pearson's and Spearman's correlation coefficients were calculated and considered as significant when P-values were ≤ 0.05. Associations between total isolates, isolates from BAA, and isolates from BAACIP were tested.

#### RESULTS

#### Isolation and Identification of Enterococcus spp.

The API R 20 STREP tests identified 70 presumed isolates to Enterococcus spp., including 36 from BAA agar and 34 from BAACIP agar. Further identification to species level by MALDI-TOF MS resulted in 64 E. faecium isolates, five E. hirae isolates and one E. casseliflavus isolate. **Table 1** shows the origin of isolates and the year of sampling. E. faecium was detected in samples from as early as the early 1980s. E. hirae was first cultivated from dust from 1989. Enterococci growing on CIP-supplemented media appeared later in 1994. E. faecium was detected in dusts from barns occupied with different animal species, whereas E. hirae was isolated from only pig barns.

#### Frequency of Antimicrobial Resistances in Enterococcus spp. Isolates

**Figure 1** shows that all isolates were resistant to one or more of the tested antibiotics. Ninety-six percent (67/70) of all isolates were resistant to three or more antimicrobial agents. Overall, isolates from fattening poultry showed higher numbers of resistances, although a single isolate from a pig barn exhibited the highest number of phenotypic resistances (n = 11). Isolates from laying hen houses were resistant to fewer antibiotics compared to isolates from pig and fattening poultry barns. Only three isolates from a cattle barn were included. However, the results in **Supplementary Table 1** show that these isolates were resistant to a minimum of three antibiotics from different drug classes.

Seven isolates obtained from dust in fattening poultry barns collected before 2000 were resistant to TYLT. However, for the dust samples collected since 2000, the percentage of resistant isolates was 61.9% (13/21) among fattening poultry. In the isolates from pig farms, the rate of resistant isolates before 2000 was 46.7% (7/15), and then it dropped to 26.7% (4/15) from 2000 onward.

The percentages of SYN-resistant isolates from samples collected before 2000 were 100% (7/7) from fattening poultry barns and 93.3% (14/15) from pig barns. For the younger isolates, the percentages were 76.2% (16/21) and 53.3% (8/15), respectively.

TABLE2|MICdistributionforantimicrobialagentsofEnterococcusspp.

from

BAA.

**303**

#### 4 December 2018 | Volume 9 | Article 3074

 breakpoint is confirmed; the range of trimethoprim/sulfamethoxazole is shown as the trimethoprim concentration. Unshaded areas show the span of the broth microdilution panels. Single vertical bars indicate a susceptibility breakpoint; double vertical bars are breakpoints for resistance. NA,

 not applicable.

#### 6 December 2018 | Volume 9 | Article 3074

 breakpoint is confirmed; the range of trimethoprim/sulfamethoxazole is shown as the trimethoprim concentration. Unshaded areas show the span of the broth microdilution panels. Single vertical bars indicate a susceptibility breakpoint; double vertical bars are breakpoints for resistance. NA,

 not applicable.

## Correlations Between Age of Isolates and Number of Resistances

Correlation analyses were carried out to investigate associations between the number of phenotypic resistances (total isolates, isolates from BAA, and isolates from BAACIP) and the age of the isolates. No correlations were found for total isolates (Spearman correlation coefficient, ρ = 0.16, p = 0.187) and isolates from BAA (ρ = 0.05, p = 0.776). A moderate monotonic relationship (ρ = 0.45, p = 0.008) was only obtained when the number of resistances and the age of isolates from BAACIP media were compared.

#### Antimicrobial Susceptibility

The minimal inhibition concentrations for 16 antimicrobial agents of Enterococcus spp. are shown in **Table 2** (isolates from BAA) and **Table 3** (isolates from BAACIP). All enterococci were sensitive to GEN and VAN. Only a few isolates were not susceptible to LZD and TGC. In contrast, relatively high resistance rates of isolates from both media were calculated for LIN, ERY, PEN, TET, SYN, and CIP. All isolates from BAACIP were resistant to CIP (>4µg/ml, **Table 3**), whereas 15 out of 32 isolates from BAA were susceptible to this antibiotic (**Table 2**). The association between resistant isolates and growing on CIP-containing media was highly significant (Fisher's exact test, p < 0.0001). Interestingly, isolates from BAA from the early and late 1980s were already resistant to CIP (**Supplementary Table 1**).

A breakpoint for daptomycin was not available, but the results indicate that most isolates were not susceptible. Obvious differences appear between the resistance rates of different animal groups in **Tables 2**, **3**. However, the numbers of isolates (pigs = 30, fattening poultry = 28, laying hens = 9, cattle = 3) varied between the groups, which hampered the comparison. Therefore, the significance of differences was calculated by a model, and significant outcomes are summarized in **Tables 4**, **5**.

## Significant Differences of Antibiotic Resistance Rates Between Animal Groups

An exact conditional logistic regression was conducted to analyze the significant differences between the resistances of isolates from different animal groups. As shown in **Table 4**, the results indicate that the resistances to four antibiotics (ERY, PEN, CIP, and TYLT) were significantly different between isolates from different animal groups. Calculations of the exact odds ratios show significant differences in pairwise comparisons of the animal groups (**Table 5**). The chances of finding resistant isolates showed a general trend. Dust from fattening poultry barns obviously more often contained isolates resistant to ERY, PEN, CIP, and TYLT. Isolates from pig holdings had a higher chance of being resistant to ERY than those from laying hen houses.

## DISCUSSION

Enterococcus spp. were isolated from dust samples using BAA. The preparation of dust samples and the subsequent cultivation had a detection limit of 1,000 cfu/g dust. The method enabled the detection of Enterococcus spp. in even the oldest sample. Considering that microbial growth in the samples was not possible under storage conditions (Schulz et al., 2016), this means that the oldest isolate (E. faecium) survived 35 years in a stored environmental sample. Analyzing more presumed isolates and using an enrichment method would have probably

TABLE 4 | Significant differences (p ≤ 0.05) of the resistances between isolates from different animal groups.

Results of the exact conditional logistic regression.

enhanced the overall detection rate (Ieven et al., 1999). However, isolating Enterococcus spp. from all time periods was possible, and we suggest that Enterococcus spp. can be suitable indicator bacteria for retrospective studies with contaminated dry material.

The main species of presumed enterococci was E. faecium. This species probably belongs to the typical microbiota in feces from farm animals (Hershberger et al., 2005). Furthermore, E. faecium tends to survive longer on dry material than other enterococci (Neely and Maley, 2000). Both of these factors may have influenced the predominant isolation of E. faecium. Other species detected were E. hirae and E. casseliflavuss. Enterococcus hirae may be part of the intestinal microbiota of pigs (Larsson et al., 2014) and E. casseliflavus was detected also in broiler flocks (St˛epien-Py ´ ´sniak et al., 2016). The survival for more than two decades in dust also indicates a high tenacity of these species.

The number of phenotypic resistances varied between one and 11. Most of the isolates (98.6%) were multidrug resistant (MDR) according to a definition by Frye and Jackson (2013). The number of antibiotic resistances may vary due to the different treatment regimes in animal husbandry. The treatment status of the sampled barns was unknown. For instance, other studies on isolates of MDR E. faecium from food animals revealed 31.7% MDR isolates from cattle, 65.8% MDR isolates from broiler chickens, and 84.6% MDR isolates from pigs (EFSA, 2015; Nowakiewicz et al., 2017). Although the studies are not directly comparable, other sets of antibiotics were tested, so the results of our study and the cited studies indicate that MDR Enterococcus spp. is probably widespread in farm animal husbandry.

It is noteworthy that the oldest isolate in the present study (isolated in 1981) was resistant to seven different drug classes. A significant association was not found between the age of the isolates and the number of resistances. It is known that bacteria of animal origin can accumulate antimicrobial drug resistances over time (Tadesse et al., 2012). In the present study, younger isolates showed not more resistances than older isolates. In this context, it must be considered very likely that the heterogeneity of the investigated samples, e.g., different numbers of samples from different time periods and different origins, influenced the results. As an example, isolates from laying hens (all sampled in 2005) showed fewer resistances than older isolates from pigs and fattening poultry.

The susceptibility of isolates to different antimicrobial agents varied greatly (**Table 2**, **3**). Enterococci were completely sensitive to VAN and high-level GEN. Only a few isolates were not susceptible to LZD and TGC. There was a relatively high rate of resistance to LIN, ERY, PEN, TET, SYN, and CIP. BAA supplemented with CIP was used to isolate enterococci from dust samples because fluoroquinolone-resistant enterococci were of special interest. Ciprofloxacin was chosen as a representative of fluoroquinolones because it is a common choice for human bacterial diseases and it is closely related to enrofloxacin, which has been was used extensively in animal husbandry (Guardabassi et al., 2008).

Bacteria show cross-resistance to ciprofloxacin and enrofloxacin (Van den Bogaard et al., 2001). Enrofloxacin was first introduced in German animal husbandry in 1989 (Guardabassi et al., 2008). Thus, the results indicate that the occurrence of CIP-resistant enterococci in the early 1980s was not influenced by the treatment of animals. Resistance to fluoroquinolones in bacteria is multifactorial (Redgrave et al., 2014), and the reason for this early occurrence remains unknown. Isolates from supplemented media were significantly more resistant to ciprofloxacin. However, the resistance among 58% of the isolates from non-selective media and the detection in pig and poultry barns and a cattle barn (**Table 2**) indicate a spread of ciprofloxacin resistance in the farm animal facilities investigated.

High-level resistance breakpoints were used for aminoglycosides because enterococci can prevent aminoglycosides from penetrating the bacterial cell membrane and thus have low-level intrinsic resistance (Zimmermann et al., 1971; EUCAST, 2016). Although high-level resistance against gentamicin was not found, nearly one-third of isolates had high-level resistance to kanamycin and streptomycin. Resistances to these antibiotics in farm animals might result from the wide and long-term usage of aminoglycosides in Europe (EMA, 2014).

Due to serious nosocomial infections, VRE invariably cause concern among researchers. VRE have been isolated in Germany as early as 1987 (Lütticken and Kunstmann, 1988). Vancomycin resistant enterococci have been isolated from food animals in Sweden, the Netherlands, and Germany (Stobberingh et al., 1999; Nilsson et al., 2009; Sting et al., 2013). However, all enterococci isolated in this study were sensitive to vancomycin.

There was a high percentage of LIN-resistant enterococci, especially in isolates from poultry farm dust. Thirty-six isolates (97.3%) of enterococci from dust in broiler, layer, and turkey houses were resistant to LIN. These findings are consistent with those from another study (Maasjost et al., 2015). Lincosamides and macrolides are important therapeutic agents for the treatment of infections in farm animals (Pyörälä et al., 2014). The resistance to ERY was notable in this study. Except for the isolates from laying hen barns, the percentages of ERYresistant enterococci were all over 60% (**Tables 2**, **3**). Isolates from pig barns and fattening poultry barns had a higher chance of being resistant to ERY than those from laying hen barns (**Table 5**). It can be assumed that laying hens are generally treated less because of the problem with residues in eggs.

There was a lower percentage of resistance to TYLT, another macrolide, than to ERY (**Tables 2**, **3**). Furthermore, the percentage of TYLT resistant isolates from poultry and pig barns decreased since 2000. It is uncertain whether this observed decrease was influenced by the ban of TYLT as a growth promoter at the end of 1998 in the European Unions (Wegener et al., 1999), but the results show that resistance was still present in isolates from 2015.

Quinupristin/dalfopristin was the first antibiotic for human VRE infections with good clinical effect (Wegener et al., 1999). Virginiamycin and SYN are streptogramins. Due to the "Precautionary Principle," virginiamycin was prohibited as an antibiotic growth promoter at the same time as TYLT (Casewell et al., 2003). A decrease in resistant isolates has been observed for SYN. However, a more comprehensive study would be necessary to confirm this downtrend.

Over 70% of Enterococcus spp. were resistant to PEN. Resistance rates of the same magnitude were detected in E. faecium isolates from poultry production environments in the United States (Hayes et al., 2004). These high resistance rates may be due to an induced, intrinsic, low-level resistance of E. faecium to PEN (Maasjost et al., 2015). A correlation between penicillin and ciprofloxacin resistance has also been observed (Adela et al., 2004).

Although the rate of CHL resistance was <20%, it was obviously higher than in other studies in Germany (Peters et al., 2003; Maasjost et al., 2015). Chloramphenicol was forbidden for use in farm animals in Europe in 1994 (Maasjost et al., 2015). However, in the present study resistant isolates occurred sporadically in poultry and pig barns after the ban.

Linezolid has been allowed for clinical use in humans in Europe since 2001 (Seedat et al., 2006). Although LZD can be used in pets, it should be prescribed only in rare cases (Wijesekara et al., 2017). The first LZD-resistant VRE was found in Germany in 2004 (Halle et al., 2004). In our study, no LZDresistant E. faecium was detected. Only one isolate of E. hirae was resistance to LZD. Almost all MIC values for LZD were in the intermediate range (**Tables 2**, **3**). Resistance to TMP/SMX was also scarce. In general, resistance to TMP/SMX seems to be rare in Gram-positive bacteria isolated from German farm animals (Schwarz et al., 2013).

All enterococci in this study were resistant to one or more antimicrobials (**Figure 1**). Approximately 75.0% of isolates from dust from fattening poultry farms were resistant to seven or more antimicrobials compared with only 26.7% from pigs. The resistances to ERY, PEN, CIP, and TYLT were significantly different between isolates from different animal groups (**Table 4**). In a second step, a statistical model revealed that Enterococcus spp. isolated from fattening poultry barns were more often resistant to these antibiotics compared to other animal groups (**Table 5**). Furthermore, isolates from fattening poultry barns showed the highest rate resistance to multiple antibiotics (**Figure 1**). These results may be related to the different antibiotic regimes in the environments investigated and suggest that more antibiotics were used in poultry barns.

Metagenomic analyses of environmental samples revealed that antibiotic resistance is an ancient, naturally occurring phenomenon (D'Costa et al., 2011). Although such studies can confirm that genes homologous to resistance genes existed in ancient bacteria, DNA fragments cannot confirm functional resistance against antibiotics (Perron et al., 2015). A study from Perron et al. (2015) revealed functional antibiotic resistance in at last 5,000 years old permafrost. However, whether bacteria survived such a long time or were part of subpopulations remained unknown. This study showed that the long-term survival of enterococci in dust enabled a retrospective view of the phenotypic antimicrobial resistances in isolates from different barns of intensive livestock farming. In comparison to a study from Schulz et al. (2016), the present study detected fluoroquinolone resistant bacteria before these antibiotics were used in farms. The resistance in the absence of fluoroquinolone pressure is likely to be related to the biology of resistance (Redgrave et al., 2014). However, this demonstrates that farm animals can be a reservoir of fluoroquinolone resistant bacteria, although animals came never into contact with these antibiotics. Moreover, it was forbidden to treat laying hens with fluoroquinolones in the European Union (Anonymous, 2002) but all isolates from laying hens in 2005 were resistant to CIP. An eradication of CIP resistant enterococci will not be as simple as prohibiting the use of these agents.

Farmers, animals, and the environment are exposed to dustbound MDR enterococci shed by carrying animals. Intervention methods such as thoroughly cleaning of all contaminated surfaces in barns are necessary to avoid transmissions. Whether animal strains can be transmitted to humans remains controversial (Donabedian et al., 2006). However, in terms of prevention, farmers might protect themselves by hygiene measures such as changing clothes, appropriate hand hygiene, and wearing dust masks.

### AUTHOR CONTRIBUTIONS

ML isolated and identified the isolates, conducted the antimicrobial susceptibility testing, and carried out the data analysis. NV programmed the statistical model and analyzed the results. JS planned the study and did the correlation analysis. ML, NK, and JS wrote the manuscript. All authors read and approved the final manuscript.

### FUNDING

ML was supported by the China Scholarship Council. The authors declare no further support or funding.

## REFERENCES

#### ACKNOWLEDGMENTS

We would like to thank Prof. Jörg Hartung for providing the sedimentation dust collection. We also thank Mrs. Kira Butenholz and Mrs. Maria Sember for their excellent technical assistance and support. Mengda Liu would like to thank his research supervisors Prof. Dr. Nicole Kemper, Prof. Dr. Karl-Heinz Waldmann and Prof. Dr. Uwe Rösler for their support of his thesis which delivered basic data for this publication.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.03074/full#supplementary-material

Supplementary Table 1 | Origin, identification, and MIC values of isolates.

**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 Liu, Kemper, Volkmann and Schulz. 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(s) 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.

# Investigation of the Dominant Microbiota in Ready-to-Eat Grasshoppers and Mealworms and Quantification of Carbapenem Resistance Genes by qPCR

Vesna Milanovic´ 1 , Andrea Osimani 1 , Andrea Roncolini 1 , Cristiana Garofalo1 \*, Lucia Aquilanti 1 , Marina Pasquini 1 , Stefano Tavoletti 1 , Carla Vignaroli 2 , Laura Canonico2 , Maurizio Ciani 2 and Francesca Clementi 1

1 Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, Università Politecnica delle Marche, Ancona, Italy, 2 Dipartimento di Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Ancona, Italy

#### Edited by:

Patrícia Poeta, Universidade de Trás-os-Montes e Alto Douro, Portugal

#### Reviewed by:

Ziad Daoud, University of Balamand, Lebanon Victor Ladero, Spanish National Research Council (CSIC), Spain

> \*Correspondence: Cristiana Garofalo<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 10 September 2018 Accepted: 26 November 2018 Published: 17 December 2018

#### Citation:

Milanovic V, Osimani A, Roncolini A, ´ Garofalo C, Aquilanti L, Pasquini M, Tavoletti S, Vignaroli C, Canonico L, Ciani M and Clementi F (2018) Investigation of the Dominant Microbiota in Ready-to-Eat Grasshoppers and Mealworms and Quantification of Carbapenem Resistance Genes by qPCR. Front. Microbiol. 9:3036. doi: 10.3389/fmicb.2018.03036 In this study, 30 samples of processed edible mealworms (Tenebrio molitor L.) and 30 samples of grasshoppers (Locusta migratoria migratorioides) were obtained from producers located in Europe (Belgium and the Netherlands) and Asia (Thailand) and subjected to PCR-DGGE analyses. The PCR-DGGE analyses showed that species in the genus Staphylococcus were predominant in the samples of mealworms from Belgium and grasshoppers from the Netherlands; species in the genus Bacillus were detected in the samples of mealworms and grasshoppers from Thailand. Moreover, Weissella cibaria/confusa/spp. was found in grasshoppers from Belgium. Since data concerning the role of novel foods such as edible insects in the dissemination of carbapenem resistance are currently lacking, the quantification of five carbapenemase encoding genes (blaNDM−1, blaVIM, blaGES, blaOXA−48, and blaKPC) by qPCR was also carried out in all the samples under study. The genes coding for GES and KPC were not detected in the analyzed samples. A very low frequency of blaOXA−48 (3%) and blaNDM−1 (10%) genes was detected among mealworms. In contrast, grasshoppers were characterized by a high incidence of the genes for OXA-48 and NDM-1, accounting for 57 and 27% of the overall grasshopper samples, respectively. The blaVIM gene was detected exclusively in two grasshopper samples from Thailand, showing only 7% positivity. The analysis of variance showed that all the effects (producers, species, and producers × species) were statistically significant for blaNDM−1, whereas for blaOXA−48 and blaVIM, no significant effects were detected for the same source of variation. Further studies are necessary to assess the possible role of edible insects as reservoirs for the resistance to carbapenems and to understand the correlation with the insect microbiota. Furthermore, an intensified surveillance plan examining the occurrence of carbapenemase encoding genes in the food chain and in environmental compartments is needed for a proper risk assessment. In such a context, the appropriate use of antimicrobials represents the main preventive action that should always be applied.

Keywords: edible insects, antibiotic resistance, PCR-DGGE, carbapenemase genes, qPCR

## INTRODUCTION

The use of insects for human consumption is a frequent practice worldwide, mainly in Thailand and other Asian countries, Africa, America, and Australia (van Huis et al., 2013; Schlüter et al., 2017). In Europe, edible insects represent an innovative and uncommon protein source, although in some European countries, especially in the Netherlands and Belgium, the rearing and the industrial production of edible insects is gradually increasing (ANSES, 2014; Schlüter et al., 2017). Indeed, several promising aspects are associated with insect consumption since insects (i) are generally characterized by a positive nutrient profile in terms of high-quality proteins and amino acids, good lipids, vitamins, minerals, and fiber, (ii) are easy to breed, and (iii) cause lower emissions of greenhouse gases and ammonia than traditional livestock (Klunder et al., 2012; van Huis et al., 2013; Schlüter et al., 2017). Due to these numerous nutritional, social, and environmental benefits, edible insects are considered the "food of the future" and categorized as novel foods by Regulation (EU) No 2015/2283 of the European Parliament and of the Council.

Among the edible insects, mealworms and grasshoppers are included within those that are already commercialized as food in EU countries (Schlüter et al., 2017) and that have been partially investigated for the presence of relevant pathogens or potentially pathogenic microorganisms (Ali et al., 2010; Klunder et al., 2012; Stoops et al., 2016; Garofalo et al., 2017; Osimani et al., 2017a, 2018a) as well as for the presence of transferable resistances to antibiotics (Milanovic et al., 2016; ´ Osimani et al., 2017b,c, 2018b).

Carbapenems are broad-spectrum β-lactam antibiotics, currently considered the last-line antibiotics for the treatment of severe human infections caused by multidrug-resistant Gramnegative bacteria (EFSA BIOHAZ Panel, 2013; Guerra et al., 2014; Woodford et al., 2014). The production of carbapenemases, which are β-lactamases capable of hydrolyzing carbapenems and almost all β-lactams, represents the main mechanism of resistance to carbapenems (Tzouvelekis et al., 2012; Doi and Paterson, 2015; Fischer et al., 2017). Among the carbapenemases, the plasmid-acquired class A serine-β-lactamases KPC (Klebsiella pneumoniae carbapenemase, 17 variants) and GES (Guiana extended spectrum, 9 variants), class B metallo-β-lactamases VIM (Verona integron-encoded metallo-beta-lactamase, 40 variants) and NDM (New Delhi metallo-beta-lactamase, 10 variants), and class D serine-β-lactamases including OXA carbapenemases (Carbapenem-hydrolyzing oxacillinase) such as OXA-48 are among the most common and important from an epidemiological point of view and are thus the main clinical concern (Queenan and Bush, 2007; Grundmann et al., 2010; Miriagou et al., 2010; Walsh, 2010; Cantón et al., 2012; Monteiro et al., 2012; Pfeifer et al., 2012; Guerra et al., 2014; Woodford et al., 2014; Fischer et al., 2017). Carbapenemases are encoded by genes that are easily transferable among bacteria by horizontal gene transfer events since they are located on mobile genetic elements, thus increasing their worldwide spread among bacteria in different reservoirs (Tzouvelekis et al., 2012; Woodford et al., 2014; Doi and Paterson, 2015; Fischer et al., 2017).  In the last decade, the rapid and global dissemination of infections caused by carbapenemaseproducing Enterobacteriaceae (CPE) in hospitals and healthcare institutions is of great concern since these outbreaks are often associated with high mortality rates due to the limited and inadequate alternative treatments (Nordmann et al., 2011; Doi and Paterson, 2015; Rossolini, 2015; Grundmann et al., 2017; Zhang et al., 2017). From all of these data, the current opinion is that the acquired carbapenemases are a primary pressing public health threat related to antibiotic resistance (AR) (Tzouvelekis et al., 2012; EFSA BIOHAZ Panel, 2013; Woodford et al., 2014; Doi and Paterson, 2015).

Although the occurrence of carbapenemases was first discovered and mainly investigated at hospitals and healthcare facilities, scientific studies reporting carbapenemase-producers, and carbapenemase-encoding genes (CEG) in animals, the environment and food are increasingly frequent.  This suggests that non-human sources may be reservoirs of CPE and CEG. Since it is widely recognized that the food chain is one of the main routes for the introduction of antibiotic-resistant bacteria and their genes into the human digestive tract, and for the diffusion and spread of AR in human pathogens (Clementi and Aquilanti, 2011; Rolain, 2013; Milanovic et al., 2017 ´ ), there is a need for intensified surveillance of the occurrence of CEG in the food chain and in different environmental compartments. This is also underscored by the European Food Safety Authority, which has recently recognized the need to improve European legislation to ensure the monitoring of carbapenem resistance in animals and food (EFSA BIOHAZ Panel, 2013).

Based on all these premises, edible insects deserve great attention in terms of safety, including the assessment of the microbiota and of the incidence of AR genes, in particular CEG.

Therefore, in order to obtain an overview of the predominant bacterial species in samples of processed edible mealworms (Tenebrio molitor L.) and grasshoppers (Locusta migratoria migratorioides) obtained from producers in Europe (Belgium and the Netherlands) and Asia (Thailand), the total microbial DNA was analyzed by culture-independent PCR-DGGE.

Concerning the AR issue, it is worth noting that currently, only a few scientific studies are available on the occurrence of transferable AR genes in edible insects (Milanovic et al., 2016; ´ Osimani et al., 2017b,c, 2018b; Vandeweyer et al., 2018), and to the authors' knowledge, none have been published on the detection of CEG.

To address this gap, the edible insect samples under study were subjected to screening by quantitative PCR (qPCR) of five among the most common carbapenem resistance genes (blaNDM−1, blaVIM, blaGES, blaOXA−48, and blaKPC) (Monteiro et al., 2012). Statistical analyses were performed to determine if edible insect species (mealworms and grasshoppers) or geographical location correlated with the occurrence of carbapenem resistance genes in this study.

#### MATERIALS AND METHODS

#### Sampling

Thirty samples of edible mealworms and 30 samples of grasshoppers (boiled, dried, and salted) were purchased via the internet from dealers located in Europe (Belgium and the Netherlands) and Asia (Thailand). Ten mealworm and ten grasshopper samples from each country were collected and marked as follows: TN1-TN10 (mealworms from the Netherlands, Producer 1), TB1-TB10 (mealworms from Belgium, Producer 2), TT1-TT10 (mealworms from Thailand, Producer 3), GN1-GN10 (grasshoppers from the Netherlands, Producer 1), GB1-GB10 (grasshoppers from Belgium, Producer 2), and GT1-GT10 (grasshoppers from Thailand, Producer 3). All the samples were provided in sealed plastic containers and delivered at ambient temperature via international shipping. No information was available on the rearing and hygiene conditions of processing, transport and storage applied to these edible insects before marketing.

#### Bacterial DNA Extraction

Total microbial DNA was extracted directly from the insect samples using PowerFood Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA) as described by Osimani et al. (2017a). The extracted DNA was quantified and checked for the purity using a NanoDrop ND 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) and then standardized to 2 ng µL −1 for qPCR assays and to 25 ng µL −1 for PCR-DGGE analysis. The effective extraction of bacterial DNA was confirmed by conventional PCR amplification of 2 µL (50 ng) of extracted DNA suspensions in a My Cycler Thermal Cycler (BioRad Laboratories, Hercules, CA, USA) using universal prokaryotic primers 27F and 1495R as described by Osimani et al. (2015).

#### PCR-DGGE Analysis

The equal portions of DNAs extracted from insects were mixed together with the goal of obtaining six pooled samples (TB, TN, TT, GB, GN, and GT), each representing an insect type (mealworms and grasshoppers) and country of origin (Belgium, the Netherlands, and Thailand). The amplification products obtained from the 27F-1495R primer pair as described above were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Buckinghamshire, UK). Subsequently, 2 µL of the purified PCR products was reamplified using the universal prokaryotic primers U968GC (added with GC clamp) and L1401 (Muyzer et al., 1993; Randazzo et al., 2002) following the PCR conditions previously described by Aquilanti et al. (2013). Following amplification, 5 µL of the PCR reaction was loaded on a 1.5% agarose gel together with a 100 bp molecular weight marker (HyperLadderTM 100 bp) to check for the expected product size of 480 bp. Twenty microliters of these PCR amplicons were analyzed by DGGE (30–60% urea-formamide denaturing gradient; 4 h at 130 V) using DCode Universal Mutation Detection System (Bio-Rad Laboratories) as described by Garofalo et al. (2015). All DGGE bands visible under UV light were excised from the gel, and the DNA was eluted overnight at 4◦C in 50 µL of molecular biology grade water (Garofalo et al., 2008) and reamplified via PCR as described above, but with the forward primer U968 lacking the GC clamp. The PCR products were sent to Genewiz (Takeley, UK) for purification and sequencing, and the obtained sequences were identified at species level as described above by Osimani et al. (2018c).

#### Reference Strains

DNA extracted from five reference strains (**Table 1**), each carrying one of the carbapenem resistance genes under study, was used as positive control in the qPCR reactions and for the construction of qPCR standard curves.

#### qPCR

Absolute quantification of each carbapenemase gene (blaNDM−1, blaVIM, blaGES, blaOXA−48, and blaKPC) in the insect samples was performed by qPCR in a Mastercycler R ep realplex machine (Eppendorf, Hamburg, Germany) using the qPCR primers and cycling conditions described by Monteiro et al. (2012). To check for product specificity, all cycles were followed by a melt curve step analysis with temperature gradually increasing from 65 to 95◦C by 0.2◦C/s. Each qPCR reaction consisted of 4 µL (8 ng) of the extracted DNA; 5 µL of Type-it 2X HRM PCR Master Mix (Qiagen, Hilden, Germany) containing HotStarTaq Plus DNA Polymerase, EvaGreen Dye, an optimized concentration of Qsolution, dNTPs and MgCl2; 0.2µM of forward and reverse primers for each gene; and nuclease-free molecular biology grade water to a final reaction volume of 10 µL. The exogenous standards for each gene were prepared by qPCR amplification of the DNA extracted from the reference strains (**Table 1**) as described above but in a final reaction volume of 25 µL. The correct melting temperatures (Tm) and sizes of the obtained PCR products were checked by melting curve analysis and

TABLE 1 | Bacterial reference strains carrying carbapenems resistance genes, used as positive controls in the qPCR reactions.

electrophoresis on a 1.5% agarose gel, respectively. The Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences) was used for the purification of the amplicons following the manufacturer's instructions. The quantity and the purity of the products were determined using the NanoDrop ND 1000 (Thermo Fisher Scientific). The calculation of each gene copy number was performed using an online calculator (www.idtdna.com) based on the mass and size of the purified qPCR products. The standard curves were created by qPCR amplification of 10-fold serial dilutions of exogenous standards. The amplification efficiencies were estimated from the slopes of the standard curves, and the correlation coefficients (R 2 ) (Stolovitzky and Cecchi, 1996) were calculated automatically by Mastercycler R ep realplex software. To determine the qPCR detection limit for each gene, the standard curves were generated in the range from ∼1 to 107 gene copies per reaction.

For the absolute quantification of CEG, the DNAs extracted from mealworms and grasshoppers were run along with the 10-fold serial dilutions of the standards prepared as described above. The gene copy number of each gene detected in the analyzed insect samples was determined using the slope of the corresponding standard curves. The baseline and threshold calculations were performed automatically by the Mastercycler R ep realplex software. In addition to melting curve analysis, the correct sizes of the amplification products were checked by electrophoresis on 1.5% agarose gels using 100 bp DNA Ladder (HyperLadderTM 100 bp, Bioline, UK) as a molecular weight marker. Moreover, the accuracy of the amplification reactions was validated by the sequencing (Genewiz) of randomly selected positive samples (TB6, GT7, and GN1 for the blaOXA−48 gene; GT2 and TT8 for the blaNDM−1 gene; GT7 and GT8 for the blaVIM gene). The resulting sequences were compared with those from the GenBank database (http://www.ncbi.nlm.nih.gov) using Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). All qPCR reactions were performed in triplicate and the results were expressed as the mean gene copy number per ng of DNA ± standard deviation for each gene.

#### Statistical Analysis

Descriptive statistics, calculated on 20 samples for each producer and on 30 samples for each insect species were carried out for the blaOXA−48, blaNDM−1, and blaVIM gene copies by computing the means ± standard deviation.

After first checking for conformance to a normal distribution and identification of outliers, analysis of variance (ANOVA) was carried out using JMP statistical software version 11.0.0 (SAS Institute Inc., Cary, NC, USA) to test the following main effects: producers (Belgium, Thailand, the Netherlands), insect species (mealworm, grasshoppers) and producer × species.

Principal component analysis (PCA) was applied to discriminate among mealworms and grasshoppers coming from different producers (Belgium, the Netherlands, and Thailand), and the presence of genes related to resistance to carbapenems (blaNDM−1, blaVIM, blaGES, blaOXA−48, and blaKPC). PCA was carried out using the Unscrambler 7.5 software (CAMO ASA, Oslo, Norway). The mean data were normalized to neutralize any influence of hidden factors. PCA provides a graphical representation of the overall differences in terms of distribution of genes between the insects coming from different producers.

## RESULTS AND DISCUSSION

In the present study, the microbiota of commercialized read-toeat grasshoppers and mealworms from different countries was investigated via PCR-DGGE, as well as the quantification and distribution of five common CEG within the same matrices, in order to have a more complete picture of some safety aspects related to edible insects.

#### Determination of Microbial Diversity

To have an overview of the predominant bacterial species found in the edible insects considered in this study, the total microbial DNA was extracted from the samples, the DNAs were mixed in order to obtain six pooled samples, each representing an insect type (mealworms, grasshoppers) and country of origin (Belgium, the Netherlands, Thailand) and then analyzed by a culture-independent PCR-DGGE method. The results obtained are reported in **Table 2**.

The dominant species found in mealworms from Belgium and grasshoppers from the Netherlands belonged to the genus Staphylococcus, and species in the genus Bacillus were found in mealworms and grasshoppers from Thailand. Grasshoppers from Belgium were positive for Weissella cibaria/confusa/spp., while bacterial species with a percentage identity below 97% were found in mealworms from the Netherlands. It is interesting to note that the predominance of lactic acid bacteria (LAB), and in particular the Weissella spp., has been reported for processed and fresh grasshoppers from Belgium and the Netherlands (Stoops et al., 2016; Garofalo et al., 2017; Osimani et al., 2017a), thus suggesting that the specific rearing conditions may have selected for this microbial group or that this bacterial species is intrinsically associated with this edible insect.

The genera Staphylococcus and Bacillus identified among the other pooled samples of mealworms and grasshoppers may contain pathogenic species such as Staphylococcus aureus and Bacillus cereus, and these data are in agreement with other studies on the microbiota in fresh and processed mealworms and grasshoppers (Stoops et al., 2016; Garofalo et al., 2017). The presence of Staphylococcus members may be due to an environmental contamination occurring during human handling or processing. Indeed, these insects were boiled and salted, and since Staphylococcus spp. is a halophile bacterium usually predominating in environments with low microbial competition, it could have found conditions suitable for growth.

The lack of detection of bacterial DNA belonging to the Enterobacteriaceae has been already reported by Osimani et al. (2018c), although it is generally reported that Enterobacteriaceae represents a predominant bacterial group in the edible insect gut microbiota (Stoops et al., 2016; Garofalo et al., 2017; Osimani et al., 2017b). These data suggest that good manufacturing practices were applied during rearing and processing of the insects and/or that a degutting step may have been applied. A further explanation is that members TABLE 2 | Sequencing results of the bands excised from the DGGE gel obtained from the amplified fragments of pooled bacterial DNA extracted directly from mealworms and grasshoppers.

TB- pool of 10 (TB1-TB10) mealworm samples from Belgium; TN- pool of 10 (TN1- TN10) mealworm samples from the Netherlands; TT- pool of 10 (TT1-TT10) mealworm samples from Thailand; GB- pool of 10 (GB1-GB10) grasshopper samples from Belgium; GN- pool of 10 (GN1-GN10) grasshopper samples from the Netherlands; GT- pool of 10 (GT1-GT10) grasshopper samples from Thailand.

aPercentage of identical nucleotides in the sequence obtained from the DGGE bands and the sequence of the closest relative found in the GenBank database.

bAccession number of the sequence of the closest relative found by a BLAST search.

of the Enterobacteriaceae family could be present within the processed insect samples but with a lower abundance in respect with other microbial groups. This latter hypotesis is supported by the fact that these microorganisms had previously found below the detection limit of microbial counts (<1 Log cfu g−1 ) in the same samples (Osimani et al., 2017b,c).

Additionally, the lack of detection of bacterial DNA belonging to Pseudomonadaceae is unusual, but it is possible, as suggested for Enterobacteriaceae, that PCR-DGGE was not able to detect members of this microbial group if they were in the minority relative to the others.

#### Quantification and Distribution of carbapenem Resistance Genes

This study represents the first report on the screening of five carbapenemase encoding genes (blaNDM−1, blaVIM, blaGES, blaOXA−48, and blaKPC) in processed edible mealworms (T. molitor L.) and edible grasshoppers (L. migratoria migratorioides) from producers located in Europe (Belgium and the Netherlands) and Asia (Thailand). The identification of the genes coding for these carbapenemases in the samples of edible insects under investigation was conducted by using qPCR. As previously underlined by Monteiro et al. (2012), molecular assays are considered the best solutions for the rapid detection of carbapenem resistance genes and for the identification of the resistance mechanism. The detection limit, defined as the lowest concentration at which 95% of the positive samples are detected was <10 gene copies per reaction for all the genes. The efficiencies of the qPCR reactions were 1.00 for the genes blaOXA−48, blaVIM, and blaGES; 1.01 for the gene blaKPC and 0.96 for the gene blaNDM−1. The R 2 was 1.000 for the genes blaOXA−48 and blaNDM−1; 0.999 for the gene blaGES, 0.996 for the gene blaKPC and 0.995 for the gene blaVIM. Moreover, the specificity of the primers used for the amplification of carbapenem resistance genes was confirmed by the results of the sequencing of randomly selected positive samples, which showed >97% similarity with the corresponding gene sequences deposited in the GenBank database. In more detail, the results of the BLAST analysis for the blaOXA−48 gene showed 98% of the similarity with the sequences deposited in GenBank such as K. pneumoniae (CP031374), Citrobacter freundii (MG430338), Enterobacter ludwigii (MG436907), Pantoea agglomerans (MG436898), Escherichia coli (NG\_055499), Shewanella sp. (NG\_055475), and Proteus mirabilis (KT175900); for the blaNDM−1 gene 99% similarity with the Enterobacter hormaechei (AP018835), K. pneumoniae (AP018834), Raoultella planticola (MH257689), E. coli (CP021206), Pseudomonas aeruginosa (KT364224), and Acinetobacter baumannii (KU180703); and for the blaVIM gene 98% similarity with the K. pneumoniae (MH071811), C. freundii (NG\_061412), Paenibacillus sp. (KR822172), E. coli (MF169879), E. hormaechei (LT991955), Kluyvera cryocrescens (MG228427), Alcaligenes faecalis (KY623659), Klebsiella oxytoca (NG\_050362), and Enterobacter cloacae (CP030081).

The results of the qPCR quantification of carbapenem resistance genes in samples of ready-to-eat edible mealworms and grasshoppers produced in the Netherlands, Belgium and Thailand are reported in **Tables 3**, **4**.

Regarding mealworms, none of the samples were positive for the genes blaGES, blaKPC, and blaVIM, while only sample TB6 was found positive for blaOXA−48 (3% positivity) and samples TN2, TN8, and TT8 were positive for blaNDM−1 (10% positivity) (**Table 3**).

Regarding grasshoppers, the genes blaGES and blaKPC were not detected in any of the analyzed samples while only two samples from Thailand (GT7 and GT8) were positive for the blaVIM gene (7% positivity). Interestingly, a high prevalence of blaOXA−48 was noted (57% positivity), followed by blaNDM−1 (27% positivity) (**Table 4**). Specifically, blaOXA−48 was prevalent in 80% of the samples from Belgium, in 50% of the samples from the Netherlands and in 40% of the samples from Thailand. The highest frequency of blaNDM−1 was found among samples from Thailand (40%), followed TABLE 3 | Results of qPCR quantification of carbapenemase genes in samples of ready-to-eat edible mealworms produced in the Netherlands (TN1-TN10), Belgium (TB1-TB10) and Thailand (TT1-TT10).

n.d., not detected. n.dr., not determined.

by samples from Belgium (30%) and the Netherlands (10%) (**Table 4**).

All the insect samples analyzed in this study were previously screened for the presence of 12 selected genes coding for resistance to tetracyclines [tet(M), tet(O), tet(S), and tet(K)], macrolide-lincosamide-streptogramin B (MLSB) [erm(A), erm(B), erm(C)], vancomycin (vanA and vanB), beta-lactams (blaZ and mecA) and aminoglycosides [aac(6′ )-Ie aph(2′′)-Ia referred as aac-aph] through PCR and nested PCR assays (Osimani et al., 2017b,c). It is interesting to note that the mealworms samples TN2 and TN8 from the Netherlands were also found to be positive for the presence of genes coding for resistance to tetracyclines [tet(M), tet(K), tet(S)] and erythromycin [erm(B), erm(C)], while sample TB6 was positive for genes coding for resistance to tetracyclines [tet(M), tet(K)], and sample TT8 from Thailand was positive for genes coding for resistance to tetracyclines [tet(K)], erythromycin [erm(B)], and aminoglycosides (aac-aph) (Osimani et al., 2017b). Moreover, among grasshoppers, almost all of the samples that were found to be positive for blaOXA−48, blaNDM−1, and blaVIM previously showed positivity for AR genes coding for resistance to tetracyclines [tet(M), tet(S), tet(K)], erythromycin [erm(B), erm(C)], aminoglycosides (aac-aph) and beta-lactams (blaZ) (Osimani et al., 2017c).

The average levels of gene copies ng−1 in the 60 samples of edible insects were as follows: 0.59 ± 2.39 with a TABLE 4 | Results of qPCR quantification of carbapenemase genes in samples of ready-to-eat edible grasshoppers produced in the Netherlands (GN1-GN10), Belgium (GB1-GB10) and Thailand (GT1-GT10).

n.d., not detected. n.dr., not determined.

minimum value of 0 and a maximum value of 16.56 for blaOXA−48, 2.94 ± 11.53 with a minimum value of 0 and a maximum value of 70.37 for blaNDM−1, 7.25 ± 50.85 with a minimum value of 0 and a maximum value of 392.25 for blaVIM.

Descriptive statistics on 20 samples from each producer are shown in **Table 5**. In addition, descriptive statistics on 30 samples from each insect species are reported in **Table 6**.

The analysis of variance (**Table 7**) showed that all the variables (producers, species, and producers × species) had significant effects (P < 0.05) on the frequency of blaNDM−1, whereas for blaOXA−48 and blaVIM, no significant effects were detected for the same source of variation.

Regarding the distribution of blaOXA−48 and blaVIM, multiple comparisons between ACC Least Square Means (LSM) carried out using the Tukey test showed no significant differences among samples from different producers or insect species. Regarding blaNDM−1, multiple comparisons (Tukey HSD) showed that insect species, but not the origin of the sample, had a significant correlation (P < 0.05) with the frequency of the gene.

PCA did not discriminate between the presence of genes encoding resistance to carbapenems among mealworms and grasshoppers coming from different producers. In contrast, in the previous studies on the occurrence of transferable ARs in ready-to-eat edible insects, PCA showed a differentiation among producers, thus suggesting that different rearing and clinical practices associated with different countries may have played a role in the variability observed (Milanovic et al., 2016; Osimani ´ et al., 2017b,c).

As reported by Schlüter et al. (2017), it is presumed that the rearing and processing conditions applied to edible insects will comply with the same food safety regulations as for livestock farming. The use of carbapenems is prohibited in food-producing animals in all countries (OIE, 2015; Webb et al., 2016). Notwithstanding, scientific studies reporting CPE and CEG in livestock and their environment are progressively more frequent (Guerra et al., 2014; Webb et al., 2016; Zurfluh et al., 2016; Fischer et al., 2017). Furthermore, in the last EFSA report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2015, the presumptive extended-spectrum beta-lactamase (ESBL)- /AmpC-/carbapenemase-production in Salmonella and E. coli was monitored in humans, meat (pork and beef), fattening pigs and calves for the first time (EFSA and ECDC, 2017). Varying occurrence/prevalence rates of ESBL-/AmpC-producers were observed between countries, and carbapenemase-producing E. coli were detected in single samples of pig meat and from fattening pigs from two Member States (EFSA and ECDC, 2017). These data indicate that other antimicrobial classes could indirectly select CPE outside the hospital setting and that the rapid dissemination of CPE is also promoted by CEG located on plasmids transmissible by horizontal gene transfer events (Tzouvelekis et al., 2012; Woodford et al., 2014). As reviewed by Caniça et al. (2015), AR comprises a dynamic network that involves several environmental niches (e.g., water, soil, and plants) and different reservoirs (e.g., husbandry, hospitals, wild animal, human settings, human hand, food and global trade in foodstuffs) in which the path of dissemination and dynamics of AR genes has to be taken into consideration in order to understand and prevent the AR transmission and spread. Therefore, it is possible to hypothesize that, irrespective of the use of carbapenems in the edible insect rearing, the CEG may derive from the substrates used for feed or from surfaces and hands of operators or from treatments applied for processing, in addition to transport and storage. It is also interesting to note that grasshoppers and mealworms have different dietary habits since grasshoppers are grass-feeders whereas mealworms are usually reared on cereal-based matrices; therefore, the differences

SD, standard deviation. Values are expressed as gene copies ng−1 .

TABLE 6 | Descriptive statistics on 30 samples for each insect species.

.

SD, standard deviation. Values are expressed as gene copies ng−1

TABLE 7 | ANOVA results for blaOXA−48, blaNDM−1, blaVIM.

Df, degrees of freedom. Significant at P < 0.05. SS, sum of squares. MS, mean square. P-value.

in terms of presence and distribution of CEG among these insect species may derive from different rearing practices and substrates.

## CONCLUSION

Edible insects such as grasshoppers and mealworms represent a novel food that deserves attention in terms of safety, including the assessment of the incidence of AR genes. The investigation of the microbiota of the mealworm and grasshopper samples in this study revealed the presence of potential pathogenic and non-pathogenic species.

Scientific studies reporting carbapenemase-producing microorganisms and CEG in animals, the environment and food are increasingly frequent. The data presented in this study is the first attempt aimed at determining the incidence of CEG among samples of commercialized ready-to-eat grasshoppers and mealworms from Belgium, the Netherlands and Thailand. Although further studies are necessary to understand the correlation of CEG with the insect microbiota and to assess the possible role of edible insects as reservoirs of resistance to carbapenems, an intensified surveillance plan examining the occurrence of CEG in the food chain and in different environmental compartments, along with a prudent use of

#### REFERENCES

carbapenems and antimicrobials in general, are primary measures that should be applied.

#### AUTHOR CONTRIBUTIONS

VM and AR carried out molecular analyses. MP, ST, LC, and MC performed statistical analyses. CG wrote the manuscript. AO, LA, CV, and FC critically analyzed the results and revised the final manuscript.

#### FUNDING

The study was partly supported by funding for the project "PSA2017-Carbapenemase-producing bacteria: from the environment to humans or vice versa?" from the Polytechnic University of Marche.

#### ACKNOWLEDGMENTS

We wish to thank the Esoteric Testing Laboratory, in the Pathology Department at Tampa General Hospital (Tampa, FL, USA) for providing the DNA extracted from reference strains, each carrying one of the carbapenem resistance genes studied in this research.

producing enterobacteriaceae isolates from rivers and lakes in Switzerland. Appl. Environ. Microbiol. 79, 3021–3026. doi: 10.1128/AEM.00 054-13

**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 Milanovi´c, Osimani, Roncolini, Garofalo, Aquilanti, Pasquini, Tavoletti, Vignaroli, Canonico, Ciani and Clementi. 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(s) 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.

# Impact on Public Health of the Spread of High-Level Resistance to Gentamicin and Vancomycin in Enterococci

Mónica Sparo1 \* † , Gaston Delpech1† and Natalia García Allende2

1 Clinical Department, Medicine School, National University of Central Buenos Aires, Tandil, Argentina, 2 Hospital Alemán, Buenos Aires, Argentina

#### Edited by:

Gilberto Igrejas, Universidade de Trás-os-Montes e Alto Douro, Portugal

#### Reviewed by:

Ana P. Tedim, Neiker Tecnalia, Spain Atte Von Wright, University of Eastern Finland, Finland

\*Correspondence: Mónica Sparo<EMAIL_ADDRESS>†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 28 June 2018 Accepted: 28 November 2018 Published: 18 December 2018

#### Citation:

Sparo M, Delpech G and García Allende N (2018) Impact on Public Health of the Spread of High-Level Resistance to Gentamicin and Vancomycin in Enterococci. Front. Microbiol. 9:3073. doi: 10.3389/fmicb.2018.03073 Antibiotic resistance has turned into a global public health issue. Enterococci are intrinsically resistant to many antimicrobials groups. These bacteria colonize dairy and meat products and integrate the autochthonous microbiota of mammal's gastrointestinal tract. Over the last decades, detection of vanA genotype in Enterococcus faecium from animals and from food of animal origin has been reported. Vancomycin-resistant E. faecium has become a prevalent nosocomial pathogen. Hospitalized patients are frequently treated with broad-spectrum antimicrobials and this leads to an increase in the presence of VanA or VanB vancomycin-resistant enterococci in patients' gastrointestinal tract and the risk of invasive infections. In humans, E. faecium is the main reservoir of VanA and VanB phenotypes. Acquisition of high-level aminoglycoside resistance is a significant therapeutic problem for patients with severe infections because it negates the synergistic effect between aminoglycosides and a cell-wallactive agent. The aac(60 )-Ie-aph (200)-Ia gene is widely spread in E. faecalis and has been detected in strains of human origin and in the food of animal origin. Enzyme AAC(60 )- Ie-APH(200)-Ia confers resistance to available aminoglycosides, except to streptomycin. Due to the fast dissemination of this genetic determinant, the impact of its horizontal transferability among enterococcal species from different origin has been considered. The extensive use of antibiotics in food-producing animals contributes to an increase in drug-resistant animal bacteria that can be transmitted to humans. Innovation is needed for the development of new antibacterial drugs and for the design of combination therapies with conventional antibiotics. Nowadays, semi-purified bacteriocins and probiotics are becoming an attractive alternative to the antibiotic in animal production. Therefore, a better understanding of a complex and relevant issue for Public Health such as high-level vancomycin and gentamicin resistance in enterococci and their impact is needed. Hence, it is necessary to consider the spread of vanA E. faecium and high-level gentamicin resistant E. faecalis strains of different origin in the environment, and also highlight the potential horizontal transferability of these resistance determinants to other bacteria.

Keywords: enterococci, clinical, foodborne, high-level resistance, gentamicin, vancomycin, clonal, transfer

## INTRODUCTION

fmicb-09-03073 December 15, 2018 Time: 15:10 # 2

Enterococci are resistant to diverse physicochemical conditions and are widespread in nature. They are capable of growing and surviving under harsh environmental conditions and have been found in soil, plants, birds, and insects (Butler, 2006; Ghosh and Zurek, 2015).

In the intestinal tract of humans and other animals, the genus Enterococcus can be found among their flora. The microbiological and ecological factors that contribute with intestinal colonization are unknown, even though up to 108 CFU/g of enterococci have been found in human feces. In addition, strains from this genus have been isolated from fermented and dairy products. Moreover, some enterococcal strains have been regarded as food biopreservants and probiotics, although their safety remains questioned (Beibei et al., 2015).

Traditionally, enterococci have not been considered as community-acquired pathogens. Usually, these bacteria do not cause infectious diseases in healthy people, except for occasional urinary tract infections; however, Enterococcus faecium as well as E. faecalis, are prevalent producers of health-care associated opportunistic infections (Woodford and Livermore, 2009). The genomic plasticity of enterococci has contributed with their adaptation to the hospital environment. Their relevance as nosocomial-infections' agents is bolstered by their natural resistance to multiple antimicrobials and an outstanding ability for acquiring and transferring genetic resistance determinants (Werner et al., 2013).

Enterococci express natural (intrinsic) resistance to antibiotics, e.g., clindamycin and trimethoprimsulfamethoxazole. In addition, enterococci show a naturally low-level resistance to gentamicin. Minimum inhibitory concentration (MIC) values to gentamicin range from 6 to 48 µg/mL (Chow, 2000).

Antimicrobials consumption constitutes an important risk factor for colonization with multi-drug resistant enterococci because of the suppression of the competitive indigenous microbiota in the gastrointestinal tract. The increased number of gut enterococci, due to the decrease of competitive gut indigenous flora, frequently precedes bloodstream infections (Ubeda et al., 2010; Reyes et al., 2017).

Antimicrobials can be used in animal husbandry with therapeutic, prophylactic/metaphylactic and growth promotion purposes. Despite the use of antibiotics as growth promoters has been forbidden in many countries, worldwide, foods supplemented with antimicrobials are freely acquired in several countries with no veterinarian control, including in Argentina. This leads to bacterial exposure to subtherapeutic concentrations of antibiotics and, hence, it may promote the expression of antibiotic resistance (Andersson and Hughes, 2014). Antimicrobials employed for human therapies and also used in animal production (in decreasing order) are tetracyclines, penicillins, macrolides, sulfonamides, aminoglycosides, lincosamides, and cephalosporins (Love et al., 2011; Kuehn, 2014). Specifically, ceftiofur, sulfamides and tetracyclines are used for prevention and treatment of pneumonia in pigs; gentamicin and neomycin are employed for the therapy of bacterial diarrhea (Dewey et al., 1999; EFSA, 2011).

The addition of antibiotics for growth promotion in animal feed became a common practice without rigorous testing. The mechanism of action in growth promotion induced by antibiotics appears to be related to the reduction of pathogenic bacteria in the intestines. The concentration of antimicrobials used for growth promotion has often been lower than that used for therapy and prophylaxis. These sub-therapeutic doses of antibiotics often create an auspicious condition for selecting antibiotic resistant bacteria (Van Immerseel et al., 2004; Dibner and Richards, 2005). Previously, McDonald et al. (2001) reported antimicrobial resistant enterococci in food produced with animals fed with antibiotics in sub-therapeutic doses.

Extensive use of antimicrobials in animal husbandry has exerted a considerable pressure for the genesis of antimicrobialresistant bacteria in the environment, such as vancomycinresistant enterococci (López et al., 2009; Ruzauskas et al., 2009; Marshall and Levy, 2011; Nieto-Arribas et al., 2011; Ribeiro et al., 2011; Sánchez Valenzuela et al., 2013).

Furthermore, enterococci, due to their characteristics of gastrointestinal colonization, environmental persistence, natural and acquired resistance to different antimicrobials and their availability to transfer genes horizontally, can be used as biomarkers of antimicrobial resistance in intensive husbandry.

#### TRANSFERABLE GENETIC DETERMINANTS OF ANTIMICROBIAL RESISTANCE

Intensive breeding of animals, especially poultry, pigs and cattle, facilitates the selection, spread and resistance determinants transfer of resistant bacteria. Increased antimicrobials resistance in colonizing bacteria from animals and food of this origin was documented (Normanno et al., 2007).

The extended and permanent use of antimicrobials for therapy purposes and growth promotion purposes in husbandry contributed with drug-resistant bacteria selection in humans. When antimicrobials are used in low doses and in prolonged cycles, a selective pressure is exerted that favors the propagation of drug-resistant bacteria (Fey et al., 2000; Graveland et al., 2010).

As a result, antimicrobial-resistant enterococci, as well as other resistant gut bacteria, can be spread in the environment by fecal residues. These bacteria can rapidly transfer their resistance to other strains through genetic determinants carried by mobile elements. Resistant enterococci are able to persist in the animal intestine, contaminate the environment and food of animal origin, and transfer determinants to human gut's isolates (Tasho and Cho, 2017). Moreover, community people can be exposed to antimicrobial resistant enterococci through direct contact.

Use of antimicrobials can enhance gene transfer between bacteria (Malhotra-Kumar et al., 2007). Gene conjugative transfer is frequent in the human gut, as well as in nature. Enterococci acquire antibiotic resistance genes, e.g., for high-level gentamicin

resistance and glycopeptides resistance determinants (Willems et al., 2011; Sparo et al., 2012).

Further, enterococci can horizontally transfer resistance genes to relevant bacteria in clinical settings, such as Escherichia coli, Staphylococcus aureus, and Listeria spp. (Verraes et al., 2013).

Generally, severe infections caused by enterococci are treated with a cell-wall active agent-aminoglycoside (mostly gentamicin) combination. The emergence of β-lactam and glycopeptide resistance and high-level resistance to gentamicin in enterococci has led to the employment of alternative antimicrobials (Arias et al., 2010; Bartash and Nori, 2017).

**Figure 1** shows a presumable bidirectional transfer of resistance determinants and/or resistant enterococci between different niches such as human and animal. This transfer can occur through direct contact, foodborne contamination, as well as in health-care settings and the environment (community).

## High-Level Vancomycin Resistant Enterococci

In enterococci, vancomycin resistance is associated with different van genotypes each corresponding with a typical Van phenotype. These genes are chromosomal or extrachromosomal encoded in transposons and/or plasmids. In human E. faecalis and E. faecium, VanA and VanB (inducible resistance) are the most relevant types. vanA gene cluster is most often found on conjugative or non-conjugative plasmids (Cetinkaya et al., 2000; Top et al., 2008). VanA is encoded by Tn1546, or closely related transposons. vanA gene is linked with highlevel resistance to vancomycin and teicoplanin, while variablelevel resistance to vancomycin is associated with a VanB phenotype. The vanB operon is found among large conjugative plasmids or in the chromosome (Cetinkaya et al., 2000). The most frequent vanB subtype, vanB2, is encoded by conjugative transposons Tn1549-/Tn5382-like. It is interesting to note that Tn1549-vanB has also been detected in anaerobes that inhabit the human gut (Dahl et al., 2000; Launay et al., 2006).

 Lester et al. (2006) have proven, in volunteers, the existence of genetic transfer in the human intestine between ingested chicken vanA-E. faecium and non-resistant to vancomycin human E. faecium. It is important to highlight that this research has been performed in a human gut model with its complexity and its diverse microbiota.

Furthermore, there is a global concern regarding plasmidmediated vanA transfer from E. faecalis to methicillin-resistant S. aureus and their co-colonization, with the likelihood of VanA-S. aureus isolation (Flannagan et al., 2003; Weigel et al., 2003).

In the last decades, vanA-E. faecium were recovered from animals and food of this origin. Initially, the European Union stated that there was a link between Veterinarian use of a glycopeptide (avoparcin) and the emergence of vancomycin resistance (Werner et al., 2008). After avoparcin's ban, glycopeptide-resistance did not disappear. López et al. (2009) reported high-level vancomycin resistant enterococci (4%) from samples of animal origin 10 years after avoparcin was forbidden. Continuous presence of vancomycin-resistant enterococci in farms and in food of animal origin suggests that is possible the co-transfer of resistance genes located in the same conjugative plasmid, such as vanA and ermB, which encodes for macrolides resistance, widely used in Veterinary medicine. Also, the presence of ABC-type transporter genes and the toxin-antitoxin system may favor the persistence of vancomycin resistance determinants (Aarestrup, 2000). In addition, deficient hygiene conditions in animal husbandry, should not be underestimated (Garcia-Migura et al., 2007). In the same period, a different situation was observed in the United States, since food of animal origin glycopeptide-resistant E. faecium were not detected but, nevertheless, they emerged in health-care settings, turning into a pathogen almost as prevalent as E. faecalis had been so far (Coque et al., 1996; Ramsey and Zilberberg, 2009). However, in Michigan, United States, vanA-E.faecium was detected in farm animals where avoparcin was not used; which supports the existence of alternative ways for spreading of van genes, their transfer or carrying isolates from humans to animals (Johnsen et al., 2011; Gordoncillo et al., 2013).

In Argentina, vanA-E. faecium from artisanal food of animal origin was reported by Delpech et al. (2012). Previously, it was observed that animal-origin vancomycin-resistant E. faecium of animal origin were ingested in meats, proving the risk of resistant bacteria colonization when meat products carrying resistant bacteria were consumed (Heuer et al., 2006).

In Argentina, since the late 19900 s vancomycin-resistant E. faecium infections have been reported. In several Argentinean hospitals, the prevalence of clonal complex (CC) 17 carrying the vanA gene was detected. Most of these enterococci also expressed high-level aminoglycoside resistance (Corso et al., 2007).

Recently, during a year-period (2013), genetic relatedness (PFGE studies) between vanA enterococci from humans, food and the hospital environment in the District of Tandil (Argentina) was investigated. vanA-E. faecium (n: 13) were recovered from human, food and hospital environment samples. vanA enterococci were distributed among seven clonal types; esp gene was detected in clinical strains. However, the clonal relationship between vanA-E. faecium of clinical and food origin was not found. The clonal relationship was observed among isolates from the hospital environment and from patients (Pourcel et al., 2017).

Bacterial conjugation provides an efficient gene transfer pathway and can be considered as the most relevant mechanism for the increase of antimicrobial resistance (Hammerum, 2012). It is possible that bacteria from food can constitute reservoirs of antimicrobial resistance.

The horizontal gene transfer of vanA-resistance between food strains and human gut microbiota becomes a possible mechanism

of resistance dissemination when enterococci do not fit in the hospital settings (Hammerum et al., 2010).

### High-Level Gentamicin Resistant Enterococci

The most prevalent mechanism of high-level aminoglycoside resistance in clinical bacteria is their enzymatic modification. Three families of aminoglycoside modifying enzymes have been recognized: phosphotransferases (APH), acetyltransferases (AAC), and nucleotidyltransferases (ANT). Genes for aminoglycoside modifying enzymes are often plasmidic, with bacteria-bacteria aminoglycoside resistance dissemination (Bassenden et al., 2016).

The following risk factors for the acquisition of infections with high-level gentamicin resistant enterococci have been identified: previous long-term antimicrobial treatment, number of prescribed antimicrobials, previous surgeries, peri-operative antimicrobial prophylaxis, hospitalization term/antimicrobial treatment, urinary catheterization and renal failure. Infections caused by E. faecalis with HLGR constitute a severe risk for patients with invasive conditions and long-term hospitalization (Miranda et al., 2001; Wendelbo et al., 2003; Ceci et al., 2015).

The most ubiquitous HLGR gene among human and food enterococci is aac (60 )-Ie-aph (200)-Ia that encodes AAC(60 )- APH(200)-Ia, with acetyltransferase and phosphotransferase activities. Enterococci with this enzyme express resistance to most of the available aminoglycosides (MIC > 2,000 µg/mL), except for streptomycin (Leclercq et al., 1992). Generally, aac(60 )-Ie-aph(200)-Ia gene is flanked by inverted repeats of IS256, composing transposon Tn5281 in E. faecalis as part of a conjugative plasmid (Rosvoll et al., 2012).

Other monofunctional genes encoding aminoglycosidemodifying enzymes have been described, such as class APH (200)-subclass I phosphotransferases, chromosomal [e.g., aph(200)- Ib y aph(200)-Id] and plasmidic [e.g., aph(200)-Ic] genes. These resistance determinants were originally found on Enterococcus species different than E. faecalis and encode enzymes which confer resistance to gentamicin and amikacin. aph(200)-Ic gene is associated with MIC for gentamicin ranging between 128 to 512 µg/mL. Nevertheless, aph(200)-Id gene, initially described in human E. casseliflavus, is linked to HLGR. This gene has been detected in clinical vancomycin-resistant E. faecalis (Ramirez and Tolmasky, 2010; Economou et al., 2017).

From 2000 to 2002, in Denmark, the proportion of high-level gentamicin resistant E. faecalis isolates increased from 2 to 6% in the pig population. Simultaneously, an emergence of HLGR E. faecalis isolates among patients with infective endocarditis was detected in the North Denmark Region (DANMAP, 2002). Afterward, Larsen et al. (2010) demonstrated that all of these isolates (human and pig origin) belonged to the same clonal group, suggesting that pigs were a reservoir for high-level gentamicin resistant E. faecalis associated with enterococcal infections.

Sparo et al. (2012) proved the spread of enterococci with HLGR from animals to humans through the food chain, and also that enterococci isolated from food of animal origin and humans carried the same aminoglycosides resistant genes, as reported, also, by other authors (Hammerum et al., 2007).

Resistance to ampicillin and vancomycin is infrequent, although E. faecalis have been shown to acquire HLGR (Kuch et al., 2012). Recently, over a 1 year period, the presence of cytolysin and HLGR in E. faecalis from human (hospital), animal (chicken feces from a farm) and food (minced meat from shops) origin were studied. Clinical samples were obtained from patients with invasive infections in Hospital Ramón Santamarina from Tandil City, Buenos Aires Province (Argentina). In all enterococci with HLGR, aac (60 ) -Ie-aph (200)-Ia gene was amplified. aac (60 )-Ie-aph (200)-Ia and cylA were detected in human, food and animal E. faecalis, proving its environmental spread (Sparo et al., 2013).

In patients presenting risk factors, a high-level intestinal colonization of E. faecalis can become a frequent precursor of human invasive infections by bacterial translocation. This event is favored by the enhanced employment of broad-spectrum antimicrobials that exert significant pressure over the intestinal microbiota, hence, resulting in a likely emergency of multiresistant enterococci. The human gut is a considerable reservoir for microorganisms potentially capable of transfer resistance to conventional antimicrobials. Moreover, the fact that bacteria isolated from food of animal origin can behave as a resistance reservoir needs to be taken into consideration. In vitro studies performed to prove genetic exchange between enterococcal strains from humans and food of animal origin, are not conclusive (Sparo et al., 2012). Therefore, in vivo models for assessing genetic transfer are needed. Research carried out in animal models with their own microbiota it will not be able to reproduce the conditions of the human intestine. The use of human colon microbiota in germ-free mice is proposed as a model for reproducing the interaction between food strains and human gastrointestinal microbiota (Hirayama, 1999). Recently, HLGR determinants transfer from food to human bacteria was proven in an animal model. Immunocompetent BALB-C mice, colonized with human feces from an infant with no previous antimicrobial treatment, were used. This study showed evidence of the likelihood of high-level gentamicin resistance horizontal transfer from food to human E. faecalis. Therefore, a gene transfer model in non-sterile mice colonized with human gastrointestinal microbiota was standardized (Sparo et al., 2012).

It is needed to highlight that the rate of HLGR in vancomycinresistant enterococci is higher than in vancomycin-susceptible enterococci strains. Mihajlovic Ukropina et al. (2011) ´ studied the frequency of antimicrobial resistance in enterococci isolated from blood cultures. HLGR was detected in vancomycin-resistant strains (87.6%) as well as in vancomycin-susceptible strains (9.9%). Hence, according to this study, HLGR in E. faecium is higher than in E. faecalis.

In an Argentinean study, E. faecalis strains with HLRG (aac (60 )-Ie-aph (200)-Ia gene) and without glycopeptide resistance were recovered from human and food samples of animal origin. PFGE patterns showed four clonal types, and also that there was a clonal relationship between E. faecalis with HLGR isolated from food and those isolated from humans (Pourcel et al., 2017).

### Clonal Complexes of High-Level Vancomycin and Gentamicin Resistant Enterococci

Worldwide, MLST E. faecium data established that the majority of the clinical strains belong to the CC17, most of which are resistant to ciprofloxacin and ampicillin, and contain virulence genes. When new algorithms such as the Bayesian analysis of population structure (BAPS) were applied, it showed that CC17 consists of two large groups with different evolutionary origin: BAPS 2-1, containing sequence-type (ST) 78 and BAPS3- 3 (ST17 and ST18). Most of the drug-resistant clinical isolates of hospital origin belong to both groups. The majority of community-origin isolates were grouped in the BAPS 2-1 group, genetically and evolutionarily different from hospital isolates and those of hospital origin are evolutionarily closer to those of farm animals. A similar trend was detected among vancomycin-resistant E. faecium, investigated in broiler flocks 15 years after the avoparcin ban, diversity was observed as well since they clustered in three BAPS populations (Willems et al., 2012; Bortolaia et al., 2015; Raven et al., 2016).

Several authors have highlighted the predominance of clonal lineages −17, −18 and −78 in human clinical isolates of E. faecium. It could be assumed that they have adapted to the intestinal environment and integrate their microbiota (Baquero and Coque, 2011; Faith et al., 2015; Tedim et al., 2015, 2017).

Nowadays, comparison of available genome sequences allowed to support the existence of two clades for E. faecium; one of the animal strains and hospital-associated enterococci (clade A) and another one of community strains (clade B), which includes human commensal isolates. Clade A has been subdivided into A1, including most of the clinical isolates (lineages ST17, ST18, and ST78) and A2, containing mainly strains of animal origin. It has also been shown that the genome of the strains included in the clade A1 has a larger size than those ones of strains belonging to A2, which seems to support the recent emergence of this clade and the importance of its recombination (Galloway-Peña et al., 2012; Tedim et al., 2015).

Unlike E. faecium, E. faecalis lack a clear structure in clades. Some clones are more frequent in hospitalized patients or in the community. Specifically, CC2 and CC9 both present high-level vancomycin resistance and have been described as highly risky due to their adaptation to the hospital environment and global dissemination (Freitas et al., 2009; Kuch et al., 2012; Guzman Prieto et al., 2016).

E. faecalis CC2, a high-risk CC, is frequently found among health-care associated isolates and represents hospital complexes linked with high-level aminoglycoside resistance (Weng et al., 2013). In addition, E. faecalis CC87, similar to CC2, expresses multi-drug resistance and can be associated with

invasive infections (Ruiz-Garbajosa et al., 2006; Tedim et al., 2015).

#### IMPACT IN HUMAN INFECTIONS AND THERAPEUTIC OPTIONS FOR RESISTANT ENTEROCOCCI

Among bloodstream infection (BSI) associated with the healthcare environment, Enterococci is the third most common one. Although vancomycin-resistant enterococci have been clinically relevant pathogens for years, the majority of clinical data is retrospective (Wisplinghoff et al., 2004). Nowadays, vancomycin-resistant enterococci are the cause of one-third of all health care associated infections in the United States and one fifth in some European countries (Hidron et al., 2008; European Centre for Disease Prevention and Control [ECDC], 2010). Furthermore, mortality rates in patients with BSIs produced by vancomycin-resistant enterococci range between 20 and 46% (Han et al., 2009; McKinnell et al., 2011; Twilla et al., 2012).

Treatment of vancomycin-resistant enterococci's BSI is particularly challenging. The therapeutic options include linezolid, daptomycin, quinupristin-dalfopristin, tigecycline, and lipoglycopeptides, such as telavancin, dalbavancin and oritavancin.

Due to limited clinical available data of lipoglycopeptides together with resistance issues in VanA enterococci, the role in systemic vancomycin-resistant enterococci infections for telavancin and dalbavancin is irrelevant. Oritavancin (the lipoglycopeptide with the broadest antibacterial coverage) has shown bactericidal activity against VanA and VanB vancomycinresistant enterococci. This drug was approved for the treatment of acute bacterial skin infections and is currently undergoing clinical trials for the treatment of bacteremia (Zhanel et al., 2010; Messina et al., 2015).

In Europe, Teicoplanin can be used for VanB phenotype infections (Svetitsky et al., 2009).

Tigecycline has not been approved for the treatment of bacteremia because it does not achieve high serum concentrations. This tetracycline can be considered as one of the first-line treatments for polymicrobial intra-abdominal infections associated with vancomycin-resistant enterococci due to its high penetration into the peritoneal space (Arias et al., 2010).

Quinupristin-dalfopristin, effective only against E. faecium, has a high molecular weight, which renders it unable to cross the blood-brain barrier. This, added to the facts that it has frequent side effects and that it easily interacts with other drugs, limits its clinical use (Rubinstein et al., 1999).

Since approval, linezolid has been widely employed for vancomycin-resistant enterococci infections. The clinical success rate can vary based on the infection site and generally range between 50 and 80%. Lower success rates are generally seen in patients with bacteremia and infections without known focus (Birmingham et al., 2003; Kraft et al., 2012; Da Silva et al., 2014; Patel et al., 2016).

Linezolid has shown utility for treating infections by vancomycin-resistant enterococci non-susceptible to daptomycin. Surveillance analysis carried out in 2012 showed 99.5% susceptibility for linezolid against enterococci in the United States health systems (Mendes et al., 2014). Prolonged use of linezolid has been associated with resistance emergency (Pogue et al., 2007; McGregor et al., 2012).

Tedizolid is a next-generation parenteral and oral oxazolidinone with a broad spectrum bacteriostatic activity against resistant Gram-positive bacteria including VanA and VanB enterococci. It has been approved for the treatment of acute bacterial skin and soft tissues infections, and, currently, clinical trials for bacteremia and pneumonia treatment are being undergone (Rybak et al., 2014).

Daptomycin has been successful for multidrug-resistant enterococci and vancomycin-resistant enterococci infections' treatment. Multiple analyses of the Cubicin Outcomes and Registry Experience (CORE) have shown a higher clinical success rate when used as first-line therapy for vancomycin-resistant enterococci bacteremia, 87–93% (Sakoulas et al., 2007; Mohr et al., 2009).

β-lactam antibiotics have been evaluated, in vitro, combined with daptomycin against vancomycin-resistant enterococci, including ampicillin, ceftaroline, ceftobiprole, and ceftriaxone, all of which produced synergistic effects even when β–lactam resistance was detected (Sakoulas et al., 2012, 2014; Hall Snyder et al., 2014; Werth et al., 2015).

For infectious endocarditis due to ampicillin susceptible and HLGR E. faecalis, ampicillin with ceftriaxone should be considered as an alternative treatment option, since it showed a similar efficacy to the observed ones for ampicillin with gentamicin, in susceptible strains, but with less nephrotoxicity. The saturation of several penicillin-binding proteins is the main reason why this combination presents a desirable bactericidal synergy (Mainardi et al., 1995; Murray, 2000; Fernández-Hidalgo et al., 2013; Economou et al., 2017).

### Alternatives/Complementary Therapeutic Options

Available evidence about infection control and prevention measures (ICP) to reduce vancomycin-resistant enterococci spread in adult hospitalized patients is insufficient. A systematic review published in 2014 (that included 9 studies with 30,949 participants) emphasized the importance of the implementation of hand hygiene program. A decrease of 47% in the vancomycinresistant enterococci acquisition rate was observed when this measure is applied. Further studies with appropriate methodological design are urgently needed to define if ICP measures have an impact in reducing the acquisition of vancomycin-resistant enterococci among hospitalized patients (De Angelis et al., 2014).

A proposal for controlling antimicrobial resistance dissemination is to reduce antimicrobials employment in animal husbandry and promoting research of novel therapeutic alternatives. Probiotics are "living microorganisms which when administered in adequate amount confer a health benefit on

the host" (Food and Agriculture Organization/World Health Organzation [FAO/WHO], 2001). These strains improve intestinal microbial balance, provide protection against gut pathogens and modulate the immune system. Probiotics are supplemented into animal feed (cattle, ducks, broilers, and chickens) and have beneficial effects on the food producing animals by enhancing weight gain, increasing egg/milk production, lowering the incidence of disease and mortality rates (Crittenden et al., 2005). Use of probiotics against pathogenic bacteria showed to be effective for reducing food-borne illnesses in consumers, in view of the absence of antibiotics in sub-therapeutic doses (Van Coillie et al., 2007).

A different approach is the use of microbial cell extracts that reduce the risks of bacterial translocation and infection (Sparo et al., 2014; Lemme-Dumit et al., 2018).

Bacteriocins are ribosomally synthesized peptides, with bacteriostatic/bactericidal activity, produced by various bacteria (Gálvez et al., 2007). The use of Gram-positive bacteriocins alone or in combination with antibiotics was proposed as a novel strategy to develop in human and veterinary medicines in order to help conventional antimicrobials against many multi-drug resistant pathogens. These combinations allow decreasing the MIC for achieving a bactericidal effect and, also, reduce undesirable sideeffects of antibiotics (Lebel et al., 2013; Naghmouchi et al., 2013; Delpech et al., 2017). Randomized controlled trials are needed for obtaining scientific evidence about the

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usefulness of these novel compounds against pathogenic enterococci.

#### CONCLUSION

Worldwide, enterococcal infections are among the most prevalent within those of nosocomial origin. Antimicrobial multi-resistant enterococci and their drug-resistant determinants spread by direct animal-human contact and/or through animal origin food. As mentioned above, the evidence is based on traditional microbiology and molecular tools, such as PFGE and MLST. Therefore, future studies combining phylogeographic methods with whole genomic sequence will provide reliable information for inferring bacteria movement between host populations.

Nowadays more countries are developing antibiotic-limiting policies, and thus arises a need of searching for an alternative or substitute for these drugs for sustainable food production, such as probiotics and bacteriocins.

#### AUTHOR CONTRIBUTIONS

MS and GD contributed to the writing of the microbiological and resistance aspects of the article, revised it and designed the Figure. NG contributed to the clinical and infectological aspects of the work.

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rates of recombination. J. Clin. Microbiol. 44, 2220–2228. doi: 10.1128/JCM. 02596-05

**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 Sparo, Delpech and García Allende. 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(s) 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.

# Advancement of the 5-Amino-1-(Carbamoylmethyl)-1H-1,2,3-Triazole-4-Carboxamide Scaffold to Disarm the Bacterial SOS Response

Trevor Selwood1,2, Brian J. Larsen3 , Charlie Y. Mo1,2, Matthew J. Culyba1,2 , Zachary M. Hostetler1,2, Rahul M. Kohli1,2 \*, Allen B. Reitz3 and Simon D. P. Baugh3 \*

1 Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States, 2 Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, United States, 3 Fox Chase Chemical Diversity Center, Inc., Doylestown, PA, United States

#### Edited by:

Gilberto Igrejas, Universidade de Trás-os-Montes e Alto Douro, Portugal

#### Reviewed by:

Giuseppe Celenza, Università degli Studi dell'Aquila, Italy Matej Butala, University of Ljubljana, Slovenia

#### \*Correspondence:

Rahul M. Kohli<EMAIL_ADDRESS>Simon D. P. Baugh<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 30 June 2018 Accepted: 16 November 2018 Published: 18 December 2018

#### Citation:

Selwood T, Larsen BJ, Mo CY, Culyba MJ, Hostetler ZM, Kohli RM, Reitz AB and Baugh SDP (2018) Advancement of the 5-Amino-1-(Carbamoylmethyl)-1H-1,2,3-Triazole-4-Carboxamide Scaffold to Disarm the Bacterial SOS Response. Front. Microbiol. 9:2961. doi: 10.3389/fmicb.2018.02961 Many antibiotics, either directly or indirectly, cause DNA damage thereby activating the bacterial DNA damage (SOS) response. SOS activation results in expression of genes involved in DNA repair and mutagenesis, and the regulation of the SOS response relies on two key proteins, LexA and RecA. Genetic studies have indicated that inactivating the regulatory proteins of this response sensitizes bacteria to antibiotics and slows the appearance of resistance. However, advancement of small molecule inhibitors of the SOS response has lagged, despite their clear promise in addressing the threat of antibiotic resistance. Previously, we had addressed this deficit by performing a high throughput screen of ∼1.8 million compounds that monitored for inhibition of RecAmediated auto-proteolysis of Escherichia coli LexA, the reaction that initiates the SOS response. In this report, the refinement of the 5-amino-1-(carbamoylmethyl)-1H-1,2,3 triazole-4-carboxamide scaffold identified in the screen is detailed. After development of a modular synthesis, a survey of key activity determinants led to the identification of an analog with improved potency and increased breadth, targeting auto-proteolysis of LexA from both E. coli and Pseudomonas aeruginosa. Comparison of the structure of this compound to those of others in the series suggests structural features that may be required for activity and cross-species breadth. In addition, the feasibility of small molecule modulation of the SOS response was demonstrated in vivo by the suppression of the appearance of resistance. These structure activity relationships thus represent an important step toward producing Drugs that Inhibit SOS Activation to Repress Mechanisms Enabling Resistance (DISARMERs).

Keywords: SOS response, antibiotic resistance, structure activity analysis, Pseudomonas aeruginosa, DNA damage

### INTRODUCTION

Antibiotic resistant bacteria represent one of the most pressing issues in infectious disease research today (Brown and Wright, 2016). An era is fast approaching when many currently treatable infections may become incurable (Boucher et al., 2009). While important efforts are underway to discover antimicrobials with different mechanisms of action (Clatworthy et al., 2007;

Thaker et al., 2013; Ling et al., 2015), the most conventional approach to overcoming resistance has involved the chemical modification of existing antibiotic scaffolds (Fischbach and Walsh, 2009). Although the resulting "next generation" antibiotics offer a respite, bacteria are likely to rapidly adapt their preexisting resistance mechanisms to counteract these gains. The limitations of conventional approaches highlight the need to pursue alternative strategies.

A promising alternative approach is to target pathways that promote acquired resistance to antibiotics. One such pathway is the bacterial DNA damage response pathway, known as the SOS response (**Figure 1**). Many antibiotics induce the SOS response, either by inducing DNA damage (e.g., fluoroquinolones) or by indirectly promoting DNA damage via targeting essential cellular and metabolic functions (Kohanski et al., 2007; Dwyer et al., 2012; Mo et al., 2016). The SOS response is well conserved across pathogens and involves numerous genes (e.g., >40 in Escherichia coli).  Thus, suppression of the SOS pathway would be predicted to compromise the response of bacteria to antibiotics.

A means to suppress the SOS pathway is to maintain repression of the SOS response. In the absence of genotoxic stress all genes of the pathway are tightly repressed by the dualfunction repressor/protease, LexA (**Figure 1**). In the presence of genotoxic stress the DNA damage sensor protein RecA forms filaments along ssDNA generated by aborted replication. The pathway is triggered when this filamentous RecA (RecA∗ ) promotes a conformational change in LexA that brings one of its protein loops into its own serine protease active site (Luo et al., 2001). Subsequent auto-proteolysis destabilizes LexA, and leads to transcriptional de-repression of SOS pathway genes (Culyba et al., 2018).

Genetic studies targeting either RecA or LexA validate the SOS response as a therapeutic target (**Figure 1**). In a murine thigh infection model an E. coli strain harboring a non-cleavable mutant of LexA abrogated resistance both to ciprofloxacin and rifampicin compared to a strain with a cleavable LexA (Cirz et al., 2005). In addition, deletion of RecA, or forced over expression of non-cleavable LexA have been shown to hypersensitize bacteria to traditional antibiotics (Lu and Collins, 2009; Thi et al., 2011; Mo et al., 2016). Furthermore, SOS inactivation in resistant bacteria resulted in re-sensitization to a fluoroquinolone (Recacha et al., 2017). Together, these studies suggest that targeting the SOS response could lead to both synergy with DNA damaging antibiotics to lower MIC values and suppression of acquired resistance (Cirz and Romesberg, 2007; Smith and Romesberg, 2007; Culyba et al., 2015).

While specifically targeting RecA has produced some important gains (Wigle et al., 2009; Alam et al., 2016; Bellio et al., 2017), we aimed to inhibit the RecA∗ -induced cleavage of LexA as this represents the key initiating step in the SOS response. To this end we developed a high throughput screen (HTS) that allowed estimation of RecA∗ -mediated LexA cleavage. Using this screen some 1.8 million compounds were evaluated for inhibition of RecA∗ -mediated LexA cleavage (Mo et al., 2018). The result of this screen was the identification of several chemotypes with the potential to modulate the SOS response (Mo et al., 2018). Herein is described the advancement of one of the chemotypes, the 5-amino-1- (carbamoylmethyl)-1H-1,2,3-triazole-4-carboxamide scaffold (**Figure 2**) via a modular synthesis that allowed for evaluation of structure-activity relationships and lead improvement to increase potency and expand the breadth of targetable pathogens. This work underscores the feasibility of developing DISARMERs (Drugs to Inhibit SOS Activation to Repress Mechanisms Enabling Resistance) – molecules that can act as adjuvants in standard antimicrobial therapies to both sensitize bacteria to antibiotics and reduce the rise of acquired resistance.

#### MATERIALS AND METHODS

#### Materials

All reagents used in chemical synthesis were purchased from Aldrich Chemical Co., (Milwaukee, WI, United States), Alfa Aesar (Ward Hill, MA, United States), or Thermo Fisher Scientific (Pittsburgh, PA, United States) and were used without further purification. Chemicals used in biochemical assays were obtained from Sigma-Aldrich (St Louis, MO, United States).

#### Compound Synthesis

Compounds were synthesized using a method that proceeds via a [3+2] cycloaddition, allowing facile, catalytic, non-moisture sensitive, and non-air sensitive syntheses of a variety of 5-amino-1-(carbamoylmethyl)-1H-1,2,3-triazole-4-carboxamides. For the majority of analogs, catalysts employed were either sodium ethoxide (synthesis A, **Table 1**) or cesium carbonate (synthesis B, **Table 1**). The base-mediated cyclization is depicted in **Figure 2**.

For reactions catalyzed by sodium ethoxide (synthesis A), a solution of sodium ethoxide (1.2 mmol) in anhydrous ethanol (10 mL) was maintained under nitrogen and cooled to 0◦C with stirring. Once cooled, the cyano component (1.1 mmol) was added to the solution. The resulting solution was stirred for 10 min at 0◦C before addition of the azido component (1.0 mmol). The resulting solution was maintained at 0◦C for a further 2 min before being allowed to warm to room temperature. Upon reaching room temperature, the solution was lightly sonicated for 20 s before the temperature was raised to 40◦C. The solution was maintained at 40◦C for 4 h before being allowed to cool to room temperature. Once the solution reached room temperature the reaction was quenched with deionized H2O (100 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic fractions were washed with deionized H2O (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to yield the crude product. The crude product was dissolved in DMF, filtered through a syringe filter and purified via reverse phase HPLC using acetonitrile in deionized H2O (with 0.1% TFA in both solvents) targeting virulence.

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to yield, after evaporation and lyophilization, the desired product.

For reactions catalyzed by cesium carbonate (synthesis B), the azido component (1.1 mmol), the cyano component (1.0 mmol) and cesium carbonate (0.25 mmol) were dissolved in DMSO/deionized H2O (7:3, 4 mL) with stirring. The reaction vial was capped and stirred for 24 h before being diluted with deionized H2O, partially concentrated in vacuo, frozen,

FIGURE 2 | Lead compound and synthetic approach. (A) The lead 1 is shown with the Areas A, B and C highlighted. These areas are the focus of diversification in analog synthesis to explore structure-function relationships in the lead series. (B) Retrosynthesis of the 5-amino-1-(carbamoylmethyl)- 1H-1,2,3-triazole-4-carboxamides is shown, with the core of Area B formed via a cycloaddition of azide 15 and nitrile 16. In Area B the 5-amino group derived from the nitrile is highlighted to help illustrate the cycloaddition mechanism.

and lyophilized to remove water and DMSO. The resulting crude material was purified via reverse phase HPLC using acetonitrile in deionized H2O (with 0.1% TFA in both solvents) to yield, after evaporation and lyophilization, the desired product.

For compound **7** a variation of synthesis A (synthesis C) was employed in which sodium methoxide was used instead of sodium ethoxide. For this synthesis a mixture of the cyano component (0.24 mmol), and sodium methoxide (0.26 mmol) in methanol (1.07 mL) was stirred for 30 min before addition of the azido component (0.21 mmol). The reaction was stirred for 16 h before treatment with methanol (0.25 mL) and stirring for 3 h. Methanol (1 mL) was added, and the reaction was heated at 95◦C for 2 h. The mixture was treated with deionized water (25 mL), concentrated HCl (1 drop), and ethyl acetate (10 mL). The aqueous layer was treated with saturated aqueous NaHCO3 (5 mL) and extracted with ethyl acetate (10 mL). The combined organic extracts were washed with saturated aqueous NaHCO3 (10 mL), brine (10 mL), and dried over anhydrous MgSO4 before concentration. The crude material was purified via reverse phase HPLC using acetonitrile in deionized H2O (with 0.1% TFA in both solvents) to yield, after evaporation and lyophilization, the desired product.

Some compounds in the Supplementary Information were synthesized by alternative methods in which an acetylene component replaced the cyano component. The alternate syntheses are described in the **Supplementary Methods**. Analogs which were not synthesized were obtained from commercial vendors ChemDiv (San Diego, CA, United States) and Vitas-M Laboratory (Champagne, IL).

All compounds were readily soluble in DMSO and were stored as 10 mM frozen (−30◦C) stocks when not in use.

#### TABLE 1 | Synthesis and inhibition by lead analogs.

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1 IC50 values are the avearge of 4 determinations and the errors are ± 1 SD. 2A-NaOEt conditions. B-CS2CO3 conditions, C-NaOMe conditions.

#### FlAsH-LexA Cleavage Assay

IC50 values were routinely determined using the E. coli FlAsH-LexA cleavage assay previously used to perform HTS (Mo et al., 2018). In this assay RecA-promoted LexA cleavage is monitored using fluorescence polarization. The E. coli FlAsH-LexA and RecA were constructed, expressed and purified as previously described (Mo et al., 2018). The conditions were 100 nM E. coli FlAsH-LexA, 200 nM RecA, 5 µM ssDNA (SKBT25: GCG TGT GTG GTG GTG TGC) (Tracy and Kowalczykowski, 1996), 5 µM ATPγS in 100 mM Tris-HCl, pH 6.5, 150 mM NaCl, 5 mM MgCl2, 0.1 mM TCEP, and 0.01% (w/v) Pluronic-F127. Reactions were performed in 384-well plates and components were added as 10 µL additions of ATPγS, ssDNA and RecA, in buffer and 10 µL of E. coli FlAsH-LexA in buffer using a Janus liquid handler (Perkin-Elmer). Compound was added as a DMSO solution using a pin tool, and the final concentration of DMSO in the reaction was 1.2%. Once the reaction components were combined, reactions were centrifuged for 1 min at 500 rpm and incubated for 30 min at room temperature. Reactions were quenched with a 10 µL addition of 50 mM EDTA and plates were read on a Tecan Infinite F200 Pro plate reader (Tecan US, Inc., Morrisville, NC, United States). The final assay conditions resulted in 100– 120 mP difference between the uncleaved and cleaved control wells, representing an approximately 60% cleavage of the E. coli FlAsH-LexA. On each plate 32 positive (-RecA) and 32 negative controls (+RecA) in which DMSO without compound was added were used to define the range of mP and calculate the fraction inhibited.

IC50 values were estimated by non-linear least squares fitting to the data using Equation 1.

$$\text{FI} = \frac{[\text{I}]^{\text{n}}}{\text{IC}\_{50}^{\text{n}} + \text{I}^{\text{n}}} \tag{1}$$

where FI = Fraction inhibited, [I] = Concentration of compound and n = Hill coefficient. Fitting was performed using Igor Pro (WaveMetrics Inc., Lake Oswego, OR, United States).

In the FlAsH-LexA cleavage assay the highest compound concentration was 111 µM and all of the compounds that demonstrated activity (**1**, **3**, **4**, **5**, **7**, **8**, **10**–**14**, **22,** and **23**) elicited normal titration curves suggesting that aqueous solubility was maintained up to 111 µM. Representative titrations for the compounds can be found in the **Supplementary Figure 1**.

### Orthogonal 32P-LexA Cleavage Assay

Full-length E. coli and P. aeruginosa LexA were engineered with a RRXS phosphorylation site on the N-terminus of the full-length protein, allowing for 32P labeling by protein kinase A to produce 32P-LexA, as described previously (Mo et al., 2018). Reactions contained 100 nM 32P-LexA, 200 nM RecA and 10 µM ATPγS and the buffer conditions were identical to those in the HTS assay. Compounds were added in DMSO and the final concentration was 2%. Reactions were incubated for 30 min at room temperature after which 2 × Laemmli buffer was added to stop the assay. The stopped reactions were subjected to 15% SDS-PAGE and the gels were visualized via phosphorimaging on a Typhoon imager (GE Healthcare Bio-Sciences, Marlborough, MA, United States). The intact and cleaved bands were quantified using Quantity One (Bio-Rad, Hercules, CA, United States) and the fraction inhibited was calculated. As for the HTS assay, controls contained DMSO and the negative controls contained RecA while the positive controls did not. Plots of fraction inhibited against compound concentration were fitted to Equation 1.

#### Electrophoretic Mobility Shift Assay

For the electrophoretic mobility shift assay (EMSA) full-length, catalytically inactive LexA-S119A was used (Mo et al., 2014). Increasing concentrations (0–1 µM) of LexA-S119A were mixed with 10 nM SOS operator DNA labeled with Cy5 in EMSA running buffer (70 mM Tris-HCl pH 7.5, 10 mM MgCl2, 150 mM NaCl, 5 mM DTT, 0.1 mg/ml BSA, 10 ng/µL ssDNA, 5% glycerol, 0.04% bromophenol blue) in the presence of 50 µM of compound (or DMSO carrier). After incubation at room temperature for 30 min, 20 µL of each reaction was subjected to 6% native PAGE. Gels were visualized on a Typhoon Imager using default fluorescence filter settings for Cy5. Gel bands were quantified using ImageJ (NIH, Bethesda, MD, United States) to determine the fraction of bound DNA at each LexA concentration. Data were fitted to a variable-slope sigmoidal dose-response curve.

#### Cell-Based SOS Reporter Assay

An E. coli MG1655 strain lacking sulA (1sulA) and the tolC transporter (1tolC) (Mo et al., 2016) was transformed with a reporter plasmid in which gfp expression was under the control of the recA promoter (pMS201 pRecA GFP) (Zaslaver et al., 2006). To perform assays overnight, cultures of the reporter strain were diluted 100-fold in M9 minimal media and grown at 37◦C with agitation to an OD595 of ∼0.6. For each reaction sample 100 µL of culture were mixed with 100 µL of M9 minimal media containing ciprofloxacin (256 ng/mL). Pre-diluted compounds were added (5 µL) in DMSO and cultures were incubated at 37◦C with agitation for 2 h after which the cells were fixed by adding 200 µL of phosphate buffered saline, pH 7.4 containing 1% paraformaldehyde. After 1 h of fixing, the cells were spun down at 4,000 rpm and re-suspended in phosphate buffered saline, pH 7.4. Fixed cells were analyzed using flow cytometry (BD FACSCalibur, Ex/Em: 488 nm/530 nm) and the mean fluorescence of 20,000 cells in each condition was recorded.

#### Frequency of Resistance

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A starter culture of 1tolC E. coli was cultured overnight at 37◦C with shaking in LB broth. The next day the culture was diluted 3 × 107 -fold in to LB broth. The dilution was used to produce four sets of twelve cultures, each containing 1 mL. To one set was added 10 µL of deionized H2O plus 10 µL of DMSO, to the second set was added 10 µL of 125 ng/mL ciprofloxacin in deionized H2O plus 10 µL of DMSO, to the third set was added 10 µL of deionized H2O plus 10 µL of a 10 mM solution of **14** in DMSO and to the fourth set was added 10 µL of 125 ng/mL ciprofloxacin in deionized H2O plus 10 µL of **14** in DMSO. The final concentration of ciprofloxacin (1.25 ng/mL) was below the MIC for ciprofloxacin which was determined to be 5 ng/mL.

The 48 cultures were incubated at 37◦C with shaking for 48 h. To determine the population size, spot plating was performed starting with 1 µL of the cultures diluted 105 -fold. A 100 µL aliquot of each 105 -fold dilution was transferred to a 96-well plate and serially diluted (10-fold dilutions) into LB broth. The dilutions (5 µL) were spotted on LB agar plates and the plates were incubated overnight at 37◦C. To determine the rifampin resistant population, 999 µL of each 1 mL culture was centrifuged at 6000 rpm for 10 min to remove the cells from solution and the cells were suspended in 100 µL of autoclaved 0.15 M NaCl. The 100 µL solutions were plated on the LB plates containing 100 µg/mL rifampin and incubated for 2 days at 37◦C. Following counting of the colonies the program bz-rates (Gillet-Markowska et al., 2015) was used to estimate mutation rates.

## RESULTS

Among the leads isolated from the HTS performed for inhibitors of RecA∗ -mediated LexA cleavage, lead **1** was selected for progression (**Figure 2A**). In the initial HTS, the parent 5-amino-1-(carbamoylmethyl)-1H-1,2,3-triazole-4-carboxamide, **1**, had an IC50 value of 32 µM (**Table 1**). This chemotype was well behaved in the HTS, producing close to 100% inhibition, and appeared to offer the most chemical tractability to allow for the construction of structure activity relationships (SARs). Furthermore, as LexA cleavage involves formation of a β-turn at the site of self-cleavage (Lee et al., 2005; Whitby et al., 2011), the structural similarity of **1** to β-turn mimetics also suggested that structure-activity relationships could inform on the possible mode of inhibitor action.

In order to better understand SARs, a modular synthesis was devised that would permit generation of informative analogs. While the construction of 5-amino-1,4-disubstituted-1,2,3-triazoles has been extensively investigated (Tome, 2004) no synthetic routes to 5-amino-1-(carbamoylmethyl)-1H-1,2,3 triazole-4-carboxamides based on **1** have yet been reported.

Initial synthetic routes that proceeded via the generation of two potential carboxylic acid intermediates followed by peptide couplings to vary the left- and right-hand portions of the final product were unsuccessful. These reactions were low yielding and/or the precursors were prone to decomposition. A more successful strategy proved to be to proceed via the simple structural intermediates, azides (**15**) for the left-hand portion and nitriles (**16**) for the right-hand portion (**Figure 2B**). These intermediates were either synthesized in 1–2 steps (Hering et al., 2005; Ju et al., 2006; Srinivasan et al., 2006; Ng et al., 2008; Xia et al., 2014) or purchased directly and could be combined via known base-mediated conditions to produce the desired aminotriazoles via a [3+2] cycloaddition.

Three sets of reagents that have been previously reported to facilitate such cyclizations were screened: stoichiometric sodium methoxide (Alfred, 1970; L'abbé and and Beenaerts, 1989; Julino and Stevens, 1998), stoichiometric sodium ethoxide (Hoover and Day, 1956; Livi et al., 1979), and catalytic cesium carbonate (Krishna et al., 2015). In most cases the choice of base between stoichiometric sodium ethoxide and catalytic cesium carbonate had little to no impact on the yield (e.g., **1**, **Table 1**). Overall most reactions were successful using the cesium carbonate conditions, however, yields using this route were affected by the time and temperature of the reaction. Using either the sodium ethoxide or catalytic cesium carbonate routes readily permitted modular access to a large variety of analogs, as demonstrated by the fact that aromatic, heteroaromatic, and non-aromatic groups for R1 and R2 were tolerated (**Table 1**). This modular approach thus allowed for systematic variation and investigation of structure activity relationships.

#### Structure Activity Relationships

Initial medicinal chemistry efforts focused on developing an understanding of the necessary features to improve potency. The three areas (A, B, and C) in **Figure 2A** were systematically investigated and the IC50 values for selected compounds are listed in **Table 1** with additional data shown in **Supplementary Table 1**. Approaches used to probe the binding of compounds of this class included amino group replacement, linker methylation and N-methylation, methyl probing of the aryl rings, homologated variations, and non-aromatic variations. IC50 values were determined using the FlAsH-LexA cleavage assay.

In the linker connecting areas A and B, both mono- and bis-methylated compounds (**17** and **18**) showed no measurable activity, suggesting that substitution was not tolerated at the methylene linker. Similarly, methylation of the amide of the linker, **19**, also abrogated activity. The inability to tolerate substitution in the linker region suggested that it likely lies in a narrow groove and that attempts to modify this area could impact the conformations accessed by the lead. With the linker area appearing not amenable to modification, the

aromatic portion of area A was investigated. Replacement of the para-ethoxy group substituted phenyl ring with an unsubstituted phenyl, **2**, benzyl, **20**, or phenethyl group, **21**, led to loss of activity. However, replacement of the phenyl ring with a cyclohexyl ring, **3**, or a cycloheptyl ring, **4**, returned activity, suggesting that aromaticity was a larger restriction than hydrophobicity. Systematic variation of methyl functionalization on the phenyl ring revealed that substitution at the meta position, **6**, was not tolerated whereas substitution on the ortho, **5**, and para, **7**, positions were preferred. Beyond the single methyl functionalization, mono-substitution at the ortho position on the phenyl showed steric preferences with activities: Me (**5**) > Et (**22**) > OMe (**23)** > OEt (**24**) = H (**2**). Bis-substitution on the aryl ring was additionally investigated with for example, **8**, showing that combination of ortho- and para-substitution was tolerated but not significantly superior to ortho-substitution alone, **5**. In summary, probing of area A revealed interesting substrate preferences but failed to produce a significant increase in potency.

Additional investigations in area B also did not reveal a means to increase potency. Replacement of the amine by hydrogen (**25**), methyl (**26**), or ethyl (**27**) all rendered compounds inactive, suggesting that the amine was making contacts essential for activity. Supporting this conclusion was the finding that monosubstitution on the amine by acetyl (**28**) was tolerated but with reduced potency.

Probing of area C proved more fruitful. As with area A, the amide linker appeared important as methylation was not tolerated (**29**). Probing of the phenyl moiety indicated that its presence and correct positioning are critical. The importance of this ring was indicated by the intolerance to replacement by cyclohexyl (**30**) or methyl (**31**) groups, and the need for correct positioning was indicated by the intolerance to the replacement of the phenyl ring by benzyl (**32**) or phenethyl (**33**). The importance of substitution on the phenyl ring was investigated by systematic methyl substitution around the phenyl ring. Consistent with the meta- and para- substitution pattern on area C of **1**, this analysis indicated that ortho substitution, **9**, was not tolerated whereas individual meta, **10**, and para, **11**, substitutions were allowed. Heteroatom inclusion was also tolerated in Area C, as shown by ester-containing variation **12**, and pyridyl derivative **13**. However, compound **14**, 5-amino-1-{2-[(4-ethoxyphenyl)amino]-2-oxoethyl}-N-phenyl-1H-1,2,3-triazole-4-carboxamide, with no substitution on the phenyl ring proved to be the most potent compound tested in this series with an IC50 of 9 µM.

Before proceeding to additional analysis, cytotoxicity testing with HG2 cells was performed for select compounds, including **1** and **14**. Both the initial lead **1** and the most potent analog **14** showed no appreciable toxicity (CC50 of 277 µM and > 500 µM, respectively). Due to the increased potency and lack of cytotoxicity, the mechanism and activity of compound **14** was examined in more detail as described below.

#### Characteristics of Compound 14

A suite of assays was utilized to examine **14** in order to confirm specific inhibition against LexA and demonstrate SOS suppression in cells. To confirm the findings from the