Specific EC23S primers were selected to quantify E. coli K12 ER2738 (Forward: 5 ′ -CATAAGCGTCGCTGCCG-3′ ; Reverse: 5 ′ -AAAGAAAGCGTAATAGCTCACTGGTC-3′ ) (Ludwig and Schleifer, 2000; Chern et al., 2011). A standard curve with these primers using the 23S rRNA gene sequences as target was obtained in LB medium at 107 to 101 cells/mL. 20µL of internal control at 5.105 E. coli/mL were added to each 1 mL sample prior to DNA isolation to obtain a concentration of 104 E. coli/mL in each sample.

#### DNA Isolation and Extraction

After AAB growth and the addition of the internal control, two DNA extraction methods were tested: the Ausubel (Ausubel et al., 1992) and Lipp methods (Lipp et al., 1999). The two methods use CTAB as Jara et al. (2008) recommend. The main differences are that the second method use chloroform to purify DNA and use a CTAB precipitation solution after cell lysis to eliminate the remaining polyphenols. Moreover, DNA was re-dissolved in 50µL sterile deionized water for the methods. When indicated, cells were centrifuged and the pellets were resuspended into lysis buffer supplemented with PVPP at a final concentration of 1% (w/v). Each bacterium was tested in growth medium supplemented with 10% (v/v) ethanol, and white and red wines. All the experiments were performed in triplicate and repeated three times.

#### AAB Real-Time PCR Amplification (qPCR)

AAB primers used to amplify the 16S rRNA gene were selected from Valera et al. (2015). The forward primer AAB-F (5′ - TGAGAGGATGATCAGCCACACT-3′ ) and the reverse primer AAB-R (5′ -TCACACACGCGGCATTG-3′ ) were synthesized by Eurogentec R (France). The PCR mixture was prepared in a total volume of 25µL with 100 nmol of each primer and 5µL DNA extract. The amplifications were done in triplicate on a CFX90 real-time PCR system (Bio-Rad) under the following conditions: 95◦C for 10 min, 40 denaturation cycles at 95◦C for 15 s, and 62◦C for 1 min. Then a melting curve was produced to check the presence of only one amplification fragment. To test PCR amplification quality, BSA, and PVP treatments during qPCR at 400 ng/µL and 0.5% (w/v), respectively, were performed (Tessonnière et al., 2009). A control without treatment was also performed. The PCR cycle in which fluorescence first occurred (quantification cycle: Cq) was determined automatically using Bio-Rad CFX Manager R software after setting the regression method.

### Volatile Acidity According to AAB Population

The acetic acid concentration and AAB population were monitored over time in synthetic medium containing 10% (v/v) alcohol. The amount of acetic acid was measured enzymatically using a Biosentec kit (Cat. No 021) according to the manufacturer's instructions, expressed in gram per liter. The AAB population was determined throughout their growth by plating on mannitol agar (CFU/mL). The same experiment was also performed in red wine from the vineyard of the University of Burgundy but no inoculation was performed; the AAB contamination was natural. In this experiment, the AAB population was determined by both qPCR and FCM. FCM provided the total bacteria population count. LAB enumeration was performed in FT80 Petri dishes to avoid overestimating the AAB population determined by FCM. Moreover, 15 red wines were chosen randomly from different wine regions to analyze the AAB populations (qPCR and FCM). In addition, acetic acid concentration was determined.

# RESULTS

#### qPCR Specificity

The specificity of the AAB primers was tested against a panel of microorganisms known to be naturally present in wine. In silico tests were performed on O. oeni, Pediococcus spp., Lactobacillus spp, Zygosaccharomyces bailii, Candida vini, Pichia membranifaciens, P. fermentans, and S. cerevisiae. AAB primers did not match with the main wine microorganisms. In vitro tests were performed in synthetic medium against O. oeni and a panel of yeasts (see above). The Cq-values were the same as the negative control for all populations of O. oeni and yeast, thus validating the specificity of the primers. The EC23S specific primers targeting the 23S rRNA gene from E.coli (internal) control did not amplify DNA of the wine microorganisms tested. E. coli has been chosen because this microorganism is not naturally present in wine.

#### Comparison of DNA Isolation Methods

DNA extraction for AAB quantification was performed after growth in ethanol medium, and white and red wines using either the Lipp or Ausubel methods with or without PVPP during cell lysis. In order to compare the DNA extraction methods, the Cq obtained from DNA extracted by both methods with or without PVPP for the three different media were compared for each bacterium.

As shown in **Table 1A**, for ethanol medium, the Lipp method without PVPP gave significantly better results, as shown by a lower Cq whatever the bacteria, except for G. oxydans, showing no significant difference in the results obtained by both methods. PVPP addition during DNA extraction using either the Lipp or Ausubel method did not improve qPCR efficiency for most of the bacteria tested. PVPP addition during extraction with the Ausubel method significantly improved Cq only for A. aceti and A. pasteurianus. Thus, the choice of DNA extraction method is essential.

For white wine (**Table 1B**), the effect of PVPP is species dependent, but the results confirmed that the Lipp method gave much better results.

Regarding red wines which are rich in qPCR inhibitor compounds, the Lipp method gave much better Cq compared to the Ausubel method, except for G. oxydans. When PVPP was added prior to DNA extraction, the Cq-values did not improve whatever the DNA extraction method, except in a few cases (**Table 1C**). However, when using PVPP, the Lipp method was always better than the Ausubel method, as demonstrated by the lower Cq-values. For example, in our study we obtained a Cq for TABLE 1 | *C*q results according to DNA extraction (Ausubel and Lipp methods) with (+) or without (−) PVPP for *A. aceti*, *A. pasteurianus*, *G. oxydans,* and *Ga. liquefaciens* in (A) growth medium supplemented with 10% (*v*/*v*) ethanol, and (B) white, and (C) red wines.

Values represent the Cq mean ± Standard Deviation (SD); n = 3; values followed by different letters within a column are statistically different at p < 0.05 (XLStat©).

A. aceti of 27.8 in red wine (Ausubel + PVPP). When using the Lipp method with PVPP, the Cq was equal to 22.4. Thus, using the suboptimal extraction method, Cq led to underestimating the real AAB population.

Another way to improve qPCR efficiency is to add either BSA or PVP in the qPCR mix (Jiang et al., 2005; Malorny and Hoorfar, 2005). These compounds are assumed to trap inhibitors in the reaction mix. **Figure 1** shows the results of PCR assays on A. pasteurianus. DNA was extracted using the Lipp extraction method with PVPP, and qPCR was run with or without either the addition of BSA or PVP in the qPCR mix compared to control. The addition of BSA or PVP in the qPCR mix did not lead to any improvement of the Cq obtained. No improvement of Cq-values was observed using these compounds and, as no difference was highlighted in red wine with the Lipp DNA extraction method between the four species, neither BSA nor PVP were added in the mix to quantify AAB using the Lipp method in red wine. However, BSA added to A. pasteurianus DNA using the Ausubel method with or without PVPP in red wine significantly improved Cq-values compared to control, but they were always significantly higher (less effective) than the Lipp method. The values from

FIGURE 1 | *C*q results for *A. pasteurianus* after DNA extraction by the Lipp method in mannitol supplemented with 10% (*v*/*v*) alcohol (containing 2.4.106 cells/mL), white wine (containing 2.7.105 cells/mL), and red wine (containing 9.9.107 cells/mL). Cell lysis was performed in triplicate with PVPP and BSA (400 ng/µL) ( ) or PVP (0.5% w/v) ( ), added to the qPCR mix and compared to control ( ). An Anova with Tukey's test was performed for each medium independently to analyze the Cq results with BSA and PVP compared to control (\*p > 0.05), (XLStat©). Error bars represent standard deviations.

the Ausubel DNA extraction method with or without PVPP were higher than 11.3 and 12.8 units, respectively, compared to the values obtained with the Lipp method. These results seem to indicate the presence of PCR inhibitor compounds in DNA extract using the Ausubel method with or without PVPP.

#### Use of *E. coli* ER2738 as Microbiological Internal Control

An E. coli standard curve from DNA isolation at various levels between 101 and 107 bacteria/mL was used to evaluate qPCR efficiency (98.8%) with a correlation coefficient of 0.998. The Tm of the product had a value of 82.5 ± 0.5◦C. The trend curve is: [E. coli]concentration(log cells/mL) = −0.2977 × Cq + 11.581. A suspension of the strain E. coli K12 ER2738 was added to obtain 104 cells/mL in ethanol medium (10% v/v), and white and red wines samples containing A. aceti, A. pasteurianus, G. oxydans, and Ga. liquefaciens. According to the standard curve, efficient DNA extraction and amplification with EC23S primers should give a Cq-value of 25.5 ± 0.4 for 4 log cells/mL of E. coli. Thus, following DNA extraction performed in triplicate of each AAB in each media with both Ausubel and Lipp methods, with or without PVPP, E. coli was quantified.

The values in **Table 2** are the averages of E. coli populations found after DNA extraction according to methods with or without PVPP. No significant differences (p > 0.05) can be seen between the two techniques used for DNA extraction with or without PVPP for E. coli enumeration in ethanol medium and white wine. No significant cell loss was highlighted for either the Ausubel or Lipp method. However, the standard deviation of the Lipp method was lower than 0.3 log E. coli/mL compared to the Ausubel method for both media.

In red wine, the mean values for E. coli concentration in the Lipp method after DNA extraction with or without PVPP were 3.7 ± 0.2 and 3.3 ± 0.2 log cells/mL, respectively, whereas Ausubel DNA extractions led to 1.8 ± 0.2 and 1.9 ± 0.5 log

TABLE 2 | Averages of *E. coli* K12 ER2738 found after initially adding 4 log cells/mL in ethanol growth medium and white and red wines containing *A. aceti*, *A. pasteurianus*, *G. oxydans,* and *Ga. liquefaciens,* and following DNA extraction.

The Lipp and Ausubel methods were used with (+) or without (−) PVPP during cell lysis. qPCR was done with EC23S primers.

An Anova representation with Tukey's test was applied to the E. coli enumeration results using as control modality of 4.0 log E. coli/mL, (XLStat©). Values followed by different letters within a column are statistically different at p < 0.05 (XLStat©).

cells/mL, respectively, with and without PVPP. Thus, as shown in **Table 2**, the Lipp method with or without PVPP led to the recovery of the internal control population, which was not significantly different from the added population. Although not significant, the Cq-value following Lipp DNA extraction with PVPP tended to be slightly lower in red wine compared to experiments performed without PVPP. On the basis of these results, the Lipp DNA extraction method with PVPP was used for all extractions in wine. AAB can be quantified only if E. coli quantification is not significantly different from the added concentration (104 E. coli/mL), since the detection of a loss of internal standard leads to an underestimation of AAB population.

#### AAB Quantification with the Presence of Other Wine Microorganisms

To verify the absence of interference by other microorganisms in naturally contaminated wine, the following procedure was implemented. AAB quantifications in red wine containing 105 A. pasteurianus/mL alone or supplemented with O. oeni and B. bruxellensis at 105 cells/ml were performed. qPCR with AAB primers was performed after validating the presence of E. coli at 104 cells/mL. The results of the samples containing A. pasteurianus alone or with other microorganisms (O. oeni and B. bruxellensis) were identical: 5.1 ± 0.1 and 5.1 ± 0.2 log A. pasteurianus/mL, respectively.

#### Linearity, Repeatability, and Reproducibility in Red Wine

After artificial contamination of the red wine by AAB and incubation allowing their growth, verified over time by flow cytometry, Lipp DNA extraction with PVPP was performed in triplicate with 3 repetitive independent experiments. The Tm of the product had a value of 82.5 ± 0.5◦C. AAB quantification could be done after confirming the presence of 4 log E. coli/mL. Efficiency, r 2 , and y-intercept are presented in **Table 3**. No significant differences between efficiency, r 2 , and y-intercept following a Tukey's test between red wines, containing each AAB species or a mix of all AAB, were observed. Thus, all the data were compiled to determine the general standard curve allowing AAB quantification in red wine whatever TABLE 3 | Statistical analysis of qPCR parameters obtained from independent DNA isolation experiments performed on *A. aceti, A. pasteurianus. G. oxydans,* and *Ga. liquefaciens* after growth in red wine in triplicate and with three repetitions of the experiment over time.

Three red wines containing a mix of AAB were also analyzed in triplicate. An Anova with Tukey's test were performed according to efficiency, r2 , and y-intercept, (XLStat©). Values followed by the same letter within a column are statistically identical at p > 0.05 (XLStat©).

the species present (**Figure 2**):

[AAB]concentration(log cells/mL) = −0.2927 × Cq +11.135.

#### Limit of Quantification (LOQ) of AAB in Red Wine

For our study, LOQ was determined using the slope, residue standard deviation, and standard deviation of the intercept obtained from linearity validation experiments (OIV, 2005). Using the Lipp method after AAB growth in red wine, the LOQ value of 5.2.103 cells/mL was obtained. These results were the mean of the three independent experiments performed in triplicate. Moreover, the sampling volume of 10 mL instead of 1 mL led to the improvement of LOQ by 0.9 ± 0.1 log cells/mL.

# Acetic Acid Monitoring According to AAB Population

Acetic acid concentration monitoring was performed after A. pasteurianus inoculation in ethanol medium [10% (v/v)] at 102 cells/mL in independent experiments performed in triplicate (**Figure 3**). The initial amount of acetic acid was 0.04 g/L. **Figure 3** shows acceptable acetic acid values of 0.04 ± 0.00 g/L for an AAB concentration from 2.2 to 5.4 log CFU/mL. The sample presented 0.52 g/L acetic acid when the A. pasteurianus population reached 6.9 log CFU/mL. The last analysis point has a bacterial population of 7.2 ± 0.1 log CFU/mL and shows acetic acid values from 2.39 ± 0.61 g/L. Thus, acetic acid production increased in ethanol medium and occurred when acetic acid bacteria exceeded 6 log CFU/mL. AAB growth was low between the analysis points of 10 and 14 days, indicating that culture reached a stationary phase. The high acetic acid concentration determined during this time seemed to highlight overproduction during the stationary phase of the culture, i.e., when AAB are in high concentration.

To confirm these hypotheses of acetic acid production beyond acceptable limits at a population higher than 6 log AAB/mL and during the stationary phase of AAB growth, AAB growth was monitored in red wine from the vineyard of the University of Burgundy, contaminated naturally over time (**Figure 4**). Both the AAB population and acetic acid concentration were determined over time. The enumeration of total bacteria in the samples was performed by FCM and LAB quantification in specific medium. Neither yeasts nor LAB were detected in the wine under study, consequently only AAB were present and enumerated. Therefore, it was possible to compare the AAB concentration determined by FCM/qPCR methods and acetic acid production. Acetic acid

concentration as a function of AAB population is presented in **Figure 4**. For the first points of analysis containing a population ranging from 2.9 to 6 log AAB/mL, quantifications by qPCR were well-correlated with quantification by FCM. The acetic acid concentrations at these analysis points did not exceed 0.29 g/L. Then, higher acetic acid concentrations were reached for higher acetic acid bacteria concentrations. The analysis point containing an AAB population of 6.8 ± 0.2 log bacteria/mL had 0.82 ± 0.01 g/L acetic acid. The last point of AAB growth monitoring in red wine presented a high AAB concentration with acetic acid levels exceeding the European limit values and an increase in the mean difference between the qPCR and FCM methods (1.2 ± 0.6 log cells/mL). **Figure 4** validates acetic acid production by AAB during the stationary growth phase, thus when AAB exceeded 6 log bacteria/mL.

The measure of acetic acid and counting of AAB by FCM/qPCR methods were then performed on 15 red wines chosen randomly from wine regions (**Figure 5**). Twelve red wines had a mean amount of acetic acid at 0.52 ± 0.15 g/L. For these same samples, the median amount of acetic acid was 0.57 ± 0.15 g/L. These wines are considered as unspoiled. The mean relative difference between FCM vs. qPCR, allowing the determination of the AAB population, was 0.5 ± 0.3 log cells/mL. Three of the fifteen red wines analyzed presented a high amount of acetic acid and were thus spoiled. These wines had an AAB concentration higher than 6 log AAB/mL.

# DISCUSSION

Cell quantification by qPCR in red wine is difficult since various qPCR inhibitors such as polyphenols and polysaccharides are abundant, thereby increasing the risk of false negative results (Demeke and Jenkins, 2009) and making the amplification of genetic material challenging. qPCR has been used to quantify AAB in wine (González et al., 2006; Andorrà et al., 2008; Torija et al., 2010; Valera et al., 2013), however, the authors of these

studies did not take into account extraction efficiency or the presence of inhibitors, nor did they use any control process (cells added to the matrix). Process control can provide information on the efficiency of extraction and on the PCR procedure. The addition of a "spike" control to ensure accurate and reliable quantification is important (Fukushima et al., 2003; Stoeckel et al., 2009; van Doorn et al., 2009) and has already been reported for food analysis (Josefsen et al., 2010; Krøjgaard et al., 2011; Ishii et al., 2013), but as far as we know only once for quantifying Brettanomyces in wine (Tessonnière et al., 2009). In order to develop an accurate and efficient qPCR method to quantify AAB, DNA extraction procedures were first tested in order to ensure good DNA preparation. Indeed, the extraction efficiency and quality of DNA must be optimized for quantitative PCR. The selection of an appropriate DNA extraction method from those available is thus crucial (Jara et al., 2008).

## DNA Extraction Method

We opted for the Lipp method (Lipp et al., 1999) since this method has proven useful for many foodstuffs, especially those rich in phenolic compounds. By comparing this method with classical CTAB extraction, we clearly demonstrated the advantage of using the former. Using our process control strain, we demonstrated that the DNA extraction method was well-suited for wine and especially red wine. Moreover, as PVPP is known to remove PCR inhibitors (Tessonnière et al., 2009) it was added in our assay and improved Cq-values. The results of our validation protocol proved the specificity of the assay. Indeed, qPCR primers showed good specificity for all the wine AAB tested and did not amplify from the other wine organisms. Moreover, our study showed that AAB population determination by qPCR with the Lipp method using PVPP was not influenced by the other microorganisms present (O. oeni and B. bruxellensis) in high concentration. Therefore, pre-amplification using a nested PCR technique (González et al., 2006) is not required to determine the AAB population in samples.

#### AAB qPCR Limit of Quantification

Our LOQ was 5.102 cells with a test sample of 10 ml. This LOQ is similar to those previously reported and was reached with AAB grown in red wines, contrary to González et al. (2006) and Torija et al. (2010). In our study, the correlation coefficient of the standard curve obtained for AAB in red wines was 0.76. This coefficient might appear low but it comes from a calibration curve used for wine. Indeed, it is essential to create calibration curves in food matrices. The creation of standard curves for the quantification of a microorganism by diluting DNA from one extraction, as has been done in most studies, should be avoided (González et al., 2006; Torija et al., 2010; Valera et al., 2013). Consequently, the influence of food matrices is not considered, resulting in an underestimation of microbial load (Cocolin and Rantsiou, 2012). Most studies have obtained a high correlation coefficient between 10-fold serial DNA dilutions, but have not performed DNA extraction from a sample containing a low bacterial population, thus biasing efficiency. DNA extraction must be performed from each dilution before running the qPCR to create a standard curve.

### Evaluation of the Method with Naturally Spoiled Wines

Little is known of the spoilage caused by AAB populations and it probably depends on the matrix (sulfites, polyphenols, etc.). Also, this topic is controversial in the literature. According to Joyeux et al. (1984), a low population can activate significant volatile acidity production when exposed to air. However, Drysdale and Fleet (1985) reported that A. pasteurianus and A. aceti occur at 101–103 CFU/mL in many wines during bulk storage in wineries without causing spoilage. Moreover, Bartowsky and Henschke (2008) have shown that 2.104 A. pasteurianus/mL in a Shiraz wine leads to an acetic acid concentration of 0.6 ±0.0 g/L. This wine was not considered spoiled because the initial wine, without microorganisms, contained 0.5 ±0.0 g of acetic acid per liter. Another wine containing 9.104 A. pasteurianus/mL had 3.5 ±1.7 g/L of acetic acid, and was thus spoiled. These results may underestimate the true population due to the uncertainty in recovering all the bacteria. In the literature, there is no clear consensus regarding AAB concentration leading to spoilage risk. Therefore, we determined the level of acetic acid concentration in ethanol medium and red wine according to the AAB population over time. In the latter, the LAB concentration was determined to avoid overestimation of the AAB population by FCM bacteria enumeration, even if the LAB is not a serious issue because AAB causes wine spoilage only during aging in the cellar and after bottling (Henick-Kling, 1993), normally without the presence of LAB. In our study, neither yeasts nor LAB were detected in these red wines. Thus, the assessment of our improved qPCR assay for AAB quantification in naturally contaminated red wine proved reliable and efficient in comparison with cytometry results. The small deviations between both methods were probably derived from the matrix, which was different to that used to write the equation. In our study, during the monitoring of AAB naturally present in red wine, red wine exhibiting an AAB population lower than 6 log bacteria/mL was not altered. However, for wines presenting an AAB population of about 6-7 log cells/mL, the acetic acid exceeded the aroma threshold (Davis et al., 1985; Eglinton and Henschke, 1999a,b; Swiegers et al., 2005) but not the European limit values for sale. For the other wines with high AAB populations and during the stationary phase, acetic acid exceeded the European limit values. Thus, the method developed in this study had an elevated LOQ without using a higher sample volume (e.g., 10 mL).

#### CONCLUSION

The method allowed AAB detection and quantification before spoilage occurred, which meets the needs and expectations of the wine industry when monitoring AAB populations on a regular basis. Therefore, in our study, we developed a qPCR method which allows the reliable quantification of AAB in red wine. We showed that the previously reported DNA extraction method was not efficient enough for precise quantification. The methods used in these studies probably led to underestimating the AAB population. Use of an internal control allows validating DNA extraction and qPCR efficiencies. No underestimation can be made if the initial concentration of the internal control added in the sample before DNA extraction is found. Moreover, the standard curve was established with AAB that proliferated in red wine. Finally, as far as we know, this is the first time a qPCR protocol allowing AAB quantification in red wines without bias (neither cell loss nor PCR inhibitor presence) has been validated. Moreover, the presence of other microorganisms in the sample did not alter AAB quantification.

Specific AAB species quantification is not possible with our method; however, our main goal was to quantify AAB in wines

#### REFERENCES

whatever the species present in order to evaluate the risk of spoilage. Furthermore, one drawback of the method is that qPCR quantifies live and dead AAB. Propidium monoazide could be used (Vendrame et al., 2013; Rizzotti et al., 2015) but cell exposure to a stress like ethanol, which is known to permeabilize membranes (Alexandre et al., 1994), can result in cells stained by propidium iodide, for example, but that are still alive (Davey and Hexley, 2011). Moreover, Shi et al. (2012) highlighted an underestimation of total yeasts, S. cerevisiae, total LAB, non-O. oeni LAB, and total AAB in wine by realtime PCR coupled with ethidium monoazide. However, they demonstrated that 40 min of incubation in recovery medium could completely cancel the underestimation of viable cell counts performed in wine, but not cell growth, which must be checked for each sample. To avoid this potential error, one way to circumvent the drawback of the method is to monitor the AAB population over time. Under these conditions, a decrease in Cq-value would reflect AAB growth and potentially the risk of spoilage.

#### AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: HA, CL, and MG. Performed the experiments: CL. Generated and analyzed the data: CL, HA, and MG. Wrote the paper: CL, HA, and MG.

#### FUNDING

This work was funded by the Regional Council of Burgundy and the Interprofessional Office of Burgundy Wines.

RNA and single copy genes. Lett. Appl. Microbiol. 52, 298–306. doi: 10.1111/j.1472-765X.2010.03001.x

**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 © 2016 Longin, Guilloux-Benatier and Alexandre. 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) or licensor 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.

# *Vibrio parahaemolyticus* Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China

Jingjiao Li 1, 2, Feng Xue1 \*, Zhenquan Yang3 , Xiaoping Zhang4 , Dexin Zeng1 , Guoxiang Chao5 , Yuan Jiang1 \* and Baoguang Li 6

1 Animal Quarantine Laboratory, Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing, China, 2 Key Laboratory of Veterinary Biotechnology, Department of Animal Science, School of Agriculture and Biology, Shanghai JiaoTong University, Shanghai, China, 3 Jiangsu Key Laboratory of Zoonosis, School of Food Science and Engineering, Yangzhou University, Yanghzou, China, 4 Beijing Kemufeng Biopharmaceutical Company, Beijing, China, 5 Yangzhou Key Centre for Disease Control and Prevention, Yanghzou, China, 6 Division of Molecular Biology, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Laurel, MD, USA

*Edited by:* Giovanna Suzzi, Università Degli Studi di Teramo, Italy

#### *Reviewed by:*

Adrian Canizalez-Roman, Autonomous University of Sinaloa, Mexico Learn-Han Lee, Monash University Malaysia, Malaysia

#### *\*Correspondence:*

Feng Xue<EMAIL_ADDRESS>Yuan Jiang<EMAIL_ADDRESS>
#### *Specialty section:*

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

> *Received:* 15 April 2016 *Accepted:* 09 May 2016 *Published:* 31 May 2016

#### *Citation:*

Li J, Xue F, Yang Z, Zhang X, Zeng D, Chao G, Jiang Y and Li B (2016) Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China. Front. Microbiol. 7:787. doi: 10.3389/fmicb.2016.00787 Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand, and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST) analysis, and 48 sequence types (STs) were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17, and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these "environmental" pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve.

Keywords: *Vibrio parahaemolyticus*, serotyping, MLST, virulence genes, TDH-related hemolysin (TRH), pandemic serotypes, epidemiology, phylogeny

# INTRODUCTION

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium that inhabits global coastal waters and rivers, and in seafood, such as fish and shellfish (Kelly and Stroh, 1988). V. parahaemolyticus was initially discovered in 1950 (Parveen et al., 2008; Letchumanan et al., 2015b). A novel serotype of O3:K6 clone was discovered in India in 1996 (Okuda et al., 1997), and since then, this clone and its serovariants have disseminated worldwide and become a pandemic clonal group (Ansaruzzaman et al., 2005; Quilici et al., 2005; Nair et al., 2007; Chao et al., 2009; Velazquez-Roman et al., 2013; Li W. et al., 2014; Flores-Primo et al., 2015).

V. parahaemolyticus can cause three major clinical syndromes: gastroenteritis, wound infections, and septicemia (Daniels et al., 2000), and is also considered as the causative agent of the most prevalent food poisoning in Asia since the outbreak in 1959 (Miyamoto et al., 1962). V. parahaemolyticus infections usually resulted from consumption of raw or undercooked seafood, mostly causing gastroenteritis (Miyamoto et al., 1969). Recently, V. parahaemolyticus has been identified as a major foodborne pathogen in food poisoning incidents in China, raising public health concern (Ma et al., 2014; Zhang et al., 2015).

V. parahaemolyticus was initially discovered in 1950 (Parveen et al., 2008). Traditionally, the identification of V. parahaemolyticus is performed by serological and biochemical tests. V. parahaemolyticus is classified by serotyping and the serotypes of V. parahaemolyticus are determined by the combination of somatic (O) antigens and capsular (K) antigens. There are 13 O serotypes and 71 K serotypes that have been reported (Iida et al., 1997; Nair et al., 2007; Chen et al., 2012). V. parahaemolyticus infections are associated with pathogenic strains of numerous serotypes (clinical); whereas non-pathogenic strains comprise an even greater variety of serotypes.

Serotypes are useful distinguishing features to identify clinical isolates (Jones et al., 2012), and serotyping has been widely used in epidemiological research and etiological diagnostics for many decades. However, the increasing genetic diversity such as emerging of new serotypes and STs among V. parahaemolyticus strains poses a challenge to this traditional way of strain identification and differentiation due to the high cost of the antisera and the potential ambiguity encountered during serotyping (Bogdanovich et al., 2003). In the last two decades, numerous DNA-based subtyping methods such as pulsed-field gel electrophoresis (PFGE; Wong et al., 1996), multilocus sequence typing (MLST; Gonzalez-Escalona et al., 2008), repetitive element PCR (Rep PCR; Wong and Lin, 2001), multilocus variable-number tandem-repeat analysis (MLVA; Kimura et al., 2008), clustered regularly interspaced short palindromic repeats (CRISPR; Sun et al., 2015), and microarray analysis (Li et al., 2015) have been developed to investigate the genetic diversity of outbreaks caused by V. parahaemolyticus and other foodborne pathogens (Li B. et al., 2014). Each of these subtyping methods has its advantages and disadvantages with respect to sensitivity, specificity, cost, and speed (Li et al., 2015). In general, MLST is the most commonly used method (Maiden, 2006; Nair et al., 2007; Gonzalez-Escalona et al., 2008), due to its high specificity, repeatability, and portability (Nair et al., 2007).

In this study, in an effort to assess the homogeneity and heterogeneity between the clinical and environmental V. parahaemolyticus isolates from Jiangsu Provence, an east coast area in China, where seafood is widely consumed, we used the traditional classification method, serotyping, to identify 72 V. parahaemolyticus strains from the food poisoning case samples and the environmental samples. The genetic diversity among V. parahaemolyticus strains were further assessed by MLST analysis and the presence of the virulence factors such as tdh, trh, orf8, GS-PCR, PGS-PCR, and HU-α. Furthermore, we analyzed the V. parahaemolyticus isolates by using eBURST and START (http:// eburst.mlst.net) to investigate the relationship between clinical and environmental V. parahaemolyticus strains based on MLST databases. Additional information on genetic variation and the distribution of virulence genes among V. parahaemolyticus strains from various cities in Jiangsu Province would enrich the MLST database and epidemiological archive and be beneficial for the development of an efficient risk assessment of this common foodborne pathogen.

#### MATERIALS AND METHODS

#### *V. parahaemolyticus* Strain Identification

Seventy-two presumed V. parahaemolyticus isolates were collected from nine different cities in Jiangsu province of China between 2006 and 2014, including 21 clinical isolates from patients with food poisoning and 51 isolates from food samples. All strains were characterized according to GB 4789.7–2013 Chinese Food Safety Standards (http://www.foodmate.net). The V. parahaemolyticus strains were inoculated onto Vibrio culture plates (CHROMagar, Paris, France) and Thiosulphate Citrate Bile salt Sucrose [(TCBS) Beijing Land Bridge, China] culture plates and incubated at 37◦C for 16–24 h. The colonies with typical contour were selected and characterized by VITEK automatic biochemical analyzer (Biomerieux, France).

#### Serotyping

The serotype of V. parahaemolyticus strains was determined using two diagnostic kits; 11 antisera targeting the O1–O11 antigens and 71 antisera for the K antigens (Denka Seiken, Tokyo, Japan) and 11 antisera for O1–O13 antigens (Tianjin Biochip Corporation, Tianjin, China). Serotyping was carried out in accordance with the GB4789.7–2013 Chinese Food Safety Standard (http://www.foodmate.net). Single colonies were selected and plated onto the surface of 3% sodium chloride peptone agar plates, incubated at 37◦C for 18 h. Bacterial suspension was obtained by washing the surface of agar with solution containing 3% NaCl and 5% glycerol.

O-antigen identification: The bacterial suspension was autoclaved at 121◦C for 1.5 h followed by centrifugation at 12,000 g for 15 min. The pellets were washed two or three times with normal saline solution and centrifuged at 12,000 g for 15 min. The final suspension was used for O antiserum agglutination and normal saline solution was used as a negative control. If the result was negative, all the above steps were repeated; thereafter, the negative was considered as unknown antigen O.

K-antigen identification: Multi-serum against the K-antigen was added to the bacterial suspension. Positive colonies were selected for further analysis using individual K-antigen antiserum. Solution of NaCl (3%) was used as negative control.

# Identification of the *tdh, trh, orf8, GS-PCR, PGS-PCR,* and *HU*-α Genes

Single colonies of V. parahaemolyticus strains were picked and inoculated into liquid culture medium containing 3% sodium chloride peptone. Cultures were incubated at 37◦C for 16 h, followed by centrifugation at 12,000 g for 10 min. The genomic DNA was isolated using E.Z.N.A.TM kit (OMEGA, Beijing, China), and the concentrations of DNA were determined by spectrophotometry to ensure the OD260/OD280 was between 1.8 and 2.0.

The primers for the tdh, trh, orf8, GS-PCR, PGS-PCR, and HU-α genes were synthesized (Chao et al., 2009; Li W. et al., 2014; **Table 1**) by Sango Biotech Co., Ltd. (Shanghai, China). PCR reactions were performed in a volume of 25µl containing 1µl DNA (50 ng/µl), 12.5µl 10 × PCR mix (Takara, Dalian, China), 1µl of the forward and reverse primers of the virulence genes, and 9.5µl of sterile distilled water. Strains ATCC33847 (tdh+trh−, isolated in US in 1973) and ATCC17802 (tdh−trh+, isolated in Japan in 1965) were used as positive controls. PCR reactions were performed under the following conditions: initial denaturation at 95◦C for 2 min, followed by 30 cycles of denaturation at 95◦C for 30 s, annealing at 50◦C for 30 s, and elongation at 72◦C for 1 min, and ending with elongation at 72◦C for 5 min. PCR products (1µl) were analyzed using Agilient 2100 analyzer (Waldbronn, Germany) to determine the PCR amplicon size.

#### MLST

Seven housekeeping genes, dnaE, gyrB, recA, dtdS, pntA, pyrC, and tnaA, were selected as target genes in this study. The primers for these genes were adopted from MLST website (http://www. pubmlst.net). The MLST primers for recA gene failed in most strains in the current study, and new primers for recA gene were adopted from a previous study (Gonzalez-Escalona et al., 2008). All primers as shown in **Table 2** were synthesized by Sango Biotech Co., Ltd. (China). Primers were diluted to 10µM and stored at −20◦C.

#### PCR Amplification of Housekeeping Genes

To sequence the housekeeping genes, the PCR reaction volume was set at 50µl. The components of PCR reactions as well as the parameters for each cycle were optimized for best performance. PCR products were visualized using Agilent electrophoresis and imaging system. The bidirectional DNA sequencing was conducted by Sango Biotech Co., Ltd.

#### Allele and Sequence Analysis

The alignment of DNA sequences was performed by uploading each of the sequences of the seven housekeeping genes of the 72 V. parahaemolyticus strains to the MLST website (http://

TABLE 1 | Primers used for the detection of *V. parahaemolyticus* virulence genes and pandemic marker genes by PCR.

aForward primer.

bReverse primer.

pubmlst.org/vparahaemolyticus/). If a novel allele or sequence was identified, the forward and reverse sequences were uploaded and submitted to the database administrator to obtain a serial number for the allele or sequence.

### Sequence Analysis by eBURST v3.0 and START v2.0

The ST types of all the 72 strains were analyzed by eBURST v3.0 (http://eburst.mlst.net) to distinguish clonal complex (CC), group, and singleton STs. The most stringent definition was adopted to identify the homeotic complexes, each of which was defined by the presence of at least six or seven identical alleles. The single locus variant (SLV) was defined by the presence of a single allele difference between any two ST types, based on eBURST v3.0 analysis. The evolution of each allele and ST type was analyzed by using START v2.0 (http://pubmlst.org/software/ analysis/start/).

# RESULTS

V. parahaemolyticus colonies were round, translucent and purplish red on CHROMagar plates measuring 2–3 mm in diameter. They were round, translucent, and smooth greencolored colonies on TCBS plates. All 72 V. parahaemolyticus isolates were confirmed by VITEK biochemical analysis.

#### Serotyping

There were 25 serotypes identified among the 72 V. parahaemolyticus isolates. The dominant serotypes were

#### TABLE 2 | Primers of housekeeping genes used in MLST and PCR conditions in this study.

The underlined lower-cased letters represent common sequences that were adapted from M13 for more efficient sequencing.

O3:K6 (n = 8) and O2:K28 (n = 8), followed by O1:KUT (n = 6), O5:K17 (n = 5), O5:KUT (n = 5), O1:K25 (n = 3), O4:K34 (n = 3), O10:KUT (n = 3), O2: KUT (n = 3), O1:K32 (n = 3). For the 21 clinical strains, nine serotypes were identifies where O3:K6 was the dominant serotype (n = 8), followed by O5:K17 (n = 5), O2:KUT (n = 3), and one strain of O1:K25, O11:K40, and O13:KUT (novel serotype). In addition, there was one strain (WX14115) that failed to react to either O or K antiserum (**Table 3**).

Of the 25 serotypes, six were novel serotypes. Out of the six novel serotypes, five new serotypes were recovered from environmental samples and one was from clinical sample. Specifically, the five environmental strains are O2:K25, O4:K9, O4:K42, O8:K39, and O11:31, and the single clinical isolate is O13:KUT (**Table 4**). In the present study, 25 strains of pandemic serotypes were identified from Jiangsu Province, including 16 clinical strains and 9 environmental strains. Specifically, the clinical strains were serotypes O3:K6 (n = 8), O5:K17 (n = 5), and environmental strains were serotypes O1:KUT (n = 6), O1:K25 (n = 3), O3:K29 (n = 1), and O3:KUT (n = 1) (**Table 3**).

# Identification of Virulence Factor Genes *tdh, trh, GS-PCR, PGS-PCR, orf8,* and *HU*-α

The virulence genes in the 72 V. parahaemolyticus isolates were assessed by PCR. There were nine tdh+ strains and four trh+

strains, accounting for 61.9% (13/21) of the clinical strains. No strain was tdh+trh+. The nine tdh+ strains included seven O3:K6, one O1:K25 strain and one O4:KUT strain. Four trh+ strains included three O5:K20 and one O13:KUT (**Table 3**). The prevalence of the other four virulence genes, GS-PCR, PGS-PCR, orf8, and HU-α, are 81.9, 38.9, 16.9, and 18.9%, respectively. GS-PCR gene showed the highest prevalence and orf8 gene showed the lowest prevalence for the four virulence genes in our collection (**Table 3**).

# MLST Analysis

All seven housekeeping genes were amplified in the V. parahaemolyticus strains using specific primers, and the PCR products were sequenced. **Seventy-two** V. parahaemolyticus strains were classified into 48 STs by MLST analysis. Of the 48 ST types, 34 were singleton and 14 were ST groups. Each singleton represented only one strain, while each ST group included two to eight strains (**Figure 1**). Twenty-one clinical strains were classified into 13 STs. The dominant ST was ST-3 with eight strains, followed by ST-79 and ST-564, each of which covered three strains. The remainder of STs was singletons. The MLST results indicated that V. parahaemolyticus strains showed genetic polymorphisms with much higher incidence rate in environmental strains than in clinical strains.

# Novel Allele and STs

The ST composition of the 72 V. parahaemolyticus strains included 32 novel STs with 32 new allele values and 34 allele spectra, accounting for 79% of total STs. All of these newly identified allele values and allele spectra were submitted to the PubMLST database (http://pubmlst.org/vparahaemolyticus/) as shown in **Table 3**. There were 13 STs among the 21 clinical strains, including seven newly identified STs accounting 53.85% (7/13) of the ST types. There were 36 STs identified among the 51 environmental strains, including 27 newly identified STs, accounting 75% (27/36) of the STs. It appears that 13 new STs were formed through allele recombination, while the other 21 new STs were created by the newly identified housekeeping gene alleles which included several types of dnaE (N = 4), gyrB (N = 6), recA (n = 7), dtdS (n = 2), pntA (n = 3), pyrC (n = 9) genes, and one tnaA gene.

# Homologous Allele Complex and Systematic Development Analysis

The system evolution diagram was plotted by eBURST v3 software, and 48 STs were divided into one clonal complex (CC), four double combinations (D), and 38 singletons (S). The CC identified in this study was the CC3 which covered seven strains of O3:K3 ST-3 and one strain of O3:KUT ST-3; these eight strains were epidemic strains from clinical samples. The D type included ST-799-ST-1108, ST-212-ST-1002, ST-996-ST-999, and ST-993- ST-968. The ST-1108, ST-1002, ST-996, ST-999, and ST-993 were newly identified in this study. Of the 38 singletons, 28 novel STs were identified in this study, which were genetically distant to the ST-3 and other STs (**Table 3**; **Figure 1**).

(Continued)

TABLE 3 | Continued

aA bold-faced number refers a novel ST.

#### Association of STs between Serotypes and Virulence Genes

The strains with identical serotype usually showed similar STs or were clustered together, such as the majority serotype of O3:K6 in the present study belonging to ST-3; only one O3:KUT strain shared the ST-3 with O3:K6 strains. On the other hand, some strains with identical serotypes have different STs, such as strains of O1:KUT. These were identified as ST-1007, ST-212, and ST-276 (**Figure 2**).

There were nine tdh+ pathogenic strains identified in this study, including eight ST-3 strains (seven O3:K6 serotypes and one O1:K25 serotype), and one ST-332 strain (serotype O4:KUT). All of these 10 strains were epidemic strains from clinical samples. There were four trh+ strains, including three ST-79 (O5:K17) strains and one ST-1114 (O13:KUT) strain. All the four trh+ strains seemed to be epidemic strains (**Table 4**).

#### DISCUSSION

V. parahaemolyticus is the major foodborne pathogen. It is widely distributed with high survival and incidence rates, especially in the coastal areas of China (Su and Liu, 2007; Chao et al., 2009; Yan et al., 2015). Based on the epidemiological surveillance data from countries in Southeast Asia, V. parahaemolyticus infections have become a majority of foodborne pathogen (Pan et al., 1997; Wong et al., 1999; Obata et al., 2001; Liu et al., 2004; Cho et al., 2008; Letchumanan et al., 2014). In the present study, 25 serotypes and 48 STs were identified among the 72 V. parahaemolyticus isolates. Of the 25 serotypes and 48 STs, six additional novel serotypes and 34 novel STs were identified, indicating the V. parahaemolyticus population in Jiangsu Province was highly dispersed. The diversity in serotypes and STs of V. parahaemolyticus, especially environmental strains, is attributed to frequent recombination events in the pathogen but not by mutation (Gonzalez-Escalona et al., 2008). One example to support this is that the serotype of epidemic strains (O3:K6), which has been continuously evolving, resulted in 21 derivative serotypes of O3:K6 such as O4:K68, O1:K25, O1:KUT, O4:K12, and O5:K17 (Nair et al., 2007). All those strains demonstrated identical genotypes and molecular spectra, therefore the O3:K6 and its derivative are called O3K6 clones or pandemic strains. MLST analysis confirmed that O3:K6 and its derivatives belong to the same genetic lineage (Chowdhury et al., 2000, 2004; Matsumoto et al., 2000). Surprisingly, the 25 pandemic strains identified in this study were not exclusively from clinical samples (n = 16), rather, some strains were recovered from environmental samples (n = 9) as well.

Serotypes of O3:K6 and O5:K17 were the most common serotypes among the 21 clinical strains whereas some serotypes such as O11:K40, O4:K8, O2:KUT, and O13:KUT are rarely reported globally. It has been shown that serotype O1:KUT is closely related to pandemic strains and is seldom detected as environmental strains (Iida et al., 1997; Mahoney et al., 2010). However, in the present study, O1:KUT was identified as a common serotype with six isolates from environmental samples. More importantly, the six O1:KUT strains demonstrated extremely high genetic diversity with five different STs among

aUT refers to untypable.

bBold faced letters and number refer to a novel serotype or ST.

the six strains (**Table 4**). Furthermore, of the five STs possessed by O1:KUT strains, three were novel STs, namely ST-1002, ST-1007, and ST-1105, suggesting these O1:KUT strains were highly dispersed and evolving rapidly in the environment. In addition, some of the O3:K6-specific pandemic markers such as GS-PCR and PGS-PCR were tested positive among most of these strains of pandemic serotypes. However, the pathogenicity of those strains needs to be further confirmed by recovering those strains from patients. Nevertheless, the risk posed to the public health in China by these "environmental" pandemic strains should not be overlooked.

The exact pathogenic mechanism of V. parahaemolyticus remains unclear, but the tdh and trh genes are considered the main pathogenic factors. Thermostable direct hemolysin (TDH), encoded by the tdh gene, manifests hemolytic, intestinal, and cardiac toxicities (Iida et al., 1997; Rosec et al., 2009; Raghunath, 2014). The trh gene is closely associated with the production of urease (Quilici et al., 2005). An epidemiological surveillance of V. parahaemolyticus in Northwest Mexico showed that up to 71.74% of the environmental isolates carried the tdh gene (De Jesus Hernandez-Diaz et al., 2015). However, numerous reports have shown that many pathogenic strains from patients were detected with neither of the two virulence factor genes, indicating more virulence factor genes are needed as markers for identification of pandemic V. parahaemolyticus strains (Garcia et al., 2009; Jones et al., 2012; Liu and Chen, 2015). Additionally, the type

III secretion system (T3SS) of V. parahaemolyticus has been identified as a potential strain virulence factor (Park et al., 2004; Broberg et al., 2011).

V. parahaemolyticus O3:K6 serogroup has group-specific gene sequences in the toxRs operon and orf8, one of the 10 known open reading frames (ORFs) which is unique to the O3:K6 filamentous phage f237. The toxRs and orf8 genes have been used as genetic markers to differentiate O3:K6 from other serogroups (Matsumoto et al., 2000; Nasu et al., 2000). Additionally, the HU-αORF, a specific biomarker for pandemic strain, which has a C-terminal amino acid sequence different from those of other strains of V. parahaemolyticus, was used to identify O3:K6 and other serotypes, such as O1:K25, O1:KUT, and O4:K68 (Matsumoto et al., 2000; Williams et al., 2004).

It has been reported that V. parahaemolyticus that lacked the tdh and trh genes were pathogenic in a study using mice (Rosec et al., 2009). In this study, most of the clinical strains (13/21) were negative for the tdh and trh genes, but possessed at least one of the four other virulence genes, GS-PCR, PGS-PCR, orf8, and HU-α. For instance, two tdh−trh− strains, OUT:KUT (WX14115, clinical) and O5:KUT (YZ0608, environmental) were positive for the other four virulence genes (GS-PCR, PGS-PCR, orf8, and HUα). Using a panel of six virulence genes as pathogenic markers, almost all the O3:K6 strains (except for two strains, WX1475 and WX14116) were positive for at least four of the six virulence genes, and only one clinical strain of O5:K17 (WX14102) was negative for all the six virulence genes among the 21 clinical strains. Thus, the results of this panel of virulence genes may more closely reflect the pathogenicity potential of those strains.

A high percentage (90.3%, 65/72) of strains were positive for at least one of the six virulence genes, tdh, trh, GS-PCR, PGS-PCR, orf8, and HU-α. Out of the six virulence genes, the GS-PCR gene showed the highest prevalence 81.9% (59/72). This result is very different from other investigators in China, where GS-PCR gene was seldom detected from environmental samples (Alam et al., 2009; Chao et al., 2009; Zhang et al., 2013). This difference might be a reflection of the genetic diversity between the indigenous isolates in this area (Jiangsu, China) and other areas or an artifact caused by sampling difference.

The GS-PCR gene has been shown to be a specific for genetic marker for the identification of pandemic V. parahaemolyticus strains (Li W. et al., 2014; Pazhani et al., 2014). The trh gene showed the lowest prevalence 5.6% (4/72), which is consistent with the results from other Asia countries (Alam et al., 2009; Chao et al., 2009; Zhang et al., 2013, 2015; Letchumanan et al., 2015a). Of note is that a number of different serotypes of tdh−trh− strains (such as O28:K28, O5:KUT, and O11:KUT) were positive for at least three of the four virulence genes, GS-PCR, PGS-PCR, orf8, and HU-α (**Table 3**). Although these strains were isolated from the environment, it is quite possible for a nonpathogenic strain to gain pathogenicity potential after acquiring several virulence genes from pathogenic strains as indicated by the evolving path of the pandemic strain O3:K6 (Nair et al., 2007).

In this study, there were 34 new STs out of the 48 ST types, and the newly defined gene spectra accounted for 70.83%, suggesting this V. parahaemolyticus group is highly dispersed. The cluster diagram of MLST analysis showed that

V. parahaemolyticus strains in Jiangsu area demonstrated a higher level of polymorphisms within environmental strains than clinical strains. ST-3 was the dominant ST among the 21 clinical strains and all ST-3 strains (n = 7) belonged to pandemic O3:K6 serotype, which is in agreement with previous reports from China and other countries (Gonzalez-Escalona et al., 2008).

It is worth noting in this study that there were a number of cases in which strains with identical serotype were classified into different STs; while strains with an identical ST possessed different serotypes. For instance, several strains of O1:KUT serotype were subtyped as ST1007, ST212, and ST276 by MLST; and the same ST strains, such as ST-3 strains, included

TABLE 5 | Possible recombination scenerios of reprentive strains of *V. parahaemolyticus* that share a serotype or a STa.

aST, sequence type; O, O-antigen; K, K-antigen.

bAn arrow refers to an O- and K-antigen or K antigen exchange between strains of different serotypes or STs.

cA blue-highlighted number indicates a new allelic gene.

O3:K6 and O3:KUT serotypes, whereas ST-276 strains included O1K:25 and O1K:KUT serotypes (**Table 5**). This observation not only indicates the advantages and limitations of serotyping and MLST analysis, but also implies that frequent mutation and/or recombination occur on the O- and K-antigens on the chromosome.

In this study, we used serotyping and MLST methods to differentiate the V. parahaemolyticus isolates and assessed the presence of six virulence factors, tdh, trh, orf8, GS-PCR, PGS-PCR, and HU-α. A total of 25 serotypes and 48 STs were identified among the 72 V. parahaemolyticus isolates; six novel serotypes and 34 novel STs were identified; and highly variable profiles of six virulence factor genes were detected among the isolates, suggesting this V. parahaemolyticus group was a highly dispersed group and was evolving rapidly. This information not only can enrich the MLST database but also can serve as a valuable set of matrices to trace the gene mutation and HGT (or recombination) among the V. parahaemolyticus population.

Data from **Table 5** indicate that the O- and K-antigens move together more often than to move independently; and the Kantigen is more likely to be swapped than O-antigen. For example, the ST-3 strains included, in addition to the seven O3:K6 strains, a single strain of O1:K25 (WX1461). TheO1:K25 strain (belonged to ST-3) can serve as an example for O- and K-antigen exchange between serotypes O3:K6 and O1:K25, i.e., the O3:K6 antigens of strain WX1461 were replaced by O1:K25 antigens as evidenced by its identical allele and virulence gene profiles with seven O3:K6 (ST-3) strains. Another example is WX1475 which is serotype O3:K6 but was subtyped as ST-1109 by MLST based on different allele and virulent gene profiles than with those other O3:K6 serotype strains (**Table 5**, lower middle). The third example is that strains with different STs acquired the O2:K28 antigens from a strain with different ST (ST-1003; Top of **Table 5**). Specifically, a serotype O2:K28 isolate (YZ0864) was subtyped as ST-1003 with another serotype O2:K28 isolate (YZ0685).

In this study, there were eight strains with serotype O2:K28 but were differentiated into six different STs, based on the high discriminatory power of MLST (**Table 5**). Serotype O2:K28 was initially found in 1977 (Libinzon et al., 1977) and is generally considered as an environmental strain (Martinez-Urtaza et al., 2004). Drastic differences were found between the results derived from the two subtyping methods. It is hard to pinpoint the exact cause(s) that led to the differences in allele profiles among those isolates during their evolution path. However, a plausible scenario is that the O- and K-antigens of the six strains (O2:K28) of different STs might have been acquired from strains such as YZ0684 (O2:K28; ST-1003) over years (**Table 5**).

Similar virulence gene profiles exist between the O2:K28 strains of ST-1003 and the six different STs strains (lacking tdh and trh genes but are positive for GS-PCR) seem to corroborate this notion (**Table 5**). In addition to the genetic evidence generated in this study, our hypothesis is also supported by the findings on bacterial antigens, virulence genes, and genetic traits of V. parahaemolyticus (Chowdhury et al., 2000; Gonzalez-Escalona et al., 2008; Chao et al., 2009; Mala et al., 2016). As shown in **Table 5**, serotype conversion (from one serotype to a different serotype) occurred more frequently than ST change (from one ST to a different ST) among the listed strains, whose serotype was shared by strains of different STs or whose ST was shared by strains of different serotypes. It seemed that O- and K-antigen conversion occurred simultaneously more often than independently, suggesting that the O- and K-antigens are actively evolving and the two antigens are mostly moving together by horizontal gene transfer (HGT) (or recombination). This presumption is well in agreement with the recent findings on V. parahaemolyticus genomic evolution that HGT is 10–1000 times more attributable than single nucleotide variants to genome diversification. This may be the underlying drive that is responsible for the high diversity among the V. parahaemolyticus studied. This hypothesis is consistent with other researchers on the diversity and pathogenicity of V. parahaemolyticus (Chowdhury et al., 2000; Gonzalez-Escalona et al., 2008; Chao et al., 2009; Mala et al., 2016). Furthermore, other studies on K-antigen and comparative genomic analysis of V. parahaemolyticus (Chen et al., 2010, 2011), have demonstrated that the O- and K-antigens are at two adjacent loci on chromosome II and, thus the O- and K-antigens could be swapped via a single recombination event to create both novel O- and K-antigens (Chen et al., 2010). Moreover, the human upper intestine is believed to be a particularly suitable niche for the intra- and inter-specific lateral transfer of genetic material necessary to enhance bacterial pathogenicity (Larocque et al., 2005; Okada et al., 2009; Hasan et al., 2010; Wang et al., 2011). Therefore, our model is supported not only by multiple lines of genetic evidence from V. parahaemolyticus, but also by the presence of suitable ecological niche that can facilitate HGT. With this notion, we can better interpret the scenarios we encountered here, i.e., the pandemic V. parahaemolyticus strains recovered not only from patients, but also from nine "environmental" seafood samples; and the high genetic diversity among the 72 isolates.

In summary, V. parahaemolyticus in Jiangsu, China, were highly dispersed and widely distributed in the environment. In light of that V. parahaemolyticus has become one of the major foodborne pathogens in China in recent years (Li Y. et al., 2014; Qi et al., 2016), the new threat to the public health posed by these "environmental" pandemic strains should not be overlooked. T. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources; the information on the genetic diversity among isolates enriches the MLST database; and our proposed possible Oand K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve. Our findings also underscores the necessity for more epidemiological studies and more comprehensive surveillances on V. parahaemolyticus in order to efficiently prevent the diseases caused by this organism.

# AUTHOR CONTRIBUTIONS

JL performed the experiments. YJ, FX, and BL conceived and designed the study. XZ, DZ, JL, FX, and BL analyzed the data. ZY and GC provided the isolates. JL, YJ, FX, and BL wrote the manuscript. All the authors reviewed the manuscript.

#### ACKNOWLEDGMENTS

We would like to thank Yanhong Wan, Lu Gao and Haibo Zhou for their useful suggestions to this work. The study was supported by the National Natural Science Foundation

#### REFERENCES

of China (31301460) the National Science and Technology Support Program of 2012BAK17B10, National 10000 Talents— Youth Top-Notch Talent Support Program, and Jiangsu Province Science and Technology Support Program of BE2013733.

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**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 © 2016 Li, Xue, Yang, Zhang, Zeng, Chao, Jiang and Li. This is an openaccess 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) or licensor 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.

# Recovery and Growth Potential of Listeria monocytogenes in Temperature Abused Milkshakes Prepared from Naturally Contaminated Ice Cream Linked to a Listeriosis Outbreak

Yi Chen1 \*, Emma Allard2 , Anna Wooten3 , Minji Hur4 , Ishani Sheth1 , Anna Laasri1 , Thomas S. Hammack1 and Dumitru Macarisin1

1 Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA, 2 College of Life Sciences and Agriculture, University of New Hampshire, Durham, NH, USA, 3 Joint Institute for Food Safety and Applied Nutrition, University of Maryland, College Park, MD, USA, 4 Department of Food Science, Gachon University, Seoul, South Korea

Edited by: Giovanna Suzzi, Università degli Studi di Teramo, Italy

#### Reviewed by:

Antonio Valero, University of Cordoba, Spain Alexandra Lianou, Agricultural University of Athens, Greece

> \*Correspondence: Yi Chen<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 29 March 2016 Accepted: 05 May 2016 Published: 18 May 2016

#### Citation:

Chen Y, Allard E, Wooten A, Hur M, Sheth I, Laasri A, Hammack TS and Macarisin D (2016) Recovery and Growth Potential of Listeria monocytogenes in Temperature Abused Milkshakes Prepared from Naturally Contaminated Ice Cream Linked to a Listeriosis Outbreak. Front. Microbiol. 7:764. doi: 10.3389/fmicb.2016.00764 The recovery and growth potential of Listeria monocytogenes was evaluated in three flavors of milkshakes (vanilla, strawberry, and chocolate) that were prepared from naturally contaminated ice cream linked to a listeriosis outbreak in the U.S. in 2015, and were subsequently held at room temperature for 14 h. The average lag phase duration of L. monocytogenes was 9.05 h; the average generation time was 1.67 h; and the average population level increase per sample at 14 h was 1.14 log CFU/g. Milkshake flavors did not significantly affect these parameters. The average lag phase duration of L. monocytogenes in milkshakes with initial contamination levels ≤ 3 CFU/g (9.50 h) was significantly longer (P < 0.01) than that with initial contamination levels > 3 CFU/g (8.60 h). The results highlight the value of using samples that are contaminated with very low levels of L. monocytogenes for recovery and growth evaluations. The behavior of L. monocytogenes populations in milkshakes prepared from naturally contaminated ice cream linked to the listeriosis outbreak should be taken into account when performing risk based analysis using this outbreak as a case study.

Keywords: L. monocytogenes, ice cream, milkshake, growth potential, temperature abuse, outbreak

# INTRODUCTION

In March 2015, a listeriosis outbreak (outbreak I) was reported from a hospital (hospital X) involving five highly susceptible elderly patients who were hospitalized for other medical conditions prior to exposure to Listeria monocytogenes (Centers for Disease Control and Prevention, 2015). DNA fingerprinting [pulsed field gel electrophoresis (PFGE) and whole genome sequencing (WGS)] of L. monocytogenes isolates from these patients and various ice cream products linked four patients to the consumption of three flavors of milkshakes (vanilla, chocolate, and strawberry) served at lunch or dinner in hospital X and prepared with the contaminated

ice cream scoops that were produced in production line A (Centers for Disease Control and Prevention, 2015; Karl Klontz, personal communication). It is expected that very little postcontamination growth occurred in ice cream because this product is kept frozen all along the production and distribution chain. Thus, enumeration of L. monocytogenes in these products would provide a relatively clear estimation of the amount of L. monocytogenes consumed by the case patients and shed some light on the risk associated with L. monocytogenes contamination and its infective dose. Therefore, Chen (2015) enumerated the levels of L. monocytogenes in 2,320 individually wrapped scoops of ice cream produced in production line A. The enumeration study demonstrated that L. monocytogenes was present in 99.4% of all tested products produced between November 2014 and March 2015, and among them L. monocytogenes was present in 100% of tested products produced between November 2014 and January 2015. The levels of L. monocytogenes in individual scoops were below 20 and 50 most probable number (MPN)/g in 92.3 and 98.4% of tested scoops, respectively; and they were homogeneously low among different production dates with a geometric mean concentration of 0.15 to 7.1 MPN/g (Chen, 2015).

However, the case patients consumed milkshakes prepared from contaminated ice cream scoops, and thus, the enumeration data cannot be directly used for risk assessment without taking into account the possible growth of L. monocytogenes in milkshakes prior to human consumption. Another listeriosis outbreak (outbreak II) linked to contaminated ice cream products occurred in another state in 2015 (Rietberg et al., 2015), involving two patients from a hospital (hospital Y) who were served milkshakes prepared from contaminated ice cream. No evidence of temperature abuse of milkshakes in hospital X or Y was reported (Centers for Disease Control and Prevention, 2015; Rietberg et al., 2015), and no samples were available from outbreak II (Rietberg et al., 2015). Milkshake is a commonly consumed commodity, and these two outbreaks called for studies on the behavior of L. monocytogenes in milkshakes prepared from ice cream products, especially those naturally contaminated with L. monocytogenes. However, such studies have never been reported. We were able to obtain ice cream samples produced from the production line that was implicated in outbreak I, and therefore, the objective of the present study was to evaluate the recovery and growth potential of L. monocytogenes in milkshakes prepared from naturally contaminated ice cream scoops linked to outbreak I. Assuming an extreme scenario that a milkshake was unintentionally left at room temperature for an extended period of time, we evaluated the recovery and growth of L. monocytogenes in the course of 14 h at room temperature.

#### MATERIALS AND METHODS

#### Ice Cream Samples

Individually wrapped scoops of ice cream produced from production line A were made available by the company. The scoops produced between November 2014 and January 2015 were used to prepare the milkshakes.

#### Preparation of Milkshakes

Vanilla, strawberry and chocolate milkshakes were prepared using the recipe from hospital X. Vanilla milkshakes were made from two scoops of ice cream (80–85 g/scoop) and 118 mL of 1% fat milk. Strawberry milkshakes were made from two scoops of ice cream, 118 mL of 1% fat milk and 15 mL of strawberry syrup. Chocolate milkshakes were made from two scoops of ice cream, 118 mL of 1% fat milk and 15 mL of chocolate syrup. Milk, strawberry syrup, and chocolate syrup were purchased from a local supermarket. Ice cream scoops were briefly left at room temperature to soften, and milkshakes were then prepared using a sterilized commercial drink mixer (Model, HMD200, Hamilton Beach Inc., Southern Pines, NC, USA). For each milkshake flavor, 10 milkshake samples were prepared from randomly picked ice cream scoops, and held at room temperature (22.5 ± 0.5◦C). To facilitate thorough mixing of the milkshakes, the entire portion of milkshakes were aseptically transferred to sterilized stainless steel laboratory blenders (Model, Waring R 7011S, Conair Corporation, East Windsor, NJ, USA) after preparation, and blended prior to each hourly sampling. The temperature increases of 10 randomly selected milkshakes used for growth curve construction were measured every half hour using thermometers certified by U.S. National Institute of Standards and Technology (Cat. #ACC10033BLSFC, Thermo Fisher Scientific Inc., Waltham, MA, USA). Ten additional vanilla milkshakes were prepared as described above, aseptically transferred to plastic cups and incubated at room temperature. Their temperatures were monitored to investigate the effect of container material and the absence of blending on the temperature increase. These 10 additional samples were not used to evaluate the growth potential of L. monocytogenes and thus were not subject to hourly blending.

## Growth Curves

Enumeration of L. monocytogenes in milkshakes was conducted hourly in the course of 14 h. During the first 10 h of sampling 2 g of each milkshake was directly plated onto 5 RAPID'L. mono agar (Cat. No. 3563694, Bio-Rad Laboratories, Hercules, CA, USA) plates (400 µl/plate) using the easySpiral R automatic spiral plater (Interscience, Inc., France) set to constant volume plating, and this plating yielded a limit of detection (LOD) of 0.5 CFU/g. After 10 h, 1 g of samples was directly plated onto 5 RAPID'L. mono agar plates (200 µl/plate), and this plating yielded a LOD of 1 CFU/g. All plates were left to dry before incubation. Representative colonies were confirmed according to the L. monocytogenes chapter of the FDA Bacteriological Analytical Manual (Hitchins et al., 2016). The L. monocytogenes level change (in CFU/g) of each sample was then used to construct the growth curve. The lag phase was determined as the time for the initial population level to increase twofold (Buchanan and Solberg, 1972). The data were not transferred to log values due to very low values (<10 CFU/g) of most of the data points. The generation time was calculated as the time required for cells to double during the exponential phase.

#### Statistical Analysis

fmicb-07-00764 May 14, 2016 Time: 12:18 # 3

Comparison of lag phase durations and generation times of L. monocytogenes in different flavors of milkshakes with different initial levels of L. monocytogenes were performed using one way ANOVA analysis (Bewick et al., 2004) or t-test (Whitley and Ball, 2002).

### RESULTS AND DISCUSSION

The growth evaluation was performed on naturally contaminated ice cream products that had varying initial levels of L. monocytogenes. Combining results from milkshakes of all three flavors, the initial L. monocytogenes levels ranged from 1 to 20.5 CFU/g with 50% (15/30) of the milkshakes having initial L. monocytogenes levels ≤ 3 CFU/g and 76.7% (23/30) of the milkshakes having initial L. monocytogenes levels ≤ 5 CFU/g. The final L. monocytogenes population after 14 h of incubation at 22.5◦C ranged from 9 to 422 CFU/g with 73.3% (22/30) of the milkshakes having final L. monocytogenes levels ≤ 100 CFU/g and 93.3% (28/30) of the milkshakes having final L. monocytogenes levels ≤ 150 CFU/g. Two samples having initial L. monocytogenes levels of 12 and 20.5 CFU/g yielded final levels of 233 and 422 CFU/g, respectively. The average level increase per sample at 14 h was 1.23 ± 0.26 (average ± standard deviation), 1.01 ± 0.19, and 1.19 ± 0.15 log CFU/g for vanilla, strawberry, and chocolate milkshakes, respectively, with an average of 1.14 ± 0.22 log CFU/g for all milkshakes (**Table 1**). This indicated that the milkshakes were in the early stage of the exponential phase, and therefore, the growth curve was presented as the change of CFU/g and lag phase duration was determined as the time required for initial cell level to increase twofold (**Figure 1**; Buchanan and Solberg, 1972). The average lag phase durations of L. monocytogenes were 8.85 ± 0.78, 9.50 ± 0.82, and 8.80 ± 0.95 h for vanilla, strawberry, and chocolate milkshakes, respectively (**Table 1**; **Figure 1**), which were not statistically different (P > 0.05) from each other. The average lag phase duration was 9.05 ± 0.88 h for all milkshakes (**Table 1**). The average generation times of L. monocytogenes were 1.65 ± 0.39, 1.79 ± 0.36, and 1.57 ± 0.18 h for vanilla, strawberry, and chocolate milkshakes, respectively (**Table 1**), which were not statistically different (P > 0.05) from each other. The average generation time was 1.67 ± 0.33 h for all milkshakes. Because of the relatively long lag phase, no growth of L. monocytogenes was observed for 7.30 ± 0.79, 8.05 ± 0.96, and 7.40 ± 0.70 h for vanilla, strawberry, and chocolate milkshakes, respectively (**Table 1**; **Figure 1**). To our knowledge, this is the first report of the recovery and growth potential of L. monocytogenes in milkshakes prepared with ice cream products that were naturally contaminated with very low levels of bacteria and produced from a production line that was implicated in a listeriosis outbreak. The lag phase duration and generation time of L. monocytogenes determined in the present study contributed to a better understanding of the behavior of L. monocytogenes in the temperature abused milkshake prepared with naturally contaminated ice cream. The data can be combined with previously generated enumeration data for risk based characterization of L. monocytogenes contamination in this commodity. There is a possibility that the average generation time of the entire exponential phase may be different from the generation time observed in the present study; however, we chose to calculate the lag phase duration and generation time within 14 h of incubation because that value was relevant in assessing the recovery and growth of L. monocytogenes, had the milkshakes been unintentionally left at room temperature during the day of serving. A probabilistic analysis between direct plating and MPN for the ice cream samples showed agreement of the two methods in 96.1% of the samples with direct plating providing an underestimate in 0.8% of the samples and MPN providing an underestimate in 3.1% of the samples. This indicated that the injury status of L. monocytogenes in these ice cream products did not prevent the cells from recovering and growing on RAPID'L. mono agar (Chen, 2015). WGS analysis using the Center for Food Safety and Applied Nutrition (CFSAN) Single Nucleotide Polymorphism (SNP) pipeline (Pettengill et al., 2014; Davis et al., 2015) showed that the randomly picked isolates from these ice cream products all matched the clinical specimen of the patients in hospital X (unpublished data).

It is very important to keep in mind that during the first few hours of the exposure to room temperature, the temperature of the milkshakes was low. Milkshakes were −2 to 0◦C when freshly prepared and reached 17 to 19◦C after being held at room temperature for 3 h (**Figure 2**) in stainless steel cups, and thus, after milkshakes reached room temperature, it took L. monocytogenes another 6.05 h to reach the exponential phase. Milkshakes in plastic cups that were not subject to hourly blending reached 17 to 19◦C after 4 h at room temperature. Therefore the use of stainless steel container and hourly blending facilitated a slightly faster temperature increase of milkshakes than plastic cups without hourly blending.

Studies which examined growth kinetics of L. monocytogenes in various enrichment broths and foods under various conditions often had the technical limitation (Gnanou Besse et al., 2006) set by the sensitivity of enumeration methods. In some cases, the variability in the enumeration of low levels of bacterial cells

TABLE 1 | Lag phase duration, generation time, time before growth was observed, level increase per sample for the 30 milkshakes (10 vanilla milkshakes, 10 strawberry milkshakes, and 10 chocolate milkshakes) analyzed in the present study.

FIGURE 1 | Growth curves of 30 milkshake samples (10 vanilla milkshakes, 10 strawberry milkshakes, and 10 chocolate milkshakes) held at 22.5◦C for 14 h. Each line represents the growth curve of a milkshake sample.

could compromise the reliability of any statistical analysis (Duffy et al., 1994). This is probably why quite a few studies used foods artificially inoculated with 102 to 104 CFU/g or even higher levels of bacteria for the convenience of subsequent enumeration and statistical analysis (Xanthiakos et al., 2006; Panagou and Nychas, 2008; Schvartzman et al., 2014; Huang et al., 2015; Luo et al., 2015). However, these inoculum sizes do not reflect the low bacterial contamination levels usually found in food. Indeed, if the bacterial levels are low, a large number of agar plates or an MPN scheme with a large number of MPN tubes per level and a large number of biological replicates would have to be used to accurately determine the lag phase duration. This would be impractical for hourly monitoring of bacterial growth, especially for an extended period of time. The milkshake samples in the present study could be homogenized with no dilutions in any buffer; and they were viscous and could stay off the edge of the plates. As a result, as much as 400 µl of milkshake could be evenly spread onto one plate with the help of the automatic spiral plater, and this allowed us to plate up to 2 g per sample and to obtain reliable data to perform statistical analysis.

The average lag phase duration of L. monocytogenes in 15 milkshakes having initial L. monocytogenes levels ≤ 3 CFU/g was 9.50 ± 0.76 h, and it was significantly higher (P < 0.01) than that of 15 milkshakes having initial L. monocytogenes levels > 3 CFU/g, which was 8.60 ± 0.78 h. Among the 15 milkshakes having initial L. monocytogenes levels > 3 CFU/g, eight milkshakes had initial L. monocytogenes levels of 3.5 to 5 CFU/g and seven milkshakes had initial L. monocytogenes levels of 5.5 to 20.5 CFU/g. Their average lag phase durations were 8.44 ± 0.78 and 8.79 ± 0.81 h, respectively, which were not significantly different (P > 0.05) from each other. The enumeration study revealed that 7.7, 1.6, and 0.2% of all tested ice cream scoops had L. monocytogenes of more than 20, 50, and 100 MPN/g, respectively (Chen, 2015). The milkshakes were made of two 80–85 g scoops and then diluted in 118 mL of milk, and thus, in order to obtain milkshakes containing L. monocytogenes of more than 30 CFU/g the addition of numeric values of the L. monocytogenes levels in the two scoops needed to exceed 100. The probability of this happening was very low, and therefore, we would not have been able to obtain sufficient biological replicates of milkshakes containing L. monocytogenes of more than 30 CFU/g even if we had prepared a much larger number of milkshakes. Robinson et al. (2001) showed that when the inoculum levels of L. monocytogenes were below 100 to 1,000 cells per sample, the lag time increased as the inoculum size decreased, especially under suboptimal growth conditions, partially due to the variability in the lag phase of individual cells; and the effect of inoculum size on lag phase duration started to disappear when the inoculum levels were higher. Aguirre et al. (2013) found that under experimental conditions, the lag phase durations of L. innocua with initial levels of 0.7 to 20 CFU/sample were higher than those with initial levels of 20 to 200 CFU/sample, and there was no statistical difference in lag phase durations among populations with initial levels between 20 and 200 CFU/sample. This phenomenon was also observed by Baranyi (1998) and Pin and Baranyi (2006) in other bacteria. The results from different studies may not be directly compared due to differences in the Listeria species/strains, growth media, and applied methodologies, however, they could partially explain why we observed a longer lag phase duration (P < 0.01) of cells in milkshakes with initial levels ≤ 3 CFU/g than that of cells with initial levels between 3 and 20.5 CFU/g, and no significant difference (P > 0.05) in lag phase durations between cells with initial levels of 3.5 to 5 CFU/g and those with initial

levels of 5.5 to 20.5 CFU/g. The finding in the present study, combined with those reported previously, suggests that under certain growth conditions the average lag phage duration of cells that are sparsely distributed in foods could be significantly longer than that indicated from foods inoculated with high levels of inoculum.

No studies have been performed on the behavior of L. monocytogenes in milkshakes made from ice cream, but a few studies analyzed artificially inoculated dairy products. Alavi et al. (1999) reported that the generation time of L. monocytogenes with an initial inoculum level of 1,000 CFU/mL in liquid whole milk stored at 22.5◦C was 1.40 h. Xanthiakos et al. (2006) reported that with an initial inoculum level of 103 to 104 CFU/mL in pasteurized milk stored at 16◦C, the lag phase duration of L. monocytogenes was 7.12 h and the generation time was 1.27 h. Panagou and Nychas (2008) found that with an initial inoculum level of 100 CFU/g, L. monocytogenes in a vanilla cream dessert-type product held at 15◦C had an average lag phase duration of 7.85 h and an average generation time of 1.45 h. Gougouli et al. (2008) studied the behavior of L. monocytogenes in artificially inoculated ice cream extensively under multiple chilling and freezing conditions. A pro-longed (500 h) chilling-freezing experiment monitoring the recovery and growth of L. monocytogenes every 20 to 40 h demonstrated that L. monocytogenes cells in ice cream did not suffer significant injury in freezing conditions up to −30◦C, and during consecutive freezing and thawing cycles. This suggested that the relatively long lag phase and generation time determined in the present study were not direct results of cell injury. Due to the overall low prevalence of L. monocytogenes in foods, most studies that used naturally contaminated samples could only yield a small number of positive samples and thus not enough biological replicates could be generated (Jorgensen and Huss, 1998; Encinas et al., 1999; Lappi et al., 2004; Beaufort et al., 2007). The 100% prevalence rate of L. monocytogenes (Chen, 2015) in the ice cream samples used in the present study allowed the use of a large number of naturally contaminated biological replicates to generate the unique and highly confident research data.

#### REFERENCES

#### CONCLUSION

This is the first report on the characterization of the behavior of L. monocytogenes in milkshakes prepared from the ice cream products naturally contaminated with low levels of L. monocytogenes. This provides relevant information for future risk assessment using the 2015 U.S. ice cream listeriosis outbreak as a case study, especially since the ice cream scoops used in the present study were produced from the production line that was implicated in the outbreak. The results demonstrated the value of using samples contaminated with very low levels of L. monocytogenes to perform recovery and growth evaluations of L. monocytogenes in foods. It is important to keep in mind that the rather long lag phase and generation time observed only pertained to the strains/samples in the present study, and our conclusions by no means undermine the critical importance of stringent temperature control by food handlers.

### AUTHOR CONTRIBUTIONS

YC is the principle investigator (PI) of the project and wrote the manuscript. TH and DM are co-PIs of the project. EA, AW, MH, IS, and AL are research scientists working on the experiments.

#### ACKNOWLEDGMENTS

This research was funded in part by the University of Maryland Joint Institute for Food Safety and Applied Nutrition through a cooperative agreement with the U.S. Food and Drug Administration (FDA), #FDU001418, and in part by an appointment to the Research Participation Program at the FDA Center for Food Safety and Applied Nutrition, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. FDA.

growth in structured media. Int. J. Food Microbiol. 110, 43–51. doi: 10.1016/j.ijfoodmicro.2006.03.002

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

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. FDA.

Copyright © 2016 Chen, Allard, Wooten, Hur, Sheth, Laasri, Hammack and Macarisin. 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) or licensor 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.

# Biofilm-Forming Capacity in Biogenic Amine-Producing Bacteria Isolated from Dairy Products

Maria Diaz, Victor Ladero\*, Beatriz del Rio, Begoña Redruello, María Fernández, M. Cruz Martin and Miguel A. Alvarez

Department of Technology and Biotechnology of Dairy Products, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa, Spain

Biofilms on the surface of food industry equipment are reservoirs of potentially food-contaminating bacteria—both spoilage and pathogenic. However, the capacity of biogenic amine (BA)-producers to form biofilms has remained largely unexamined. BAs are low molecular weight, biologically active compounds that in food can reach concentrations high enough to be a toxicological hazard. Fermented foods, especially some types of cheese, accumulate the highest BA concentrations of all. The present work examines the biofilm-forming capacity of 56 BA-producing strains belonging to three genera and 10 species (12 Enterococcus faecalis, 6 Enterococcus faecium, 6 Enterococcus durans, 1 Enterococcus hirae, 12 Lactococcus lactis, 7 Lactobacillus vaginalis, 2 Lactobacillus curvatus, 2 Lactobacillus brevis, 1 Lactobacillus reuteri, and 7 Lactobacillus parabuchneri), all isolated from dairy products. Strains of all the tested species - except for L. vaginalis—were able to produce biofilms on polystyrene and adhered to stainless steel. However, the biomass produced in biofilms was strain-dependent. These results suggest that biofilms may provide a route via which fermented foods can become contaminated by BA-producing microorganisms.

#### Edited by:

Giovanna Suzzi, University of Teramo, Italy

#### Reviewed by:

Giorgia Perpetuini, University of Teramo, Italy Fausto Gardini, University of Bologna, Italy

> \*Correspondence: Victor Ladero<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 24 February 2016 Accepted: 11 April 2016 Published: 12 May 2016

#### Citation:

Diaz M, Ladero V, del Rio B, Redruello B, Fernández M, Martin MC and Alvarez MA (2016) Biofilm-Forming Capacity in Biogenic Amine-Producing Bacteria Isolated from Dairy Products. Front. Microbiol. 7:591. doi: 10.3389/fmicb.2016.00591 Keywords: biofilm, biogenic amines, histamine, tyramine, putrescine, lactic acid bacteria, dairy industry

# INTRODUCTION

Food safety is a major priority in today's food industry. Bacterial biofilms on industrial surfaces are a cause for concern since they may act as reservoirs of contaminating microorganisms (Winkelströter et al., 2014). Dairy products in particular are susceptible to such contamination (Srey et al., 2013), with equipment surfaces one of its major sources (Kumar and Anand, 1998). Stainless steel type 304 is the most common material in contact with food in the dairy industry (Zottola and Sasahara, 1994). While inert, easy to clean and highly resistant to corrosion, it can, however, develop small cracks and crevices where biofilm formation is facilitated (Winkelströter et al., 2014). In addition, some parts of food processing equipment may have inaccessible areas where bacteria can evade cleaning treatments. The main biofilm-related risk is the growth of pathogens and spoilage microorganisms.

The capacity of biogenic amine (BA)-producers to form biofilms has not been investigated. BAs are low-molecular weight organic compounds derived from their corresponding amino acids via enzymatic decarboxylation. Although, BAs play an important role in human physiology, the ingestion of food containing them in large quantities can have toxicological effects on the digestive, circulatory, and respiratory systems (ten Brink et al., 1990; Shalaby, 1996; Ladero et al., 2010a). The most important BAs (both qualitatively and quantitatively) in foods and beverages are histamine, tyramine, and putrescine. Together with fish and wine, dairy products—especially cheese can develop BA concentrations that may exceed 1000 mg kg−1 (Linares et al., 2012).

BAs form in food via the activity of bacteria with aminoacyl decarboxylase activity (Halasz et al., 1994). Their appearance in dairy products has mainly been attributed to Gram positive bacteria of the lactic acid bacteria (LAB) group. These can be present in the microbiota of milk, as part of the starter culture, or be introduced by contamination during manufacturing (Linares et al., 2011), with equipment surfaces a potentially important source of contamination (Novella-Rodríguez et al., 2004). The post-ripening processing of cheese, particularly grating, extends the contact of food with equipment surfaces, increasing the number of histamine-producing bacteria present in the final product (Ladero et al., 2009) and therefore the histamine concentration that may be reached.

The dairy histamine-producing species Lactobacillus parabuchneri has been reported to produce biofilms (Diaz et al., 2016b), but this capacity has not been studied in other BA producers. The aim of the present work was to test the ability of BA-producing bacteria isolated from different cheeses to form biofilms on polystyrene and adhere to stainless steel surfaces.

#### MATERIALS AND METHODS

#### Bacterial Strains and Culture Conditions

In this work we have examined the biofilm-forming capacity of 56 BA-producing strains belonging to three genera and 10 species (12 Enterococcus faecalis, 6 Enterococcus faecium, 6 Enterococcus durans, 1 Enterococcus hirae, 12 Lactococcus lactis, 7 Lactobacillus vaginalis, 2 Lactobacillus curvatus, 2 Lactobacillus brevis, 1 Lactobacillus reuteri, and 7 Lactobacillus parabuchneri), all isolated from dairy products. The ability of all (except of the Lactobacillus parabuchneri strains) to produce BAs was known from previous work (Ladero et al., 2010b,c, 2011a,b, 2012a,b; Diaz et al., 2015; del Rio et al., 2015). Given the reported ability of L. parabuchneri to form biofilms (Diaz et al., 2016b), and the apparent importance of the species in the accumulation of histamine in cheese (Diaz et al., 2016a), seven new strains isolated from cheese (following the protocol of Diaz et al., 2015) were included among those examined. These were identified at the species level by 16S rRNA sequencing (Diaz et al., 2015) and their ability to produce histamine checked by UHPLC (Redruello et al., 2013).

Lactobacilli were grown at 37◦C in MRS (Oxoid, Basingstoke, UK), while enterococci and lactococci were grown at 30◦C in M17 (Oxoid) supplemented with 0.5% (w/v) glucose (GM17). To test the individual capacity of the L. parabuchneri strains to produce histamine, the culture medium was supplemented with 5 mM histidine.

# Analysis of Biofilm Formation on Polystyrene

The ability of the test strains to produce a biofilm on polystyrene was performed as described by Diaz et al. (2016b). Briefly, MRS or GM17 overnight cultures were diluted to approximately 106 cfu mL−1 with fresh medium and used to fill polystyrene 96-well microtitre plates (NuncTM MicroWellTM Plates with a NunclonTM Delta Surface; Thermo Fisher Scientific, Waltham, MA, USA).  All plates were then incubated at 30 or 37◦C depending on the species. Biofilm biomass was determined using the crystal violet staining method (CV assay) (Kubota et al., 2008). After 24, 30, or 48 h of incubation, the supernatant was removed and the wells rinsed with PBS buffer to eliminate non-adhered cells. The potential biofilm present was then stained with 0.5% (w/v) CV in distilled water (dH2O); the excess dye was removed with dH2O. The bound dye was then extracted using acetone/ethanol (80:20, v:v) and quantified by absorbance at 595 nm using a Benchmark Plus microplate spectrophotometer (BioRad, Hercules, CA, USA). The mean ± SD of the optical density (OD) of three replicates was calculated for each strain. ANOVA with post-hoc Bonferroni correction was used to analyse all data. Significance was set at p < 0.05. All statistical calculations were undertaken using SPSS v.15.0 software (SPSS Inc., IL, USA). Biofilm production capacity was expressed using cut-off values (Extremina et al., 2011). The cutoff value between biofilm-producers and non-producers (ODc) was defined as the mean of the negative controls (ODnc) plus three SDs. The strains were then classified as belonging to one of the following categories: ODc < OD≤ 2 × ODc = weak biofilm producer, 2 × ODc < OD ≤ 4 × ODc = moderate biofilm producer, and OD > 4 × ODc = strong biofilm producer.

#### Analysis of Bacterial Adherence to Stainless Steel

The test surfaces used were 1 cm2 stainless steel (type AISI 304) coupons. These were washed with soap and dH2O, rinsed with dH2O, and then immersed in acetone for 30 min to remove any grease or fingerprints. They were then rinsed once again in dH2O, autoclaved, and immersed singly in tubes containing MRS or GM17 broth inoculated with 106 cfu mL−1 of the assayed strain (performed in triplicate). Each coupon was then incubated at 30 or 37◦C for 24 or 48 h before removal using sterile forceps. Non-adhered cells were removed by rinsing the coupon three times in PBS buffer. The coupons were then reimmersed in 5 mL PBS buffer, and the adhered cells detached from the coupon by sonication in an ultrasonic bath (Ultrasons-H, Selecta, Spain) for 15 min. The bacterial suspension produced was serially diluted in PBS, and 100 µL of 100 , 10−1 , 10−2 , 10−3 , and 10−4 dilutions were plated on MRS or GM17 and incubated for 48 h (Kruszewski et al., 2013). Three replicates were performed for each strain using independent bacterial cultures. Bacterial counts were expressed as log10 cfu cm−2 (mean ± SD of three replicates). To confirm the tolerance of the cells to sonication, bacterial suspensions of all the examined strains were sonicated for 15 min. Pre- and post-sonication suspensions were serially diluted in PBS, plated, incubated for 48 h, and the cells enumerated; no significant differences were seen between preand post-sonication cell counts confirming that no cells were killed by this procedure.

# Scanning Electron Microscopy of Cells Adhered to Stainless Steel

The method of Kubota et al. (2008) was followed, with some modifications, to observe by scanning electron microscopy (SEM) the cells adhered to the stainless steel coupons. Briefly, the latter were rinsed twice in PBS and then fixed in 2.5% glutaraldehyde (Sigma-Aldrich) in PBS for 16 h at room temperature. The fixed bacteria were then dehydrated using a graded series of acetone solutions (50–100% v/v), and the coupons dried with CO2 using a CPD-030 critical point dryer (Bal-Tec AG, Balzers, Liechtenstein). They were then coated with gold using a SCD 004 Sputtering Coater (Bal-Tec AG, Balzers, Liechtenstein) and observed using a JSM-6610LV SEM (JEOL USA, Inc, Peabody, MA, USA).

# RESULTS

#### Biofilm Formation on Polystyrene

Maximum biofilm biomass values were obtained at different times of incubation depending on the species. For all the Enterococcus strains assayed, biofilm biomass was maximal at 30 h; for all the L. lactis, L. curvatus, and L. brevis strains, maximum values were reached at 24 h; and for the L. reuteri and L. parabuchneri strains, maxima were recorded at 48 h. All incubation time results shown are those at which maximum biomass was reached.

All the E. faecalis strains were able to form biofilms on polystyrene. Six were classified as strong biofilm producers, and six as moderate biofilm producers (**Figure 1**). The only E. hirae strain assayed was a weak biofilm producer (**Figure 1**). All the E. durans strains tested were able to form biofilms; four were strong biofilm producers, and two were weak producers (**Figure 1**). Of the six E. faecium strains analyzed, three were strong biofilm producers, one was a weak producer, and two were unable to form a biofilm (**Figure 1**).

All the L. lactis strains were able to produce biofilms on polystyrene (**Figure 2**). Three L. lactis subsp. cremoris and two L. lactis subsp. lactis were strong biofilm producers. The remaining strains—two L. lactissubsp.cremoris and five L. lactissubsp. lactis strains—were weak producers.

None of the L. vaginalis strains were able to form a biofilm (**Figure 3**). All the L. curvatus, L. brevis, and L. reuteri strains were, however, strong biofilm producers (**Figure 3**). Two out of seven L. parabuchneri strains were strong biofilm producers, while the remaining strains were only weak producers (**Figure 3**).

# Bacterial Adherence to Stainless Steel

The strains selected for this assay were the strongest biofilm producers in the polystyrene surface assay, i.e., E. faecalis 15a, E. hirae 268, E. durans 411, E. faecium AA3, L. lactis subsp. cremoris CECT 8666, L. lactis subsp. lactis 1AA59, L. curvatus VI6, L. brevis 3811, L. parabuchneri IPLA 11150, and L. reuteri IPLA 11078. Although, no L. vaginalis strain was able to form a biofilm on polystyrene, L. vaginalis IPLA 11064 was tested with the steel surface. Two incubation times (24 and 48 h) were tested, but no differences were observed. **Figure 4** shows the number of adhered

cells of each strain after incubation for 48 h (>104 cfu cm−2 for all strains assayed).

## Scanning Electron Microscopy Imaging of Cells

SEM photomicrographs of cells adhered to the stainless steel coupons were captured for the same strains as used in the previous assay. Although, no differences were seen in the counts at different incubation times (24 and 48,h), differences in aggregation type and bacterial ultrastructure were observed.

E. faecalis 15a (**Figure 5A1**), E. faecium AA3 (**Figure 5B1**) and E. durans 411 returned clearer images after 24 h of incubation (data not shown) and appeared uniformly spread on the coupons. No adhering E. hirae 268 cells were observed at either 24 or 48 h. The E. faecalis cells were observed embedded in an extracellular matrix (**Figure 5A2**); this was not observed for the other two species. Structures that might be involved in anchoring to the surface were observed on E. faecium cells (**Figure 5B2**).

L. lactis subsp. cremoris CECT 8666 and L. lactis subsp. lactis 1AA59 returned clearer images after 24 h of incubation. The strain 8666 was uniformly spread across the coupon surface (**Figure 6A1**), while 1AA59 formed more compact aggregates (**Figure 6B1**). In both cases, an extracellular matrix was observed, but with a different appearance (see **Figures 6A2,A3,B2**). Similar structures to those observed in E. faecium, and that might be involved in anchoring to the surface, were also observed for both L. lactis strains (**Figures 6A3,B3**).

L. parabuchneri IPLA 11150, L. reuteri IPLA 11078, and L. brevis 3811 returned clearer images after 48 h of incubation. L. vaginalis IPLA 11064 and L. curvatus VI6 showed no adhered cells at either 24 or 48 h. L. parabuchneri was distributed across the coupon, showing aggregations with an extracellular matrix (**Figures 7A1,A2**). L. reuteri was distributed across the coupons in small aggregations (**Figure 7B1**) in a clear extracellular matrix (**Figure 7B2**). L. brevis was seen only in the fissures of the coupon (**Figures 7C1,C2**).

# DISCUSSION

The formation of BA in fermented foods by bacteria, especially cheese, is a cause of much concern. The industrial equipment used in cheesemaking and processing is a source of contamination, and the ability of microorganisms to adhere to the surfaces of such equipment increases their contamination potential. In this work, the ability of 56 BA-producing dairy LAB strains to adhere to a polystyrene surface was examined using the CV assay, a technique that allows easy quantification of total biofilm biomass. The strongest biofilm producers of each species were then examined for their ability to adhere to stainless steel coupons, and the adhered cells observed by SEM.

The formation of biofilms by clinical Enterococcus isolates has been much studied since it affects pathogenic potential (Langsrud, 2009). However, enterococci are also a cause of concern for the food industry, and the biofilm-forming ability of several food-related Enterococcus species has also been studied (Gomes et al., 2008; Jahan and Holley, 2014; da Silva Fernandes et al., 2015). In the present study, the biofilm-forming capacity of tyramine- or tyramine and putrescine-producing Enterococcus species was examined. The results of the CV assay showed that all the strains analyzed, except for two E. faecium strains, were able to form biofilms, although the total biomass produced differed. These results agree with those of previous studies that showed several foodborne E. faecalis and E. faecium isolates to be either weak, moderate or strong biofilm producers, while some isolates formed no biofilm at all (Gomes et al., 2008; Jahan and Holley, 2014). E. durans has also been described as a strong or moderate biofilm producer (Amel et al., 2015; Pieniz et al., 2015). The only strain of E. hirae analyzed in the present work was a weak biofilm producer; to our knowledge, the capacity of this species to form biofilms has not been previously studied.

All the Enterococcus strains selected to see whether they could adhere to stainless steel did so, with counts reaching 104 cfu cm−2 . The capacity of E. faecalis, E. faecium, and E. durans to adhere to stainless steel has been previously reported (Amel et al., 2015; da Silva Fernandes et al., 2015). The Enterococcus strains that adhered to the stainless steel coupons were observed by SEM, except for E. hirae 268; - this strain attached only weakly to the metal surface and was unable to resist the treatment required prior to observation. Of the strains that covered the surface of the steel coupons (**Figure 5**), none formed the complex three-dimensional structures reported

FIGURE 3 | Biofilm-producing capacity on polystyrene of the biogenic amine-producing Lactobacillus strains incubated at 37◦C. The L. vaginalis, L. curvatus and L. brevis strains were incubated for 24 h. The L. parabuchneri and L. reuteri strains were incubated for 48 h. Data represent means ± SD (error bars) of three experiments. Values marked with the same letter do not differ significantly (p > 0.05 according to the Bonferroni post-hoc test). (\_\_\_) Cut-off line (ODc); (\_\_ \_\_ \_\_) 2 × ODc; (.......) 4 × ODc.

by da Silva Fernandes et al. (2015). Some aggregations of E. faecalis 15a cells were observed (**Figure 5A2**), while E. faecium AA3 cells appeared separated from one another, but all cells were sufficiently well adhered not to be detached by the PBS washes or the treatment required prior to SEM observations. To our knowledge, this is the first time that images of E. durans cells adhered to stainless steel have been captured.

The ability of L. lactis to form a biofilm on a surface has been related to the latter's physicochemical properties (Giaouris et al., 2009; Oxaran et al., 2012). However, few studies have ever been performed on L. lactis biofilm formation. In the present work, 12 putrescine-producing L. lactis strains were shown by the CV assay to be either weak or strong biofilm producers. The two strains that most strongly formed biofim on polystyrene—L. lactis subsp. cremoris CECT 8666 and L. lactis subsp. lactis 1AA59—also adhered to stainless steel, reaching counts of 104 cfu cm−2 . SEM images showed cells of both strains to be embedded in an extracellular matrix and to be clearly adhered to the coupons. This is the first time that L. lactis adhered on stainless steel have been observed by SEM. Unlike that seen for enterococci, the L. lactis CECT 8666 cells showed tridimensional structures including filamentous ones that might help them adhere to the surface (**Figures 6A2,A3**). The extracellular material formed a pod-like covering over the cells (**Figures 6A2,A3**). L. lactis 1AA59 cell clusters also appeared to be attached via the extracellular matrix (**Figure 6B2**). For both L. lactis strains (**Figures 6A3,B3**) examined, and E. faecium AA3 (**Figure 5B2**), small protuberances of the cell surface were observed, which may have helped anchor the cells to the coupons. Certainly, aggregation and biofilm formation in L. lactis has been associated with the production of functional pili (Oxaran et al., 2012). Although, the adhesion of the putrescine-producing strains analyzed in this work would be undesirable for the food industry, allowing the development of L. lactis biofilms has been proposed as a means of preventing the growth of pathogens on food industry surfaces (Leriche et al., 1999; Zhao et al., 2004).

The presence of several Lactobacillus species (L. curvatus, Lactobacillus fermentum, Lactobacillus delbruekii, Lactobacillus paracasei, Lactobacillus plantarum, and L. reuteri) on the surfaces of dairy equipment has been reported (Somers et al., 2001; Scatassa et al., 2015). In the present work, two tyramine and putrescine-producing L. brevis strains were strong biofilm producers on polystyrene. This agrees with previous results for L. brevis strains isolated from onions (Kubota et al., 2008). The two tyramine-producing L. curvatus strains tested were also strong biofilm producers. Biofilm formation by L. curvatus has been previously described, although it was found to be a weaker biofilm producer than in the present work (Pérez Ibarreche et al., 2014). Fifteen histamine-producing strains, seven belonging to L. vaginalis, seven to L. parabuchneri and one to L. reuteri, were examined by the CV assay. The seven histamine-producing L. vaginalis strains were unable to form biofilms. All the L. parabuchneri strains were able to form biofilms, and were either weak or strong biofilm producers, depending on the strain. This agrees with the results of previous reports (Diaz et al., 2016b). The L. reuteri strain examined was a strong biofilm producer. Numerous studies have described the formation of biofilms by L. reuteri, some strains of which are considered probiotics (Leccese Terraf et al., 2014). A system regulating biofilm formation in L. reuteri was recently characterized (Su and Ganzle, 2014). In the present work, L. reuteri IPLA 11078, along with L. vaginalis IPLA 11064, L. curvatus VI6, L. brevis 3811 and L. parabuchneri IPLA 11150 were all able to adhere to the stainless steel coupons, and reached counts of over 104 cfu cm−2 . However, when the coupons were observed under the SEM, only L. parabuchneri

IPLA 11150 (**Figure 7A**) and L. reuteri IPLA 11078 (**Figure 7B**) cells were seen attached. For L. parabuchneri IPLA 11150, the cells aggregated into clumps composed of long chains of undivided cells. A previous study on Pseudomonas aeruginosa showed that elongated cells are inclined to form cohesive clumps (Yoon et al., 2011). According to the CV assay, the present L. parabuchneri and L. reuteristrains were strong biofilm producers. However, while the same assay suggested L. curvatus VI6 and L. brevis 3811 to be strong biofilm producers, the absorbance measured was very close to the lower limit for such classification. The few cells seen attached to the coupons might be the result of them not being able to resist the treatment required prior to SEM observation. This would appear to be supported by the fact that L. brevis 3811 was not homogenously attached to the surface of the coupons, but seen in their fissures. This is an important finding for the food industry since steel surfaces do develop cracks and these appear able to protect bacteria from cleaning procedures.

The present results show all the species examined, except L. vaginalis, had BA-producing strains able to form biofilms and that they could adhere to stainless steel, a material commonly used to make equipment in the food industry. Although, the cells attached to the stainless steel coupons commonly showed none of the three-dimensional structures reported by da Silva Fernandes et al. (2015), counts of over 104 cfu cm−2 were recorded in all cases, with E. faecalis, E. faecium, E. durans, and L. parabuchneri exceeding 105 cfu cm−2 . Given the large surface area of industrial equipment, BA-producing bacteria that can adhere to steel clearly pose a food contamination threat. This problem is of particular concern in the dairy industry since post-ripening treatments such as cheese grating bring food into close and prolonged contact with equipment surfaces. In fact, it has already been shown that cheese that has undergone post-ripening processing (cutting, slicing, or grating) has higher histamine levels than non-processed cheese (Ladero et al., 2009).

It is therefore important that our knowledge of the adhesion and biofilm forming capacities of BA-producers be improved, to prevent food contamination by these spoilage bacteria and eventually the accumulation of biogenic amines in food.

# AUTHOR CONTRIBUTIONS

MD carried out the experiments and drafted the manuscript; BR, VL collaborated in conducting some experiments; VL, Bd, and MF participated in the study design and writing of the manuscript; MC, MA provided the general concept, designed the experiments, and supervised the experimental work and the manuscript. All authors contributed to the discussion of the research and approved the final manuscript.

# ACKNOWLEDGMENTS

The authors are very grateful to Prof. Christian Magni for providing some of the strains used in this work, the Unidad de Microscopía Electrónica y Microanálisis of the University of Oviedo for assistance with the SEM techniques, and Adrian Burton for language assistance. This work was funded by the Spanish Ministry of the Economy and Competitiveness (AGL2013-45431-R) and the Plan for Science, Technology and Innovation 2013–2017 financed by the European Regional Development Fund and the Principality of Asturias (GRUPIN14-137). MD is a beneficiary of an FPI fellowship from the Spanish Ministry of the Economy and Competitiveness.

# REFERENCES

dairy environment. J. Dairy Sci. 84, 1926–1936. doi: 10.3168/jds.S0022- 0302(01)74634-6

**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 © 2016 Diaz, Ladero, del Rio, Redruello, Fernández, Martin and Alvarez. 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) or licensor 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.

# Prevalence, Molecular Characterization, and Antibiotic Susceptibility of *Vibrio parahaemolyticus* from Ready-to-Eat Foods in China

#### Tengfei Xie1, 2, Xiaoke Xu2 , Qingping Wu2 \*, Jumei Zhang2 and Jianheng Cheng2

*1 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China, 2 Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology Southern China: Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou, China*

#### *Edited by:*

*Maria Schirone, University of Teramo, Italy*

#### *Reviewed by:*

*Aoife Boyd, National University of Ireland Galway, Ireland Learn-Han Lee, Monash University Malaysia, Malaysia*

> *\*Correspondence: Qingping Wu<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 16 January 2016 Accepted: 04 April 2016 Published: 21 April 2016*

#### *Citation:*

*Xie T, Xu X, Wu Q, Zhang J and Cheng J (2016) Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus from Ready-to-Eat Foods in China. Front. Microbiol. 7:549. doi: 10.3389/fmicb.2016.00549* *Vibrio parahaemolyticus* is the leading cause of foodborne outbreaks, particularly outbreaks associated with consumption of fish and shellfish, and represents a major threat to human health worldwide. This bacterium harbors two main virulence factors: the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Additionally, various serotypes have been identified. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant *V. parahaemolyticus.* In the current study, we aimed to determine the incidence and features of *V. parahaemolyticus* in ready-to-eat (RTE) foods in China. We found 39 *V. parahaemolyticus* strains on Chinese RTE foods through investigation of 511 RTE foods samples from 24 cities in China. All isolates were analyzed for the presence of *tdh* and *trh* gene by PCR, serotyping was performed using multiplex PCR, antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing and multilocus sequence typing (MLST). The results showed that none of the isolates were positive for *tdh* and *trh.* Most of the isolates (33.3%) were serotype O2. Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (89.7%), cefazolin (51.3%), and ampicillin (51.3%). The isolates were grouped into five clusters by ERIC-PCR and four clusters by MLST. We updated 10 novel loci and 33 sequence types (STs) in the MLST database. Thus, our findings demonstrated the presence of *V. parahaemolyticus* in Chinese RTE foods, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of methods of microbiological risk assessment in RTE foods.

Keywords: *Vibrio parahaemolyticus,* ready-to-eat foods, antibiotic resistance, ERIC-PCR, MLST, serotype, virulence gene

# INTRODUCTION

Vibrio parahaemolyticus is a gram-negative halophilic bacterium that naturally occurs worldwide in estuarine environments. This microorganism is recognized as one of the leading causes of foodborne illness worldwide and has been shown to cause acute gastroenteritis in humans. Complications such as septicemia can sometimes lead to death in patients with V. parahaemolyticus infection (Qadri et al., 2005; Lopatek et al., 2015). Previous studies have focused on the prevalence of V. parahaemolyticus in shellfish, oysters, water, seafood, and shrimp (Khouadja et al., 2013; Lopatek et al., 2015). However, no reports have identified isolates of V. parahaemolyticus on ready-to-eat (RTE) foods such as cooked meat, roasted poultry, and cold vegetable dishes in sauce, that are highly popular in China. Various foodborne pathogens may be present in RTE foods and may cause illnesses in consumers because RTE foods do not require heat treatment or other forms of curing before eating (Shi et al., 2015). Moreover, the high genetic diversity on RTE foods can facilitate identification of strain relatedness and epidemiological investigations.

Traditionally, V. parahaemolyticus is considered susceptible to antimicrobials. However, during the past few decades, antimicrobial resistance has emerged and evolved in many bacterial genera owing the excessive use of antimicrobials in human, agriculture, and aquaculture systems (Cabello, 2006; Kang et al., 2016). For example, tetracyclines are recommended as antibiotics in the treatment of severe Vibrio infections, and thirdgeneration cephalosporins with doxycycline or fluoroquinolone alone are also used on occasion. Moreover, antibiotics are commonly used to treat fish (Han et al., 2007; Devi et al., 2009). Some V. parahaemolyticus isolates from seafood and other environments are commonly resistant to ampicillin, aminoglycosides (streptomycin and gentamicin), ciprofloxacin, chloramphenicol, and other antibiotics (Oh et al., 2011; Raissy et al., 2012; Shaw et al., 2014; Lopatek et al., 2015). Therefore, the potential presence of antibiotic-resistant V. parahaemolyticus on RTE foods may be an important public health problem related to disease management and control.

The virulence of V. parahaemolyticus is mainly attributed to the production of two major factors: thermo-stable direct hemolysin (TDH) encoded by the tdh gene and TDH-related hemolysin encoded by the trh gene (Honda and Iida, 1993). Clinical strains commonly contain either these genes, and the presence of these genes is associated with the pathogenicity of the strain in humans (Su and Liu, 2007; Jones et al., 2012; Pazhani et al., 2014). Detection of V. parahaemolyticus virulence factors is typically based on molecular biological analysis and amplification of V. parahaemolyticus-specific sequences (Shirai et al., 1990; West et al., 2013). To date, there are 13 O-serogroups and over 70 K-serogroups, differentiated on the basis of somatic (O) and capsular (K) antigens in V. parahaemolyticus (Ishibashi et al., 2000; Jones et al., 2012). The emergence of the first pandemic strain belonging to serovar O3:K6 (Okuda et al., 1997) supported the view that the serotype of V. parahaemolyticus is correlated with virulence. A multiplex polymerase chain reaction (PCR)-based O-antigen serotyping method has been adopted because other antiserum-based approaches are time consuming, expensive and can be associated with the risk of cross-reactions (Chen M. et al., 2012; Xu et al., 2014).

Molecular typing of V. parahaemolyticus, including pulsed field gel electrophoresis (PFGE) (Marshall et al., 1999), repetitive extragenic palindromic sequence PCR (REP-PCR) (Wong and Lin, 2001), and ribotyping (Bag et al., 1999), has been shown to be a useful tool for providing genetic relatedness information (Olive and Bean, 1999). Enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) has been proven useful for subtyping of V. parahaemolyticus strains with highly conserved repetitive intergenic consensus sequences (De Bruijn, 1992; Chen W. et al., 2012). Additionally, multilocus sequence typing (MLST), which is based on sequence analysis of selected housekeeping genes (e.g., recA, gyrB, dnaE, dtdS, pntA, pyrC, and tnaA), is becoming an important method for investigation of the evolution and epidemiology of V. parahaemolyticus owing to its high repeatability (González-Escalona et al., 2008; Banerjee et al., 2014).

V. parahaemolyticus is not frequently found in RTE foods, but is still considered hazardous in humans. Therefore, the objectives of the current study were as follows: (i) to determine the prevalence and contamination level of V. parahaemolyticus in RTE foods in China and (ii) to determine the genetic variation and phenotypic characteristics of V. parahaemolyticus isolates from RTE foods. The information generated in this study will provide insights into the distribution and population of V. parahaemolyticus across Chinese RTE foods and differentiation of V. parahaemolyticus strains on different RTE foods.

# MATERIALS AND METHODS

### Sample Collection

From November 2011 to May 2014, a total of 511 RTE food samples were collected from retail markets in 24 cities, covering most provincial capitals of China (**Figure 1**). The samples consisted of 371 deli meat samples, 97 cold vegetable dishes or noodles in sauce, and 43 fried rice or noodle samples. The samples were placed in sterile sealed plastic bags, transported to the laboratory in a cold box below 4◦C, and analyzed immediately.

#### Qualitative Detection

The bacteriological media used herein, unless indicated, were purchased from Guangdong Huankai Co. Ltd. (Guangzhou, China). For qualitative detection, the V. parahaemolyticus was isolated according to GB 4789.7-2013 for food microbiological examination of V. parahaemolyticus (National Food Safety Standards of China) with minor modifications. In brief, 25 g of each sample was homogenized for 60 s in a stomacher bag (Huankai Co. Ltd., Guangzhou, China) with 225 mL of alkaline peptone water (APW) containing 3% NaCl. Homogenates were incubated at 37◦C for 16−18 h. After incubation, a loopful from the top 1 cm was streaked onto thiosulfate-citrate-bile saltssucrose (TCBS) agar plates and incubated at 37◦C for 18−24 h. Presumptive colonies (green or blue green colonies, 2−3 mm in diameter) were streaked onto Chromogenic Vibrio Medium and

incubated at 37◦C for 24 h. The mauve colonies were selected for further V. parahaemolyticus identification by analysis of oxidase activity, Gram staining, 3.5% NaCl triple sugar iron (TSI) tests, halophilism tests, and API 20E diagnostic strips (Biomerieux Company, France).

#### MPN Enumeration (MPN) of *V. parahaemolyticus*

Enumeration of V. parahaemolyticus in RTE food samples was performed using the MPN method according to the Bacteriological Analytical Manual standard method (Kaysner and Depaola, 2004) and a previous study (Xu et al., 2014).

#### Detection of tdh and *trh* Genes