To evaluate whether easy-to-use dried reagent can replace the standard liquid reagent we compared the two methods using 14 reference strains. The method using the liquid reagent or using dried reagent were equally sensitive and specific when the results were judged using the turbidimetric system (**Table 1**). In addition, when the product of the reaction was judged by eye, the results using the dried reagent were clearer, due to the color change of the rehydrated reagent from brown to green, than that using the liquid reagent (**Figure 4A**). Furthermore, it was more difficult to judge by eye the results of *trh* (*trh*1 and *trh*2) detection than *tdh* detection using the liquid reagent. Conversely, judgment of color by eye of the results for all these genes was equally easy when the dried reagent was used (data not shown). Further, the positive results in the *tdh* LAMP assays obtained after the dried LAMP reagent was exposed to different temperatures (30, 40, 50, and 60◦C), showed the dried reagent was equally stable at all of these temperatures during the examination period (15 days).

### Stage II: Evaluation of the Enrichment Procedure Using Shellfish Change in the Enrichment Step for MPN Determination

We explored the possibility of finding a factor affecting the protocol in the enrichment step. We compared the MPN values after each of the three treatments (**Figure 1**, Steps 1–3) in the new protocol including the above modifications. Twenty-eight shellfish samples purchased in southern Thailand were examined for the MPN of *tdh*+ *V. parahaemolyticus* as described above (**Table 2**). The data clearly shows *tdh*+ *V. parahaemolyticus* is prevalent and the concentration is very high in these samples,

TABLE 1 | Comparison of liquid reagent and dried reagent for detection of the *tdh*, *trh*1 and *trh*2 genes in *Vibrio parahaemolyticus* using the turbidimetric system.

∗*Yamazaki et al. (2010).*

reagents. DNA template obtained using the boiling method from: (A) the pure culture of control strains examined for LAMP component at Stage I (Figure 2); (B) retail Thai shellfish samples examined at Stage III (Figure 2) by the recommended MPN-IMS-LAMP protocol (Figure 1). Positive (+) and negative (−) LAMP reaction directly judged by eye.

with the log MPN/10 g value ranging from 1.0 to 5.4. Hard clams gave higher log MPN than the other two shellfish. Regardless of the kind of the shellfish, the values in Step 3 were lower than those of the other two steps. When the MPN values of Steps 1 and 2 are compared, three samples (**Table 2**, sample no. 8–10) were higher in Step 1, two samples (**Table 2**, sample no. 1 and 2) were higher for Step 2 and 23 samples were the same. The average MPN values for all samples between Steps 1 and 2 did not differ significantly.

### Stage III: Evaluation of the Recommended MPN-IMS-LAMP

#### Sensitivity and MPN Accuracy Determination

Shellfish homogenates artificially contaminated with a reference strain (*tdh*+) at known concentrations were examined by the recommended protocol. Positive tubes in the threetubes MPN format were 0-0-0, 3-0-0, and 3-3-0, for the homogenates contaminated at 0.1, 1.0 and 10.0 CFU/ml, respectively. The sensitivity of the recommended MPN-IMS-LAMP was therefore determined to be 1 CFU/ml. Based on the 3-tubes MPN format results, the MPN values obtained were *<*0.03 (range: *<*0.005–0.09) MPN/ml, 0.23 (range: 0.04–1.2) MPN/ml, and 2.4 (range: 0.36–13.0) MPN/ml, respectively.

Examination of Retail Shellfish in Thailand and Japan Twenty-eight Thai samples were examined using liquid LAMP reagent in Stage II. Three (**Table 2**, sample designations 4, 21, and 25) of the 28 samples representing each shellfish group were also examined using dried LAMP reagent as in the new recommended protocol. The MPN values obtained at this stage were exactly the same, indicating the dried reagent had the same sensitivity as the liquid reagent as judged by the turbidimetric system. However, we confirmed that judgment by eye is easier when dried reagent is applied (**Figure 4B**).

Sixteen Japanese shellfish samples were also examined by the recommended new protocol (**Table 3**).

ples, with the MPN values being below the detection limit (*<*3 MPN/10 g). Two of the samples were positive for the *tdh* gene but the MPN values (15 and 23 MPN/10 g) were much lower than those obtained with Thai samples.

### Discussion

Our international research group attempted to develop an easy and sensitive method for quantitative detection of *tdh*+ *V. parahaemolyticus* in molluscan shellfish that can be applied in any

The *tdh* gene was not detected in 14 of the 16 sam-

TABLE 2 | Comparison of the MPN values of *tdh***+** *V. parahaemolyticus* obtained at the three Steps of the modified protocol**∗**.

∗*Explained in Figure 2.* † *MPN/10 g that differs between Steps 1 and 2.*

TABLE 3 | Examination of the Japanese shellfish for the MPN of *tdh***+** *V. parahaemolyticus*.

*The numbers in the parentheses indicate the numbers of the samples examined.*

part of the world; being practical and feasible even in resourceslimited and/or tropical countries. We previously reported an MPN-IMS-LAMP method that was shown to be more sensitive than the MPN-PCR-based method (Hara-Kudo et al., 2003; Jones et al., 2012; Tanaka et al., 2014). However, a problem of underestimation of the MPN values for some samples in a tropical environment was found as well as the technical practical inconvenience of the liquid LAMP reagent. In the current study, we improved the previously reported method using a series of technical modifications in the MPN and IMS components (**Figure 2**). In addition, we replaced the liquid reagent by the dried reagent in the LAMP component. As a result, much of the technical problems were solved (**Tables 2** and **3**; **Figure 4**).

While our study was in progress, a LAMP (targeting the *tlh* gene) and IMS (using nanoparticules targeting flagella) detection method for *V. parahaemolyticus* was reported (Zeng et al., 2014). However, CODEX 2011 recommends quantitative detection of pathogenic rather than total *V. parahaemolyticus* for risk assessment of *V. parahaemolyticus* in seafood (Food and Agriculture Organization World Health Organization of the United Nations [FAO/WHO], 2011). Along this line, our IMS method screens for clinically important *V. parahaemolyticus* by targeting all established K antigens and our LAMP method targets the *tdh* gene. Though, primer sets for *trh*1 and *trh*2 are available, variation in the *trh* gene sequence is widely observed in environmental strains is of major concern (Kishishita et al., 1992). A new *trh* LAMP primer set to overcome this issue is currently under development (Escalante-Maldonado and Nishibuchi, unpublished data).

The IMS component is an essential part of our protocol, therefore we needed to improve the efficiency of the IMS performance. *In vitro* experiments in Stage I confirmed that IMB incubation with constant shaking (Nou et al., 2006) for 30 min (Tanaka et al., 2014) enhances IMB-target association (data not shown). **Figure 3** shows that increasing the PickPen Operation time up to 3 min (1–3 and 2–3 min) increased CE value significantly. Increase in operation time after 3 min showed no significance difference in CE value where further extension of PickPen Operation time increases the workload. Taken together, we judged 3 min is the most effective PickPen Operation time. *In vivo* experiments conducted in Thailand employing the new protocol confirmed that these two modifications are a valuable contribution to the IMS performance (**Table 2**). The MPN values obtained after IMS application in this study are higher than the under-estimated MPN values reported in our previous study where similar shellfish samples were examined (Tanaka et al., 2014).

Another very valuable contribution to our recommended protocol is the application of the dried LAMP reagent. We utilized the dried reagent included in the commercially available kit for detection of two other pathogens. Results of the comparative experiments in Stage I indicate that both dried and liquid reagents are equally sensitive and specific for the LAMP assays for *V. parahaemolyticus* regardless of the primer set (**Table 1**). Evaluation of the stability of the dried reagent at different temperatures (30–60◦C) confirmed the dried reagent is very stable even at hightemperatures during the examination period, corroborating its utility even in tropical coastal areas of the world. Likewise, the dried reagent was proven useful because its transportation and storage do not necessarily require refrigerated or frozen conditions as confirmed during experiments in southern Thailand in Stage III.

Prevalence of infection by *tdh*+ *V. parahaemolyticus* was reported previously in southern Thailand. This prevalence is due to consumption of under-cooked molluscan shellfish, which is very common in southern Thailand (Laohaprertthisan et al., 2003). A risk assessment study conducted in southern Thailand reported low concentration of *tdh*+ *V. parahaemolyticus* in the bloody clam sold in the evening market (Yamamoto et al., 2008). However, pre-incubation of shellfish samples prior to examination may allow growth of *tdh*+ *V. parahaemolyticus* to a detectable level (Hara-Kudo et al., 2003; Yamamoto et al., 2008). Our previous study indicated that molluscan shellfish kept overnight and sold at the morning market contained relatively high levels of *tdh*+ *V. parahaemolyticus* (Tanaka et al., 2014). Under this condition, the under-estimation problem of the MPN values was raised. The current study examining similar shellfish samples solved the under-estimation problem. Comparative experiments in Stage II showed the problem is due at least in-part to the addition of the SPB enrichment step (**Figure 1**, Step 3). Among the MPN values observed at the three different steps, those of Step 3 were lower for all seafood samples examined (**Table 2**). SPB enrichment probably supported preferentially the growth of competing bacterial population rather than that of *tdh*+ *V. parahaemolyticus.*

The method yielding the highest MPN values is presumed to be most sensitive. We therefore compared the values obtained at Steps 1 and 2. The averages of the MPN values for these two steps were indistinguishable (**Table 2**). Difference in MPN values between Step 1 and Step 2 were observed only with five of 12 bloody clam samples. Two samples require special attention. They showed lower MPN values at Step 1 which were close to the detection limit. Application of IMS could avoid under-estimation of these values and assure the detection of the target.

### Conclusion

Modifications in the three components of the protocol (**Figure 2**) were critical to improve the MPN-IMS-LAMP. The underestimation problem was solved by modifying the IMS and excluding the SPB from the enrichment step. Introduction of the dried LAMP reagent made the method quicker, easier and allows its use at high environmental temperatures. We therefore recommend the modified MPN-IMS-LAMP for detection and quantitation of *tdh*+ *V. parahaemolyticus* as a universal method useful in tropical, subtropical, and temperate zones of the world.

### Author Contributions

Conceived and designed the experiments: OE-M and MN. Performed the experiments and analyzed data: OE-M and AK.

### References

Technical assistance: WY, VV, and YN. Interpretated and wrote the paper: OE-M and MN.

## Acknowledgments

We are grateful to Kensuke Ozawa, Fumio Gondaira, and Akira Oshima for valuable comments and technical assistance; and to Yohko Takeda, Jetnapang Kongrueng, Natthawan Sermwittayawong, and Pharanai Sukhumungoon and their graduate students for technical assistance. This research was supported, in-part, by Kakenhi Grant-in-Aid for Scientific Research (KAKENHI 24249038) and from the Japan Society for the Promotion of Sciences and a fund for Research on international cooperation in medical science, Research on global health issues, Health and Labor Science Research Grants, the Ministry of Health, Labor, Welfare of Japan (H26-KOKUI-SITEI-001), a fund from the GSS Program, a Kyoto University Program for Leading Graduate Schools, supported by the JSPS and MIXT, and a fund from Yakult Honsha Co. Ltd., Japan.

**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 © 2015 Escalante-Maldonado, Kayali, Yamazaki, Vuddhakul, Nakaguchi and Nishibuchi. 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.*

# Examination of clinical and environmental *Vibrio parahaemolyticus* isolates by multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA)

#### *Edited by:*

Indrani Karunasagar, Karnataka Veterinary Animal and Fisheries Sciences University, India

#### *Reviewed by:*

Volker Gürtler, RMIT University, Australia Swapan Banerjee, Health Canada, Canada

#### *\*Correspondence:*

Jessica L. Jones, Gulf Coast Seafood Laboratory, Division of Seafood Science and Technology, Food and Drug Administration, 1 Iberville Dr, Dauphin Island, AL 36528, USA<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received:* 27 March 2015 *Accepted:* 22 May 2015 *Published:* 10 June 2015

#### *Citation:*

Lüdeke CHM, Gonzalez-Escalona N, Fischer M and Jones JL (2015) Examination of clinical and environmental Vibrio parahaemolyticus isolates by multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA). Front. Microbiol. 6:564. doi: 10.3389/fmicb.2015.00564 Catharina H. M. Lüdeke1, 2, Narjol Gonzalez-Escalona3 , Markus Fischer 2 and Jessica L. Jones 1 \*

1 Gulf Coast Seafood Laboratory, Division of Seafood Science and Technology, Food and Drug Administration, Dauphin Island, AL, USA, 2 Hamburg School of Food Science, University of Hamburg, Hamburg, Germany, 3 Division of Microbiology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA

Vibrio parahaemolyticus is a leading cause of seafood-borne infections in the US. This organism has a high genetic diversity that complicates identification of strain relatedness and epidemiological investigations. However, sequence-based analysis methods are promising tools for these identifications. In this study, Multi-Locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) was performed on 58 V. parahaemolyticus isolates (28 of oyster and 30 of clinical origin), to identify differences in phylogeny. The results obtained by both methods were compared to Pulsed-Field Gel Electrophoresis (PFGE) patterns determined in a previous study. Forty-one unique sequence types (STs) were identified by MLST among the 58 isolates. Almost half of the isolates (22) belonged to a new ST and added to the MLST database. A ST could not be generated for 5 (8.6%) isolates, primarily due to an untypable recA locus. Analysis with eBURST did not identify any clonal complex among the strains analyzed and revealed 37 singeltons with 4 of them forming 2 groups (1 of them SLV, and the other a DLV). An established MLVA assay, targeting 12 total genes through three separate 4-plex PCRs, was successfully adapted to high resolution melt (HRM) analysis with faster and easier experimental setup; resulting in 58 unique melt curve patterns. HRM-MLVA was capable of differentiating isolates within the same PFGE cluster and having the same ST. Conclusively, combining the three methods PFGE, MLST, and HRM-MLVA, for the phylogenetic analysis of V. parahaemolyticus resulted in a high resolution subtyping scheme for V. parahaemolyticus. This scheme will be useful as a phylogenetic research tool and as an improved method for outbreak investigations for V. parahaemolyticus.

Keywords: *Vibrio parahaemolyticus*, phylogenetics, MLVA, MLST, HRM

### Introduction

Vibrio parahaemolyticus can cause acute gastroenteritis associated with consumption of raw or undercooked seafood (Nishibuchi and DePaola, 2005). Presently, this bacterium represents the most common cause for seafood-associated infections in the United States (Iwamoto et al., 2010). V. parahaemolyticus has shown a high rate of recombination and mutation which leads to a high genetic diversity (Gonzalez-Escalona et al., 2008). V. parahaemolyticus isolates are frequently characterized for their virulence gene profile, serotype, ribotype, and/or Pulsed-Field Gel Electrophoresis (PFGE) pattern for research studies and epidemiological investigations (Broberg et al., 2011; Jones et al., 2012; Paranjpye et al., 2012; Banerjee et al., 2014; Xu et al., 2015). However, there are several phylogenetic and evolutionary methodologies for differentiation of V. parahaemolyticus, such as Multi-Locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA), which may provide greater discrimination.

For this study, we selected two sequence-based typing methods, MLST and MLVA. MLST is based on sequence diversity of loci which are generally well-conserved, or under purifying selection, as is the case with housekeeping genes. MLST is a frequently used typing method for many organisms and the development of a public database (PubMLST) has simplified sequence analysis and identification of evolutionary relationships within bacterial species (Maiden et al., 1998; Perez-Losada et al., 2013). MLST was selected to examine the current strain collection based on the previous success of the technique for characterizing diverse environmental and clinical V. parahaemolyticus isolates (Gonzalez-Escalona et al., 2008) and to add a diverse set of isolates to the MLST database. MLVA has also been used to distinguish isolates with little genetic variation. MLVA uses PCR for amplification of size polymorphisms in several Variable-Number Tandem-Repeat (VNTR) loci (Lindstedt, 2005). The VNTRs are highly polymorphic and are well suited for differentiation of bacterial isolates (Lindstedt, 2005; van Belkum, 2007). MLVA was selected for use in this study as it is able to differentiate between indistinguishable PFGE patterns for V. parahaemolyticus (Hayat et al., 1993; Harth-Chu et al., 2009) or identical MLST sequence types (STs) in other organisms (Maiden et al., 1998, 2013). Recently, MLVA was used for epidemiological analysis for discrimination of clinical and environmental V. parahaemolyticus isolates with indistinguishable Direct Genome Restriction Enzyme Analysis (DGREA) patterns (Harth-Chu et al., 2009; Garcia et al., 2012). Generally, the PCR products from MLVA are separated by agarose gel or capillary electrophoresis (CE) (Lindstedt et al., 2013). However, differentiation of amplification products using high resolution melt (HRM) analysis has been described for MLVA assays in other organisms (Fortini et al., 2007).

The objective of this study was to evaluate the combined use of MLST and the HRM-MLVA sequence-based methods for discrimination of environmental and clinical V. parahaemolyticus isolates previously characterized by other fingerprinting methods, such as PFGE (Ludeke et al., 2014). MLST was selected to identify phylogenetic relationships while MLVA was applied with the hypothesis that it will further discriminate isolates with identical STs. In order to achieve this objective, an HRM-MLVA protocol for rapid and simple characterization of V. parahaemolyticus isolates was developed. This is the first study, to the authors' knowledge, to use the combined approach of the subtyping methods MLST, HRM-MLVA, and PFGE for differentiation of V. parahaemolyticus isolates.

### Material and Methods

### Bacterial Strains

For the MLST and MLVA analysis, 58 V. parahaemolyticus isolates were selected; among those were 28 environmental (oyster) and 30 clinical isolates. Isolates were selected to represent multiple collection states and serotypes (**Table 1**). Each isolate was inoculated into Luria Bertani broth with 1% NaCl and incubated with shaking overnight at 35◦C. Afterwards, 1 mL of the overnight culture was transferred to a 1.5 mL microcentrifuge tube, heated to 100◦C for 10 min, and placed in ice for 5 min. The samples were stored at −20◦C until used as a PCR template.

### Multi-Locus Sequence Typing (MLST)

MLST was performed as described in the protocol for Vibrio parahaemolyticus (Gonzalez-Escalona et al., 2008). The PCR products were purified using a PCR purification kit (Qiagen, Valencia, CA) with a total elution volume of 25µL. The purified samples were sequenced on an ABI 3730 × l sequencer at McLab (South San Francisco, CA). Sequences were analyzed with BioEdit software 7.1.9 (Abbott, Carlsbad, CA). The allelic and sequence type (ST) identification was determined using the MLST database (http://pubmlst.org/vparahaemolyticus). In the cases where whole genome sequence data was available, sequences were de novo assembled using CLC Genomics Workbench software 7.0.3 (CLCbio, Germantown, MD) and consensus assemblies submitted to the MLST database for allelic and ST identification.

For identification of clonal complexes, eBURST version 3 was used (http://eburst.mlst.net/). As reported previously, two different STs were considered single-locus variant (SLV) when they differed by a single locus; a double-locus variant (DLV) has two different loci (Gonzalez-Escalona et al., 2008). To be part of a clonal complex, isolates needed to share at least six out of seven alleles.

The minimum evolution tree of the concatenated sequences of the seven loci was built based on the method of Kimura-2 parameter in Mega 6 (Tamura et al., 2013). The ratio between the number of synonymous and non-synonymous substitutions, showing the type of selection at each locus, was calculated using the method of Nei and Gojobori in Mega 6. The hypotheses of neutrality (dS = dN), purifying selection (dS/dN > 1), and positive selection (dS/dN < 1) were tested.

### Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA)

Three multiplex real-time-PCR assays using HRM curve analysis were performed with the LightCycler R 480 High Resolution

#### TABLE 1 | Isolates used in this study and their sequence types (ST).

(Continued)

#### TABLE 1 | Continued

aut = untypable.

bPEI = Prince Edward Island.

cnovel allele type and novel ST in bold.

Melting Master Kit (Roche, Indianapolis, IN). The primer sequences utilized in this study are listed in **Table 2** and have been previously described (Harth-Chu et al., 2009). These primers were utilized as unmodified oligonucleotides since HRM was used as the detection method rather than CE. Each reaction mixture for the multiplex PCR A and B (Multi A and B) had a final volume of 20µL and consisted of: 1X master mix solution (Roche), 2 mM MgCl2 (Roche), 0.2µM each primer (Integrated DNA Technologies; IDT, Coralville, IA), and 5µL boiled template. For multiplex PCR C (Multi C), reaction concentrations were the same as for Multi A and B, except for primer pair VP2-07; 0.4µM of each primer was used. The temperature program was as described previously for Multi A and B (Harth-Chu et al., 2009): Initial denaturation at 95◦C for 15 min, followed by 20 cycles of a touchdown PCR consisting of denaturation at 94◦C for 30 s, annealing starting at 63◦C and decreasing 0.2◦C per cycle for 1.5 min, and elongation at 72◦C for 1 min. A final 10 cycles of denaturation at 94◦C for 30 s, annealing at 59◦C for 1.5 min, and elongation at 72◦C for 1 min was used. The cycling program of Multi C included the same initial denaturation as for Multi A and B; cycling consisted of 30 cycles of denaturation at 94◦C for 30 s, annealing at 61◦C for 1.5 min, and elongation at 72◦C for 1 min. A final annealing step at 60◦C for 30 min completed both cycling programs. The melt curve analysis was performed using the Roche High Resolution Melting Kit protocol. The temperature program for the HRM analysis started at 95◦C for 1 min with a ramp of 4.4◦C per s, followed by 40◦C for 1 min with a ramp of 2.2◦C per s, 60◦C for 1 s with a ramp of 4.4◦C per s and a continuous step at 95◦C. For the PCR amplification and HRM analysis a LightCycler R 480 (Roche) was used.

Due to overlapping peaks in Multi A and C, the presence of the target loci VNTR5 and VNTR7, VNTR3, and VP2-07 were confirmed using simplex real-time-PCR for each gene with the same PCR reaction conditions and cycling parameters as described for the multiplex, but with omission of the three other primer sets. The melt curves were analyzed with BioNumerics software 6.6 with a customized script (Applied Maths, Austin, TX). This script compares the melting curves of each multiplex PCR, as well as a combination of all curves from the three multiplex PCRs. The combined dendrogram of all three multiplex PCR was built based on the Pearson correlation of average trend curves in BioNumerics as well. This dendrogram was also converted to a rendered rooted tree.

### Results

### Multi-Locus Sequence Typing (MLST)

From the 58 V. parahaemolyticus isolates analyzed, MLST resulted in 41 different STs (**Table 1**). Four (6.9%) and one (1.7%) of the isolates were untypeable for recA and pntA, respectively. For those strains, no ST could be assigned. Twelve (42.9%) of the oyster and ten (40.0%) of the clinical isolates were a novel ST. The most frequently identified STs were ST36 (13.3%) and ST3 (10.0%) in clinical isolates and ST32 (10.7%), ST313 (7.1%), and ST676 (7.1%) in oyster isolates.

All loci showed ratios of synonymous and non-synonymous substitutions (dN/dS) below 1 and therefore under purifying selection, as expected for housekeeping genes. eBURST analysis divided the 53 isolates for which a ST could be identified into 37 singletons and two groups: one SLV and one DLV (data not shown). No clonal complexes could be identified; demonstrating that none of the STs identified in this study share more than six alleles and, therefore, belong to different V. parahaemolyticus lineages.

A minimum evolution tree was constructed using the concatenated sequences of each allele (**Figure 1**). The isolates grouped into two main clusters, or lineages (I and II), with each lineage containing ST of clinical and oyster isolates. Isolates with the same ST generally had the same serotype; ST631 isolates possessed serotype O11:Kut, ST676 were serotype O8:Kut, ST36 were serotype O4:K12 or O4:Kut, and ST313 were serotype O1:Kut. However, the three ST3 isolates had all different serotypes.

### Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA)

The 58 V. parahaemolyticus isolates from this study were further analyzed by MLVA with HRM analysis. Three multiplex PCRs covering twelve different loci were used. Each multiplex PCR generated reproducible melting curve profiles of select isolates (data not shown). **Table 3** shows the percentage of isolates from which each target sequence in the MLVA scheme was amplified. From the Multi A, VP2-03, VPTR7, or VP1-11 was not amplified in 10% and 14.3%, 46.6% and 39.3%, or 3.3% and 3.6% of clinical and oyster isolates, respectively; VPTR5 was amplified from all isolates. In Multi B, VP1-10 or VPTR8 was not present in 96.7% and 17.9%, 23.3% and 17.9% of clinical and oyster isolates, respectively; VPTR1 and VP1-17 was amplified in all isolates with the exception of 6.7% of clinical isolates not amplifying VPTR1. From Multi C, VPRT6 was not amplified in 83.3% and 85.7% of clinical and oyster isolates, respectively. All isolates amplified VPTR4, VPTR3, and VP2-07, with the exception of 3.6% of oyster isolates for VPTR4.

Using the trend curves of each individual multiplex PCR, dendrograms were constructed (data not shown). Each multiplex PCR generally clustered the isolates based on their serotypes and ST. The individual multiplex PCR dendrograms demonstrated the ability of MLVA to differentiate between the same ST (data not shown).

#### Comparison of MLST, MLVA, and PFGE

Based on the hypothesis MLVA can differentiate isolates with the same ST and PFGE pattern, these isolates' MLVA patterns were compared to the MLST data, as well as previously published PFGE results (Ludeke et al., 2014). To compare these methods, dendrograms were built of the combined melting curves from the three MLVA multiplex PCRs and correlated to the PFGE cluster and ST of each isolate. MLVA allowed further differentiation of isolates with identical STs and PFGE clusters (**Figure 2**). Specifically, the isolates with ST3 and ST36 share the same PFGE cluster, but were distinguishable by MLVA melting curve profiles (**Figure 3**). The dendrogram with only ST3 and ST36 isolates showed ST-specific clusters, but separation within those clusters based on the combined melting curves of MLVA.

### Discussion

This study analyzed 58 V. parahaemolyticus isolates by MLST and a newly-developed HRM-MLVA assay to investigate the

relatedness of the isolates. The seven gene MLST protocol reported in a previous study was employed in this study (Gonzalez-Escalona et al., 2008). A different MLST method using ten housekeeping genes was described (Yan et al., 2011). Both methods report the same level of discrimination; however, the seven gene protocol was selected due to the availability of a public repository for the data (http://pubmlst.org/vparahaemolyticus), which allows comparisons to be made with isolates analyzed by other researchers. With 22 novel STs, this study substantially contributed to the diversity in the MLST database. Four of our isolates were untypeable for recA. In a recently published study, a V. parahaemolyticus strain contained a recA gene that was fragmented by a 30 kb DNA insertion (Gonzalez-Escalona et al., 2015). It is possible a similar insertion exists in the recA gene of

some of the strains in the current study, but further analysis is needed to confirm.

ST3, ST32, and ST36 were the STs that occurred most often in our isolates, as well as in the public database. In our study, the fourth most frequent ST observed was ST676, which is one of the novel STs reported here. Two of these STs (ST3 and ST36) have been reported as part of clonal complexes CC3 and CC36, respectively (Gonzalez-Escalona et al., 2008), and correlated with outbreaks in multiple countries, including the US and Chile (Fuenzalida et al., 2006; Martinez-Urtaza et al., 2013). ST3 was identified as the ancestral ST of CC3 with ST27, ST42, and ST51 as SLVs. None of other STs from CC3 were identified in this study. A previous study using MLST on a set of clinical and environmental isolates from the Pacific Northwest Region of the United States showed that some environmental isolates were of ST3, suggesting a higher potential for virulence than other environmental isolates (Turner et al., 2013). In this study, three clinical isolates were ST3 and no direct relationship to environmental or other clinical clades were observed.

We developed an HRM-MLVA method, based on an existing MLVA method that uses CE, for subtyping of V. parahaemolyticus and to differentiate between similar PFGE patterns or STs. The CE method provides the actual number of tandem repeats while HRM does not. However, the HRM analysis still recognizes allelic variants and is able to distinguish between otherwise indistinguishable strains. For example, the ST3, ST32, and ST36 strains in this study also shared common PFGE profiles, but each isolate produced a unique HRM curve combination. This resolving power of HRM-MLVA is similar to previous reports of CE-MLVA, where Chilean isolates which shared a DGREA pattern and were ST3 could be differentiated by CE-MLVA (Gonzalez-Escalona et al., 2008; Harth-Chu et al., 2009). These data demonstrate that the HRM-MLVA method developed in this study provides similar discrimination as previously reported for the CE-MLVA method and is suitable examination of V. parahaemolyticus isolates. Additionally, the use of HRM-MLVA on the LightCycler R 480 saves the step of electrophoretic detection, thus minimizing the potential for cross contamination by PCR amplicons during that additional handling step.

The loci amplified in this study by MLVA are coding proteins such as a putative hemolysin (VPTR4) and putative collagenase (VPTR3) (Kimura et al., 2008). Most of these genes, could be amplified from the current strain selection with the exception of VPTR7 (Multi A), VP1-10 (Multi B), and VPTR6 (Multi C). Nearly all clinical isolates failed to amplify the VP1-10 locus and approximately half failed to amplify the locus VPTR7. A failure to amplify VPTR7 from some shellfish isolates has been reported previously (Harth-Chu et al., 2009). Fewer than 20% of isolates amplified VPTR6 in this study. Additionally, a previous study found VPTR6 to be one of the few loci with high genetic diversity (Harth-Chu et al., 2009). Together, these data suggest that these two loci might not be suitable targets for future MLVA studies, especially for environmental isolate screening. Nonetheless, the HRM-MLVA method successfully discriminated between otherwise indistinguishable V. parahaemolyticus isolates.

Previous studies have employed multiple methods for characterization and subtyping of V. parahaemolyticus isolates from various sources. For example, DePaola et al. used a combination of serotyping and ribotyping to identify types more highly associated with clinical isolates (DePaola et al., 2003). Turner et al. utilized REP-PCR, as fingerprint-based subtyping method, followed by MLST to identify region-specific clades of V. parahaemolyticus (Turner et al., 2013). Banerjee et al. employed PFGE, MLST, serotyping, and ribotyping to examine clinical V. parahaemolyticus isolates and provided combinatorial analysis to determine relatedness (Banerjee et al., 2014). However, none of these previous studies utilized the combination of two highly discriminatory, sequence-based methods as does the current study.

This combined method approach described here using PFGE, MLST, and MLVA has not been previously reported for discrimination of V. parahaemolyticus isolates, but is similar to approaches used for other organisms: for example, the Centers for Disease Control and Prevention has used a combination of PFGE followed by MLVA for epidemiological investigations of STEC O157 to discriminate between closely related isolates (Hyytia-Trees et al., 2006). Also, a combination of MLST and MLVA has been used as an epidemiological tool for distinguishing between clones of Listeria monocytogenes (Chenal-Francisque et al., 2013).

This study used a combined method approach to increase the discrimination of V. parahaemolyticus isolates. MLST was able to determine the identity and the phylogenetic relatedness of the isolates in this collection. As hypothesized, the developed HRM-MLVA method further refined the relationship of isolates by being able to distinguish between isolates with indistinguishable PFGE groupings or STs. Our data demonstrates

correlation and the unweighted pair group method using arithmetic averages (UPGMA). Isolates originated from oysters starting with "FDA," isolates from clinical origin labeled with "CDC." The PFGE pattern designations are as previously reported (Ludeke et al., 2014).

that a combination of PFGE, MLST, and HRM-MLVA would be the most suitable approach for outbreak and evolutionary investigations of V. parahaemolyticus, due to the high resolution provided. In instances where further discrimination is needed, and if available, next generation sequence data could be used to determine relatedness or to generate subtyping results in silico.

### References

### Acknowledgments

This project was supported by an appointment of CL to the Research Fellowship Program for the Center for Food Safety and Applied Nutrition administered by the Oak Ridge Associated Universities through a contract with the FDA.

environmental Vibrio parahaemolyticus isolates. Foodborne. Pathog. Dis. 11, 520–528. doi: 10.1089/fpd.2013.1728

**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 © 2015 Lüdeke, Gonzalez-Escalona, Fischer and Jones. 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.

## Isolation, characterization, and antibiotic resistance of *Vibrio* spp. in sea turtles from Northwestern Mexico

Alan A. Zavala-Norzagaray 1, 2, A. Alonso Aguirre3 , Jorge Velazquez-Roman4 , Héctor Flores-Villaseñor 4 , Nidia León-Sicairos 4, 5, C. P. Ley-Quiñonez 2 , Lucio De Jesús Hernández-Díaz 1 and Adrian Canizalez-Roman4, 6 \*

#### *Edited by:*

Pendru Raghunath, Dr. VRK Women's Medical College, India

#### *Reviewed by:*

Sheng Chen, Hong Kong Polytechnic University, Hong Kong Learn-Han Lee, Monash University Malaysia, Malaysia

#### *\*Correspondence:*

Adrian Canizalez-Roman, Research Unit, School of Medicine, Autonomous University of Sinaloa, Campus 2. Ave. Cedros y Calle Sauces. Fracc. Los Fresnos, 80246 Culiacán, Mexico<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received:* 08 March 2015 *Accepted:* 12 June 2015 *Published:* 25 June 2015

#### *Citation:*

Zavala-Norzagaray AA, Aguirre AA, Velazquez-Roman J, Flores-Villaseñor H, León-Sicairos N, Ley-Quiñonez CP, Hernández-Díaz LDJ and Canizalez-Roman A (2015) Isolation, characterization, and antibiotic resistance of Vibrio spp. in sea turtles from Northwestern Mexico. Front. Microbiol. 6:635. doi: 10.3389/fmicb.2015.00635 1 Programa Regional Para el Doctorado en Biotecnología, Facultad de Ciencias Químicas Biológicas, Universidad Autonoma de Sinaloa, Culiacán, México, 2 Departamento de medio ambiente y desarrollo comunitario, Instituto Politécnico Nacional, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional - Sinaloa, Guasave, Mexico, 3 Department of Environmental Science and Policy, George Mason University, Fairfax, VA, USA, 4 Research Unit, School of Medicine, Autonomous University of Sinaloa, Culiacán, Mexico, 5 Pediatric Hospital of Sinaloa, Culiacán, Mexico, 6 The Sinaloa State Public Health Laboratory, Secretariat of Health, Culiacán, Mexico

The aerobic oral and cloacal bacterial microbiota and their antimicrobial resistance were characterized for 64 apparently healthy sea turtles captured at their foraging grounds in Ojo de Liebre Lagoon (OLL), Baja California Sur (BCS), Mexico (Pacific Ocean) and the lagoon system of Navachiste (LSN) and Marine Area of Influence (MAI), Guasave, Sinaloa (Gulf of California). A total of 34 black turtles (Chelonia mydas agassizii) were sampled in OLL and eight black turtles and 22 olive ridley turtles (Lepidochelys olivacea) were sampled in LSN and MAI, respectively from January to December 2012. We isolated 13 different species of Gram-negative bacteria. The most frequently isolated bacteria were Vibrio alginolyticus in 39/64 (60%), V. parahaemolyticus in 17/64 (26%), and V. cholerae in 6/64 (9%). However, V. cholerae was isolated only from turtles captured from the Gulf of California (MAI). Among V. parahaemolyticus strains, six O serogroups and eight serovars were identified from which 5/17 (29.4%) belonged to the pathogenic strains (tdh+ gene) and 2/17 (11.7%) had the pandemic clone (tdh+ and toxRS/new+). Among V. cholerae strains, all were identified as non-O1/non-O139, and in 4/6 (66%) the accessory cholera enterotoxin gene (ace) was identified but without virulence gene zot, ctxA, and ctxB. Of the isolated V. parahaemolyticus, V. cholerae, and V. alginolyticus strains, 94.1, 33.4, and 100% demonstrated resistance to at least one commonly prescribed antibiotic (primarily to ampicillin), respectively. In conclusion, the presence of several potential (toxigenic) human pathogens in sea turtles may represent transmission of environmental microbes and a high-risk of food-borne disease. Therefore, based on the fact that it is illegal and unhealthy, we discourage the consumption of sea turtle meat or eggs in northwestern Mexico.

Keywords: antibiotic resistance, bacterial diversity, *Chelonia mydas agassizii*, *Lepidochelys olivacea*, Mexico, sea turtles, *Vibrio spp.*

### Introduction

Sea turtles are air-breathing, marine reptiles of the order Testudines. The advances in their medical management, the studies on causes of morbidity and mortality during stranding events, and the efforts to conserve them, have increased in recent years. Despite these efforts, six of the seven species of sea turtles are classified as threatened or endangered by IUCN Red List of Threatened Species (IUCN, 2014). Sea turtles can be considered excellent sentinel species of marine ecosystem health due to their ecological and physiological characteristics, including long life spans, long period of time to reach sexual maturity, and high site fidelity to near-coastal feeding habitats (Aguirre and Lutz, 2004). Sea turtles appear to be highly susceptible to biological and chemical insults despite their robust appearance (Lutcavage et al., 1997). As with other marine vertebrate species, sea turtles are threatened by increasing anthropogenic activities including fisheries bycatch; illegal traffic of meat, eggs, and their parts; coastal development; various forms of plastic; global environmental change; and, environmental pollution (Aguirre and Lutz, 2004).

The presence of many contaminants in Northwestern Mexico is related to agricultural runoff. For example, methoxychlor, endrin, and heptachlor levels in the Navachiste-Macapule lagoon system suggested that these compounds were continuously applied although their use is forbidden (Montes et al., 2012). Several recent studies evaluating heavy metals and their potential impact on sea turtles in Northwestern Mexico have been reported (Frías-Espericueta et al., 2006; Ley-Quinonez et al., 2011; Ley-Quiñónez et al., 2013; Zavala-Norzagaray et al., 2014). The region encompasses the northern nesting and feeding distribution for black (Chelonia mydas agassizii) and olive ridley (Lepidochelys olivacea) turtles (Ley-Quiñónez et al., 2013; Aguilar-Gonzalez et al., 2014; Zavala-Norzagaray et al., 2014). The effects of heavy metals and other environmental contaminants in sea turtles have been previously documented as one of the potential synergic etiologies of marine turtle fibropapillomatosis (Aguirre et al., 1994, 2006; Lutcavage et al., 1997; Aguirre and Lutz, 2004). In addition, contaminant loads can increase the incidence of other diseases and could affect various functional processes (Camacho et al., 2013) representing a serious threat to dwindling sea turtle populations (Garcia-Fernandez et al., 2009).

Although there is no food safety microbiology for the consumption of sea turtle meat or eggs, this illegal practice is common in countries with coastal areas worldwide. The health effects of humans consuming sea turtles infected with zoonotic pathogens have been reported (Aguirre et al., 2006). For example, Vibrio mimicus in Costa Rica (Campos et al., 1996), V. cholerae in China (Lu et al., 2006), and Salmonella chester in Australia (O'grady and Krause, 1999) were associated to human disease by consumption of sea turtle meat and/or eggs. Therefore, it is important to effectively communicate accurate information regarding the potential human health hazards associated with the consumption of sea turtles and their eggs in areas where this practice is common (Aguirre et al., 2006).

Many bacteria have been identified as the cause of diseases in marine turtles kept in captivity (Chuen-Im et al., 2010; Arena et al., 2014). In addition, many of these bacteria may be pathogenic to humans (Warwick et al., 2013). Other bacteria including Aeromonas hydrophila, V. alginolyticus, Pseudomonas fluorescens, Flavobacterium spp., and Bacillus spp. are common bacterial microbiota in sea turtles from Hawaii and Australia and are associated with other diseases such as ulcerative stomatitis, obstructive rhinitis-pneumonia complex and fibropapillomatosis (Glazebrook and Campbell, 1990; Glazebrook et al., 1993; Aguirre et al., 1994). Among these bacteria, Vibrio spp. are commonly found, naturally, in aquatic environments and can cause infections to humans (Chowdhury et al., 1989; West, 1989; Chakraborty et al., 1997). Particularly, V. parahaemolyticus infections have increased globally; they are usually associated with eating raw or undercooked sea products (Nair et al., 2007; Velazquez-Roman et al., 2012, 2014; Hernández-Díaz et al., 2015). To the best of our knowledge, there are no reports of the presence of the bacterial diversity linked to disease in sea turtles in Mexico.

The aim of this study was to identify, characterize, and determine antibiotic resistance of potentially pathogenic bacteria isolated from oral and cloacal swabs from black turtles and olive ridley turtles in northwestern Mexico.

### Materials and Methods

### Study Site

During January to December 2012, sea turtle surveys were conducted in selected feeding grounds in the states of Baja California Sur (BCS) and Sinaloa (SIN). Ojo de Liebre Lagoon (OLL) (27.7500◦ N, 114.2500◦ W) is located on the Pacific coast near the border between BCS and Baja California and it is part of the El Vizcaino Biosphere Reserve. Eelgrass (Zostera marina) and several species of benthic macroalgae are abundant in the lagoon (Lopez-Castro et al., 2010). The highly productive waters of BCS have been revered for decades and recognized for centuries for the abundance and diversity of charismatic megafauna they attract (Lopez-Castro et al., 2010; Micheli et al., 2012). SIN has 16 coastal lagoons with mostly surrounded by irrigation districts where commercial fisheries and aquaculture farms represent important economic activities (Hernández-Cornejo et al., 2005; Gonzalez-Farias et al., 2006; Aguilar-Gonzalez et al., 2014). The Navachiste-Macapule lagoon (25.4–25.7◦ N and 108.85–108.55◦ W) is a complex coastal system with an approximate area of 24,000 ha. It is located in the municipality of Guasave, in the southeast coast of the Gulf of California (Montes et al., 2012). This lagoon has a great ecological and economic importance, as it supports a variety of oyster (Crassostrea virginica), clam (Protothaca staminea), mullet (Mugil cephalus), mojarra (Gerres cinereus), puffer (Arthron hispidus), snapper (Pagrus auratus), jewfish (Argyrosomus hololepidotus), and snook (Centropomus undecimalis) fisheries, as well as intensive shrimp (Penaeus vannamei) aquaculture activities (Orduna-Rojas and Longoria-Espinoza, 2006; Aguilar-Gonzalez et al., 2014).

### Sample Collection

Sea turtles were captured unharmed using fishing nets and snorkel equipment (Ley-Quiñónez et al., 2013) and transported to a "floating dock" for examination, sampling and determination of morphometric parameters. All turtles were released alive and unharmed. Two nasopharyngeal and cloacal swabs were collected from each turtle for microbiology. The swabs were placed in alkaline peptone water at pH 8.5 (APW) for Vibrio spp. and in buffered peptone water pH 7.2 (BPW) for Enterobacteriacea; then transported to the School of Medicine laboratory at the Autonomous University of Sinaloa for bacteriological isolation and identification.

### Isolation and Identification of Bacterial Strains

For Vibrio spp., all the nasopharyngeal and cloacal swabs were placed in APW and streaked onto thiosulfate citrate bile salts sucrose agar (TCBS; Becton-Dickinson, USA), and CHROMagar Vibrio, (CHROMagar Paris, France). The plates were incubated overnight at 37◦C. From each plate, green and yellow colonies in TCBS or blue and violet colonies in CHROMagar Vibrio exhibiting diverse morphology were transferred to TSA-2% NaCl agar for purity. These plates were incubated overnight at 37◦C and proceeded with identification using a single isolated colony. Each colony was examined by using the oxidase test and all biochemical tests described in the Bacteriological Analytical Manual of the Food and Drug Administration for Vibrio (Kaysner and De Paola, 2004; Canizalez-Roman et al., 2011). At least three typical colonies of V. parahaemolyticus and V. cholerae were isolated from each plate and subjected to identification by biochemical test and PCR. After identification of V. parahaemolyticus, V. cholera, and V. alginolyticus a single colony from each sample was used to continue the analysis (serotyping, virulence genes or antibiotic susceptibility testing). For Enterobacteriacea, specimens were placed in BPW and streaked onto Salmonella-Shigella, Hektöen and McConkey agar (Becton-Dickinson, USA). The plates were incubated overnight at 37◦C. The presumptive colonies were transferred to TSA agar for purity. These plates were incubated overnight at 37◦C and proceeded with identification using a single isolated colony. Each colony was examined by using the biochemical test (Citrobacter freundii, E. coli, Edwarsiella spp., Aeromonas, Plesiomonas, Morganella or Proteus, and Providencia) described in the Bacteriological Analytical Manual of the Food and Drug Administration (Andrews and Jacobson, 2013; Andrews et al., 2014; Feng et al., 2014).

### PCR Assays

PCR assays were performed in a 25µL volume consisting of 1X GoTaq green master mix (Promega), primers targeting the tl gene, the pR72H plasmid and the tdh and trh genes, the toxRS and orf8 pandemic marker genes for Vibrio parahaemolyticus as previously described (Velazquez-Roman et al., 2012; Hernández-Díaz et al., 2015). Vibrio cholerae O1 and O139 were further confirmed for the presence of VC, rfbO1, O139, genes, and the ctxA, ctxB, zot, and ace toxigenic genes (Albert et al., 1997; Sarkar et al., 2002; Di Pinto et al., 2005; Goel et al., 2007) and 0.5µg of purified genomic DNA template, with the remaining volume consisting of molecular biology grade water. PCR was routinely conducted in a Thermal Cycler C1000 (Bio-Rad Laboratories, Hercules, California). Ten microliter aliquots of each amplification product were separated by electrophoresis in a 2% agarose gel. Ethidium bromide staining (0.5 mg/ml) allowed for the visualization of DNA fragments with a digital imaging system (model E1 logia 100 imaging system; Kodak). The sizes of the PCR fragments were compared against a 50-bp DNA ladder (Promega DNA step ladder). To further identify diarrheagenic E. coli strains within our E. coli isolates, a protocol of sequential multiplex, duplex and single PCR reactions was used according to a previously published protocol work (Canizalez-Roman et al., 2013).

### Serotyping

V. cholerae serotyping was performed by using V. cholerae O1–specific polyvalent rabbit antiserum and O139-specific polyvalent rabbit antiserum obtained from the National Institute of Epidemiological Reference (InDRE), Mexico. The microagglutination test was used to determine the serogroup O1, Ogawa and Inaba, and serogroup O139 as described in the Bacteriological Analytical Manual of the Food and Drug Administration for Vibrio (Kaysner and De Paola, 2004). Furthermore, serotyping of V. parahaemolyticus isolates was performed by using a commercially available V. parahaemolyticus antiserum test kit (Denka Seiken, Tokyo, Japan) with O1–O11 antisera and 71 K antisera according to the manufacturer's instructions. Briefly, strains were grown overnight at 37◦C on LB agar containing 3% NaCl. A pool of colonies was suspended in 1 mL of saline and then split in two 500µl aliquots. For serotyping, an aliquot was heated up to 121◦C for 1 h for O serotyping; if the serotype could not be obtained, the bacterial lysate was heated for an additional hour and then used for O serotyping. The second aliquot was used for serotyping based on the K antigen.

### Antibiotic Susceptibility Testing

All isolates of V. parahaemolyticus, V. cholerae, and V. alginolyticus were tested for antimicrobial susceptibility by a standard disc diffusion method on Mueller–Hinton agar. The protocol was performed as follows: fresh cultures were inoculated into LB broth and incubated until they reached an optical density equal to a MacFarland 0.5 standard. Bacterial cultures were then plated onto Mueller–Hinton agar and, then antibiotic disks (BD BBL, Franklin Lakes, NJ) were placed in a sterile environment. The plates were incubated at 37◦C for 18–20 h. The diameters (in millimeters) of clear zones of growth inhibition around each antimicrobial agent disks were measured using a precision digital caliper (Absolute, Mitutoyo, Japan). Each bacterial species was classified as Resistant (R), Intermediately Resistant (I), or Susceptible (S) according to guidelines developed by the Clinical Laboratory Standard Institute (CLSI, 2011). The following antibiotics sensidisc (BD BBL, Sensi-Disc, Becton, Dickinson and Company, USA), with their concentrations given in parentheses, were tested including ampicillin (10µg), tetracycline (30µg), trimethoprim– sulfamethoxazole (1.25µg/23.75µg), chloramphenicol (30µg), nalidixic acid (30µg), ciprofloxacin (5µg), ceftazidime (30µg), gentamicin (10µg), and cefotaxime (30µg). The following V. parahaemolyticus strains were used as a control organism: ATCC 17802, (tdh−) and multidrug resistant strain 727 (Leon-Sicairos et al., 2009).

### Results

### Identification of Bacterial Isolates

From January to December 2012, 34 black turtles (C. mydas agassizii) were sampled in OLL, and eight black and 22 olive ridley (L. olivacea) turtles were captured in LSN and MAI, respectively (**Figure 1**). A total of 82 bacterial isolates (42 from black turtles and 40 from olive ridley turtles) were identified (**Table 1**). According to the geographic site of sampling, 33 (40.3%) and 49 (59.7%) bacterial isolates from BCS and from SIN were detected, respectively (**Table 1**).

Nine different microorganisms were isolated from olive ridley turtles including Citrobacter freundii, E. coli, Edwarsiella spp., V. alginolyticus, V. cholerae, V. fluvialis, V. furnisii, V. parahaemolyticus, and Vibrio spp. Eight different microorganisms were isolated from black turtles in BCS including Aeromonas or Plesiomonas, Citrobacter, E. coli, Morganella, and Proteus, Providencia, V. alginolyticus, V. parahaemolyticus, and Vibrio spp. Only three microorganisms were isolated from black turtles including V. fluviales, V. alginolyticus, and V. parahaemolyticus in Sinaloa (**Table 1**).

Overall, the predominant isolates in descending order of frequency were: V. alginolyticus (47.5%, 39/82), V. parahaemolyticus (20.7%, 17/82), V. cholerae (7.3%, 6/82) and other bacteria (25.6%, 21/82) (**Table 1**). Vibrio alginolyticus and V. parahaemolyticus were isolated in 62% (21/34) and 3% (1/34) from black turtles captured in BCS, respectively. However, in SIN, V. alginolyticus were isolated in 75% (6/8) and 55% (12/22), and V. parahaemolyticus were isolated in 25% (2/8) and 63% (14/22) from black turtles and olive ridleys, respectively. Interestingly, V. cholerae was only isolated in 27% (6/22) of olive ridley turtles (**Table 1**).

### Serovars and Detection of Virulence Genes

V. parahaemolyticus was screened for the presence of virulence genes and serovars. There were 6 O serogroups, 4 different K types, and 8 serovars that could be identified in the 17 strains recognized or not by both O and K currently available antisera in V. parahaemolyticus isolates, (**Table 2**). A total of 47% (8/17) of

TABLE 1 | Bacterial species isolated from buccal cavity and cloacae of black turtles (*Chelonia mydas agassizii*) and olive ridley turtles (*Lepidochelys olivacea*).

BCS, Baja California Sur.

the strains were not recognized by O antisera, while 82% (14/17) were not recognized by K antisera, and eight of these latter strains did not react to O:K antisera (OUT:KUT). Serogroups O1, O2, O4, O5, and O10 were identified. In BCS, one serotype OUT:KUT was isolated from black turtles. In Sinaloa, serotypes recognized were O1:KUT, O4:K10, O5:KUT, O10:KUT, OUT:KUT, O1:K32, O2:KUT, and O4:K13, isolated from olive ridleys and OUT:KUT, isolated from olive ridley and black turtles (**Table 2**).

Based on the presence or absence of virulence genes, the V. parahaemolyticus isolates were classified into three groups: pandemic (tdh+, toxRS/new+, and/or orf8+), pathogenic (tdh+ and/or trh+), and non-pathogenic strains (tdh− and trh−). Among V. parahaemolyticus strains, two strains (11.7%) were identified as pandemic isolates. One of these strains belonged to serotype OUT:KUT and carried the tdh, trh, and toxRS/new genes (isolated from black turtles in BCS), whereas one pandemic O4:K10 strain carried the tdh and toxRS/new genes (isolated from olive ridley turtles in Sinaloa). A total of 29.4% (5/17) of isolates carried the virulence tdh gene and therefore were considered pathogenic strains (**Table 2**). Approximately 58.8% (10/17) of V. parahaemolyticus isolates were non-pathogenic.

Serological characterization of the six V. cholerae strains isolated from olive ridley turtles in Sinaloa, revealed that all strains belonged to the non-O1 and non-O139 serogroup. PCR studies revealed that four of the six isolates of V. cholerae non-O1/non-O139 harbored the ace gene; however, all strains tested were negative for the ctxA, ctxB, and zot genes (**Table 2**).

#### Antibiotic Resistance Profiles

Antimicrobial susceptibility testing of V. parahaemolyticus isolated shown that all strains were resistant to ampicillin (94.1% resistant and 5.9% intermediate resistance), but >82% of isolates demonstrated susceptibility to tetracycline (88.2%), ceftazidime (82.3%) and chloramphenicol (88.2), and >64% to gentamicin and cefotaxime (**Table 3**). There were no strains resistant to ciprofloxacin, nalidixic acid, and sulfamethoxazole/trimethoprim (SXT) although inter-mediate zone sizes were observed in 23.5, 41.2, and 17.7% of the strains, respectively. Regarding overall antibiotic resistance, most V. parahaemolyticus strains (94.1%; 16/17) were nonsusceptible to at least one antibiotic and 35.3% (6/17) of strains were resistant to two or more drugs (multidrug-resistant) (**Table 3**).

All strains of V. cholerae isolated were susceptible to ciprofloxacin, SXT, tetracycline, and chloramphenicol (**Table 3**). There were no strains resistant to nalidixic acid, ceftazidime, and cefotaxime although inter-mediate zone sizes were observed in 16.7% of the strains for each antibiotic. Low resistance (16.7%) was observed for gentamicin and ampicillin. In relation to antibiotic resistance, only the 33.4% (2/6) of strains were resistant to one antibiotic (**Table 3**).

On the other hand, almost all the V. alginolyticus strains (87.2%) exhibited resistance to ampicillin antibiotic, but low resistance was observed for tetracycline (7.7%), chloramphenicol (10.3%), nalidixic acid (5.1%), and to SXT (7.7%) (**Table 3**). There were no strains inter-mediate zone sizes observed to tetracycline and ampicillin. A high proportion of resistance and intermediate resistance were noticed among V. alginolyticus strains to gentamicin (82.1%), ciprofloxacin (76.9%), and cefotaxime (74.4%). Regarding overall antibiotic resistance, all V. alginolyticus strains were non-susceptible to at least one antibiotic and 69.2% (27/39) of strains were resistant to two or more drugs (multidrug-resistant). The resistance to 3– 6 antibiotic was observed in 19 strains representing 48.7% (**Table 3**).

#### TABLE 2 | Serological and virulence gene characteristics of *Vibrio parahaemolyticus* and *Vibrio cholerae.*

+, Presence; −, absence.

### Discussion

Sea turtles are long-distance, migratory animals and occupy niches in different marine environments and geographical regions throughout their different life cycle stages, usually ranging from pelagic environments, as hatchlings, to several coastal areas in their juvenile and adult stages. Due to migratory habits, sea turtles are susceptible to threats in both offshore and coastal environments (Bolten, 2003). Five of the world's seven sea turtle species occur along the Pacific coast of Mexico, making this region very important from a biological and socioeconomic point of view (Senko et al., 2009, 2011; Aguilar-Gonzalez et al., 2014). Black turtles that have grown large enough to reside in benthic environments have a nearly exclusive herbivorous diet consisting of selected macroalgae and sea grasses. They are found in the Mexican Pacific during all life history stages and the coastal waters of the eastern Pacific and Gulf of California provide important feeding and developmental habitats (Cliffton and Felger, 1982; Seminoff et al., 2002a,b; Senko et al., 2009, 2011; Aguilar-Gonzalez et al., 2014). Olive ridley turtles have a large range within the tropical and subtropical regions in the Pacific and Indian Oceans as well as the Southern Atlantic Ocean. This species spends most of its time within 15 km of shore, preferring shallow seas for feeding and sunbathing; however, this species is also observed in the open ocean (Eckert et al., 1999).

In Mexico, sea turtles have traditionally been an important resource for many coastal communities for centuries and have been used throughout the region for food, medicine, and decoration especially in BCS and SIN (Senko et al., 2009, 2011; Aguilar-Gonzalez et al., 2014). Coastal communities that consume sea turtles generally utilize the entire animal. While turtle meat is eaten directly (on the grill or stew), internal organs such as kidney and liver are used for soup.

The human impacts on the world's oceans have devastated populations, species, and ecosystems at a rapid scale (Aguirre et al., 2002; Aguirre and Lutz, 2004; Aguirre and Tabor, 2008). There are several zoonotic agents spilling over from terrestrial reservoirs to marine species and, on the other hand, zoonotic pathogens spill back to humans and domestic animals with severe consequences to wildlife health. Several bacterial species have been isolated from sea turtles, including Salmonella, Mycobacterium, Vibrio, and E. coli, which have been identified as potentially pathogenic to humans (Raidal et al., 1998; O'grady and Krause, 1999; Orós et al., 2005; Lu et al., 2006). In our study, a total of 82 bacterial isolates were identified including C. freundii, E. coli, Edwarsiella spp., V. alginolyticus, V. cholerae, V. fluvialis, V. furnisii, V. parahaemolyticus, and Vibrio spp. Aeromonas or Plesiomonas, Morganella or Proteus, and Providencia. All of these bacteria have been considered to be potentially pathogenic and opportunistic in sick sea turtles (Aguirre and Lutz, 2004; Orós et al., 2004, 2005). Besides, the predominant isolates from sea turtles were, V. alginolyticus, V. parahaemolyticus, V. cholerae, which are considered pathogenic to human health.

TABLE 3 | Antibiotic resistance among *Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio alginolyticus* strains isolated from black turtles and olive ridley turtles.

a Susceptible, b Intermediate, c Resistant. Vibrio parahaemolyticus strains with resistance to 3, 6, 7, 8, and 9 drugs were not detected. Vibrio cholerae no-O1 strains with resistance to 2, 3, 4, 5, 6, 7, 8, and 9 drugs were not detected. Vibrio alginolyticus strains with resistance to 0, 7, 8, and 9 drugs were not detected.

V. parahaemolyticus, are halophilic, Gram negative bacteria, that naturally inhabit marine and estuarine environments (Gutierrez West et al., 2013; Haley et al., 2014). The pathogen has emerged as a worldwide pandemic causing gastroenteritis related to consumption of sea products in recent years (Nair et al., 2007; Velazquez-Roman et al., 2014). Also, it has been demonstrated that the existence of the tdh and/or trh gene in a strain is associated with its ability to cause gastroenteritis (Nishibuchi and Kaper, 1995). Interestingly, in SIN, we have reported the presence of these bacteria in environmental samples and invertebrates (seawater, sediment, and shrimp) as well as fecal samples, identifying a high serodiversity and prevalence of pathogenic (tdh+) and pandemic (O3: K6, tdh+, and toxRS/new+) strains (Velazquez-Roman et al., 2012; Hernández-Díaz et al., 2015). However, in this study, we identified a novel serovar (04:K13) and the serotype O4:K10 with pandemic features in a different area from our previous investigations conducted during 2004– 2013 (Velazquez-Roman et al., 2012; Hernández-Díaz et al., 2015). These novel serovars were isolated from olive ridley turtles, suggesting that this species could contribute to V. parahaemolyticus clones migrations in several ecosystems.

Importantly, olive ridley turtles nest from BCS (Lopez-Castro et al., 2004) to Peru (Kelez et al., 2009) with nesting reported from July to March. The scientific communities are coming to the conclusion that ballast discharge, global trade, and climate change represent the major underlying mechanisms for the global spread of pandemic V. parahaemolyticus, particularly clone O3:K6 (Velazquez-Roman et al., 2014). This spreading of pandemic V. parahaemolyticus is still a speculative question that requires further investigation since the pandemic and pathogenic strains can potentially migrate through sea turtles from Mexico to Peru or others countries including the United States. Due to the wide geographic distribution that sea turtles cover during their life cycle, they can serve as meaningful "sentinels" for overall ecosystem health (Aguirre and Lutz, 2004), and because of this, it is especially important to document and understand any factors that might affect the dissemination of pathogens.

To date, ca. 200 serogroups of V. cholerae have been recorded, and two (O1 and O139) have been associated with major cholera epidemics. The other serogroups, referred as non-O1/non-O139, have not been associated with epidemics but rather can cause sporadic diarrhea and occasional outbreaks (Chatterjee et al., 2009). In this study, V. cholerae was detected in six (9%) specimens. All isolates were non-O:1/non-O:139 serotypes and negative for ctxA, ctxB, and zot gene, but positive for ace gene by PCR. In China, turtles and their breeding environment have been reported major reservoirs of V. cholerae and responsible for many cholera outbreaks (Liu et al., 2006; Lu et al., 2006; Chang et al., 2007). In Zhejiang Province, the incidence of O1 serogroup of V. cholerae was found to be high (9%) in turtles and cholera epidemics in this region are associated with consumption of infected turtles (Lu et al., 2006). Identification of the non-O1/non-O139 serogroups of V. cholerae carrying virulence genes (ace gene) in olive ridley turtles is very important since these new toxigenic strains with epidemic potential may emerge in the future if illegal consumption continues.

V. alginolyticus is pathogenic to a wide range of marine life including fish, mollusks, crustaceans, cnidarians, and sea turtles (Orós et al., 2004, 2005). In our study, V. alginolyticus were the predominant isolates from sea turtles and has been associated as an opportunistic pathogen for sea turtles in exudative bronchopneumonia and/or granulomatous pneumonia, traumatic skin lesions, granulomatous nephritis, renal abscesses, and necrotizing and/or granulomatous hepatitis and have been considered important causes of mortality among sea turtles (Orós et al., 2004, 2005). To the best of our knowledge, the present study is the first record of especially of V. parahaemolyticus, V. cholerae, and V. alginolyticus in black and olive ridley turtles in BCS and SIN, Mexico.

Another important contribution of this study was the investigation of susceptibility, or resistance of the isolated V. parahaemolyticus, V. cholerae, and V. alginolyticus strains to firstline antibiotics utilized in the region. All V. parahaemolyticus isolates were resistant to ampicillin, which was not a surprise as non-susceptibility to ampicillin is very common in V. parahaemolyticus strains isolated from environmental and clinical samples (Okuda et al., 1997; Wong et al., 2000; Roque et al., 2001; Sun et al., 2013; Letchumanan et al., 2015). We detected 23.5% of resistance to cefotaxime in V. parahaemolyticus from sea turtles, a similar prevalence of resistance to cefotaxime (20%) has also been reported in strains isolated in Italy from shellfish and clinical samples (Ottaviani et al., 2013). A number of studies have been reported low (Ceccarelli et al., 2015) and high (Jagadeeshan et al., 2009) prevalence of V. cholerae non-O1/non-O139 strains resistant to numerous antibiotics, isolated from environmental samples. In this study, only a few of the non-O1/non-O139 V. cholerae isolates from sea turtles were resistant to ampicillin or gentamicin. Most of the isolates were sensitive to all antibiotics tested.

### References

All V. alginolyticus strains isolated in this study, were resistant to at least one antibiotic. Several studies reported a wide range of resistance for V. alginolyticus (Snoussi et al., 2008; Scarano et al., 2014). Although a high proportion of resistance and intermediate resistance were noticed to gentamicin (82.1%), ciprofloxacin (76.9%), and cefotaxime (74.4%). These results are in accordance with other studies which found high rates of resistance to gentamicin, ciprofloxacin, and cefotaxime (Snoussi et al., 2008; Lajnef et al., 2012).

Despite a federal ban on turtle hunting, consumption and trade in Mexico since 1990 (Gardner and Nichols, 2001; Senko et al., 2009; Aguilar-Gonzalez et al., 2014), sea turtles are captured by bycatch or incidentally mostly in the summer, precisely when consumption presents the greatest potential hazards to human health (Senko et al., 2009; Aguilar-Gonzalez et al., 2014). And when consumed, may have adverse human health effects, such as extreme dehydration, vomiting, diarrhea, and even death, due to the presence of bacteria, parasites, and environmental contaminants found in these animals (Aguirre et al., 2006; Senko et al., 2009; Aguilar-Gonzalez et al., 2014).

In conclusion, although fishermen from northwestern Mexico recognize that sea turtles might be contaminated and infected with potential pathogens, and that eating them could cause health problems (Aguirre et al., 2006; Senko et al., 2009; Aguilar-Gonzalez et al., 2014), they and their families continue consumption. In this study, we found the presence of several potential toxigenic and drug-resistant or multi-drug resistance human pathogens in sea turtles. Therefore, this information is important on possible health risks for humans in hope of behavioral changes that could benefit sea turtle conservation.

### Acknowledgments

This work was supported by a grant from PROFAPI-UAS 2013/032 to AC. LH was supported by a scholarship from CONACYT No. 290225. We would like to Elizandra Quiñonez for their technical help. This research was performed under Mexican regulations and laws under permit provided by SEMARNAT (Secretaría de Medio Ambiente y Recursos Naturales) No. of permission SGPA/DGVS/04568/11.

metals in tissues from loggerhead turtles (Caretta caretta) from the southwestern Mediterranean (Spain). Ecotoxicol. Environ. Saf. 72, 557–563. doi: 10.1016/j.ecoenv.2008.05.003

**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 © 2015 Zavala-Norzagaray, Aguirre, Velazquez-Roman, Flores-Villaseñor, León-Sicairos, Ley-Quiñonez, Hernández-Díaz and Canizalez-Roman. 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.

# Strategies of *Vibrio parahaemolyticus* to acquire nutritional iron during host colonization

*Nidia León-Sicairos1,2\*, Uriel A. Angulo-Zamudio3, Mireya de la Garza4, Jorge Velázquez-Román1, Héctor M. Flores-Villaseñor1 and Adrian Canizalez-Román1*

*1 Unidad de Investigación, Facultad de Medicina, Universidad Autónoma de Sinaloa, Culiacán, Mexico, 2 Departamento de Investigación, Hospital Pediátrico de Sinaloa "Dr. Rigoberto Aguilar Pico", Culiacán, Mexico, 3 Maestría en Ciencias de la Salud, Facultad de Medicina, Universidad Autónoma de Sinaloa, Culiacán, Mexico, 4 Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico, Mexico*

#### *Edited by:*

*Pendru Raghunath, Dr. VRK Women's Medical College, India*

#### *Reviewed by:*

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

#### *\*Correspondence:*

*Nidia León-Sicairos, Unidad de Investigación, Facultad de Medicina, Universidad Autónoma de Sinaloa, Cedros y Sauces, Fraccionamiento Fresnos, Culiacán, Sinaloa 80246, Mexico<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 14 March 2015 Accepted: 26 June 2015 Published: 09 July 2015*

#### *Citation:*

*León-Sicairos N, Angulo-Zamudio UA, de la Garza M, Velázquez-Román J, Flores-Villaseñor HM and Canizalez-Román A (2015) Strategies of Vibrio parahaemolyticus to acquire nutritional iron during host colonization. Front. Microbiol. 6:702. doi: 10.3389/fmicb.2015.00702* Iron is an essential element for the growth and development of virtually all living organisms. As iron acquisition is critical for the pathogenesis, a host defense strategy during infection is to sequester iron to restrict the growth of invading pathogens. To counteract this strategy, bacteria such as *Vibrio parahaemolyticus* have adapted to such an environment by developing mechanisms to obtain iron from human hosts. This review focuses on the multiple strategies employed by *V. parahaemolyticus* to obtain nutritional iron from host sources. In these strategies are included the use of siderophores and xenosiderophores, proteases and iron-protein receptor. The host sources used by *V. parahaemolyticus* are the iron-containing proteins transferrin, hemoglobin, and hemin. The implications of iron acquisition systems in the virulence of *V. parahaemolyticus* are also discussed.

Keywords: *Vibrio parahaemolyticus*, iron, virulence, host iron proteins, mechanism of acquisition

## Introduction

*Vibrio parahaemolyticus* is a halophilic Gram-negative bacterium that naturally inhabits marine and estuarine environments (Gavilan et al., 2013). The more recent distribution of this species has been driven by climatic conditions and the eutrophication of regional waters throughout the world (Baumann et al., 1984). *V. parahaemolyticus* can survive in a wide variety of niches in a free-swimming state, and its motility is conferred by a single polar flagellum. Alternatively, this bacterium can be found in a sessile state, attached to inert or animate surfaces, such as suspended particulates, zooplankton, fish, and shell-fish (McCarter, 1999). Many strains of *V. parahaemolyticus* are strictly environmental, though small subpopulations can be opportunistic pathogens that may cause gastroenteritis, wound infection, and septicemia (Joseph et al., 1982; McCarter, 1999). As with all bacteria, *V. parahaemolyticus* reproduces via binary fission without a systematic exchange of genes with other individuals of the same species, leading to essentially clonal reproduction (Garcia et al., 2013); however, only some of these clones cause diarrhea in humans. Strains of *V. parahaemolyticus* belong to different serogroups and can produce a number of different lipopolysaccharide (O) and capsular (K) antigens, constituting the primary basis for strain classification (Nair et al., 2007; Garcia et al., 2013).

Since its discovery in 1950 by Tsunesaburo Fujino of Osaka University after an outbreak due to the ingestion of contaminated seafood, *V. parahaemolyticus* has been recognized as a leading cause of intestinal infection throughout coastal countries worldwide (Fujino et al., 1953). Following the consumption of raw or undercooked seafood, individuals infected with virulent strains may present clinical symptoms that include diarrhea, vomiting, nausea, abdominal cramping, and low-grade fever. Exposure to *V. parahaemolyticus* can also lead to wound infection and septicemia in certain medical conditions, such as immune deficiency (Su and Liu, 2007).

The infections and outbreaks caused by *V. parahaemolyticus* prior to 1996 were geographically isolated and associated with a diversity of serotypes (Wong et al., 2000; Chowdhury et al., 2004). However, the later increase in outbreaks was linked to the occurrence of gastroenteritis throughout Asia associated with a new and unique clone of serotype O3:K6 that emerged in Kolkata, India, in 1996 (Matsumoto et al., 2000; Ansede-Bermejo et al., 2010; Pazhani et al., 2014). This clone rapidly spread throughout the majority of Southeast Asian countries in the same year (Okuda et al., 1997b; Ansede-Bermejo et al., 2010; Gavilan et al., 2013). This clone, referred to as the pandemic clone, has since spread globally from Southeast Asia to Europe, Africa, and the Americas (Okuda et al., 1997a; Nair et al., 2007; Paranjpye et al., 2012; Yano et al., 2014). In particular, some countries on the American continent have reported cases of gastroenteritis due to the pandemic O3:K6 strain and its serovariants; the pandemic strain was first detected in Peru, subsequently spread to Chile in 1998, to the U. S. in the same year, to Brazil in 2001 and to Mexico in 2004 (Velazquez-Roman et al., 2012, 2013). Based on various molecular methods, these widespread pandemic O3:K6 strains have been found to be genetically closely related and appear to constitute a clone that differs significantly from the non-pandemic O3:K6 strains that were isolated prior to 1996 (Okuda et al., 1997b; Wong et al., 2000; Lan et al., 2009), with several serovariants apparently emerging since 1996 (Nair et al., 2007; Velazquez-Roman et al., 2012, 2013; Al-Othrubi et al., 2014; Pazhani et al., 2014; Letchumanan et al., 2015). The global occurrence of *V. parahaemolyticus* strains emphasizes the importance of understanding their many virulence factors as well as the mechanisms used to acquire nutrients from the environment and the effects on human hosts.

*Vibrio parahaemolyticus* possesses a wide variety of virulence factors that cause damage such as adhesins, toxins, and secreted effectors involved in attachment, cytotoxicity, and enterotoxicity (Broberg et al., 2011; Zhang and Orth, 2013). The two main factors are a thermostable direct hemolysin (TDH) and a thermostable direct hemolysin-related hemolysin (TRH) encoded by the *tdh* and *trh* genes, respectively (Honda et al., 1982; Nishibuchi et al., 1992; Vongxay et al., 2008). Nonetheless, the isolation of *V. parahaemolyticus* strains lacking functional *tdh* and *trh* genes from human infection cases and the analysis of the genome sequence of *V. parahaemolyticus* strain RIMD2210633 suggest that other virulence factors also play a role in the disease caused by this bacterium (Makino et al., 2003; Okada et al., 2009). The genome of *V. parahaemolyticus* contains two sets of type III secretion system (T3SS) gene clusters that function in the secretion and translocation of virulence factors into eukaryotic cells. These appear on each of the two chromosomes (Makino et al., 2003; Caburlotto et al., 2009). T3SSs utilize a needle-like apparatus to translocate into host cells effectors that target and hijack multiple eukaryotic signaling pathways. Indeed, T3SSs are essential virulence machines used by numerous bacterial pathogens, including *Yersinia, Salmonella, Shigella*, and pathogenic *Escherichia coli* (Makino et al., 2003).

Other systems or mechanisms that play an important role as virulence factors in *Vibrio* and all pathogenic microorganisms are those that confer the ability to acquire nutrients from the environment in which they live. These systems ensure that pathogens successfully reproduce and become established in a host. For example, the capacity to acquire nutrients such as iron (Fe) from a host is an ability obtained by pathogenic microorganisms during evolution. In fact, it has been speculated that the evolutionary pressure for microbes to develop pathogenic characteristics was to access the nutrient resources supplied by animals (Tanabe et al., 2012). The environment inside the colonized host has led to the evolution of new bacterial characteristics to maximize such new nutritional opportunities (Rohmer et al., 2011; Tanabe et al., 2012).

Currently, access to host nutrients is regarded as a fundamental aspect of an infectious disease. During the invasion of the human host, pathogens encounter complex nutritional microenvironments that could change, for example; the increase in inflammatory response due to the infection, local hypoxia in some tissues (Payne, 1993; Cassat and Skaar, 2013). The host can limit microbial access to nutrient supplies as a defense mechanism against the pathogens, however, the pathogens can counteract this by developing metabolic adaptations or improved mechanisms of nutrient acquisition to successfully exploit available host nutrients for their proliferation (Payne, 1993). Recent studies have pointed out an emerging paradigm that has been designated as 'nutritional virulence' (Abu Kwaik and Bumann, 2013). Although this term is applied to the acquisition of amino acids and carbon sources, certain nutritional ions or metals that are essential for cellular growth and other metabolic processes could be considered as part of this paradigm. As one of the most fundamental aspects of infectious diseases is the microbial acquisition of nutrients *in vivo*, which positively impacts in virulence as well as antibiotic resistance (Santic and Abu Kwaik, 2013), we suggest that the process of iron acquisition systems used by pathogenic microorganisms may be considered in the concept of 'nutritional virulence.'

Iron (Fe) is an essential element for almost all cells, including most bacteria because it serves as a cofactor for metabolic processes, such as redox reactions, nucleic acid synthesis, and electron transfer (Tanabe et al., 2012). Iron is the fourth most abundant element on the Earth's crust. In nature, there are two states of iron: (1) ferrous iron (Fe2+), which is toxic because it leads to the production of hazardous reactive oxygen species (ROS), including superoxide, in the presence of oxygen; and (2) ferric iron (Fe3+), which is insoluble under normal physiological conditions. Fe is bound to ligands, primarily proteins, in iron-dependent organisms, and trace Fe concentrations are necessary for all organisms, ranging between 0.4 and 4 µM in the majority of both eukaryotic and prokaryotic cells (Weinberg, 1974). However, there are bacteria such as some lactobacilli that are iron independent because they utilize manganese (Mn) and other cations as cofactors in their enzymes (Imbert and Blondeau, 1998).

The pathogenic microorganisms that infect mammalian hosts encounter diverse and changing environments. For example; the pH within the human body is usually neutral (7.4), but it can range from 1.0 in the stomach to 8.0 in urine. Also, if they move deeper into host tissues at mucosal surfaces, such as those from the lumen, the multilamellar mucus, and the epithelial cells of the stomach, pathogens confront drastically different or hostile environments. Some mucosal surfaces are well oxygenated, but others possess areas of low oxygen tension, for example the oral cavity, large intestine, female genital tract, abscesses and damaged tissues (Rohmer et al., 2011). The level of free Fe in mammalian bodily fluids is variable (∼10−18 M) but always far below the concentrations required for optimal bacterial growth (10−6 M), causing bacteria to rely on their own strategies or mechanisms for obtaining this metal. In an infected site there are numerous physiologically specialized environments that bacteria might encounter or colonize. For example, within the small intestine, there are variable conditions different from those found between caecum and colon (Rohmer et al., 2011). For all these reasons mentioned above, pathogens move through multiple diverse environments throughout their life cycle, and to accomplish this they require the regulation, coordination, and utilization of multiple bacterial metabolic pathways. Bacteria often use metabolic cues in order to regulate their metabolism and virulence functions to be successful as pathogens (Rohmer et al., 2011). Because they depend upon Fe as a vital cofactor that enables a wide range of key metabolic activities, bacteria must therefore ensure a balanced supply of this essential metal; accordingly, they invest considerable resources into its acquisition and employ elaborate control mechanisms to alleviate both iron-induced toxicity and Fe deficiency (Rangel et al., 2008).

The Fe concentration in coastal waters ranges from 1.3– 35.9 nM up to 23.1–573.2 nM (Gledhill and Buck, 2012), a concentration that may be sufficient to support the growth of *V. parahaemolyticus*; however, *V. parahaemolyticus* also infects humans. The human body contains 3.8 g (in men) and 2.3 g (in women) of Fe. 20 mg of Fe is required daily for the production of hemoglobin (Hb) for new erythrocytes in order to preserve Fe homeostasis. Iron absorption from the diet, however, supplies only 1–2 mg daily, and the remaining Fe is derived by recycling Fe from senescent red blood cells (Ganz and Nemeth, 2006). Most bodily Fe is found in heme proteins (Hb, myoglobin, cytochromes, and multiple enzymes), and the second largest Fe pool is found in ferritin (also in hemosiderin). The remaining Fe is found in other proteins, such as iron-sulfur cluster enzymes, Fe-chelating proteins (Tf and Lf), and a pool of accessible Fe ions called the labile Fe pool (LIP), all of them constitutes the iron-containing proteins involved in metabolic pathways from hosts.

Inside the human body the solubility of iron is extremely low, because the Fe exists in insoluble mineral complexes, or under aerobic, aqueous, and neutral pH conditions, that difficult the access of bacteria to this element. Besides, Fe is bound to mammalian high-affinity iron-binding proteins such as Tf, Lf, and Ft and in consequence, many bacteria have developed highaffinity Fe transport systems to acquire Fe from sources in their niches (Rangel et al., 2008; Jin et al., 2009; Tanabe et al., 2012). The Fe sources available in the different environmental niches of *V. parahaemolyticus* are described and discussed in **Figure 1**.

## Role of Iron in the Virulence of *Vibrio parahaemolyticus*

Iron regulates virulence factors of *V. parahaemolyticus* and almost all pathogenic bacteria. Inside a host the Fe concentration is very low, so many pathogens uses this (low-iron conditions) for inducing expression of genes involved in the virulence (Litwin and Calderwood, 1993). The presence of ferric Fe in bacterial growth media has been found to increase the adherence intensities of virulent *V. parahaemolyticus* strains to human fetal intestinal (HFI) cells *in vitro* (Hackney et al., 1980). Intraperitoneally injection with *V. parahaemolyticus* in the presence of ferric ammonium citrate in mice increased the bacterial proliferation, thus enhancing the lethality toward infected mice. *V. parahaemolyticus* cultures in low-iron conditions showed better proliferation than iron-rich cultures in response to the addition of supplementary Fe. Also, the production of thermostable direct toxin (TDH) by the hemolytic strains of *V. parahaemolyticus* was higher in iron-limited cultures than in iron-rich cultures, though the production of TDH by both iron-limited and iron-rich cultures was inhibited by the addition of Fe. In conclusion, the enhancement of *V. parahaemolyticus* virulence in the model mice likely occurred through the increase of bacterial proliferation *in vivo* and not the stimulation of TDH production. The *V. parahaemolyticus* precultured under iron-limited conditions may be more adaptable to the *in vivo* environment (Wong and Lee, 1994; Funahashi et al., 2003; Gode-Potratz et al., 2010).

The effect of lysed blood on the virulence of *V. parahaemolyticus* in mice was also investigated, and a factor released by erythrocyte lysis was found to greatly reduce the 50% lethal dose of *V. parahaemolyticus* in mice. Similar effects were observed with ferric ammonium citrate and Mn sulfate. Authors conclude that Fe from the lysed blood is involved in the virulence of *V. parahaemolyticus* (Karunasagar et al., 1984).

In a recent work, Gode-Potratz et al. (2010) demonstrated that metal ions play distinct roles in modulating gene expression and behavior in *V. parahaemolyticus*. In this work, high-calcium and low-iron growth conditions stimulated the induction of swarming and T3SS regulons from *V. parahaemolyticus* (Gode-Potratz et al., 2010). Swarming is a particular adaptation of many bacteria to grow in surfaces. In *V. parahaemolyticus* swarming is done by lateral flagella that enable the bacteria move over and colonize surfaces (Gode-Potratz et al., 2010). The authors concluded that swarming plays a signaling role with global

*Vibrio parahaemolyticus*. *V. parahaemolyticus* is an obligate halophilic organism, meaning that it requires salt to live. This organism is naturally occurring and found worldwide. It can commonly be found free swimming or attached to underwater surfaces and is found at

Humans can acquire *V. parahaemolyticus* infection from infected seafood; once infected, established *V. parahaemolyticus* can acquire iron from different iron-containing proteins, such as hemoglobin (Hb) and transferrin (Tf).

consequences on the regulation of gene sets that are relevant for surface colonization and infection and that stimulation depends on the level of Fe present in the environment (Gode-Potratz et al., 2010).

Iron also regulates virulence factors in other members of the genus *Vibrio*. Based on the description of the virulenceenhancing effect of ferric ammonium citrate on *V. cholerae* by Joo (1975), other work has supported the role and importance of Fe in the virulence of *Vibrio* sp. (Joo, 1975). It has been demonstrated that the iron overload in humans increases the virulence of pathogens *in vivo*. For example in patients with liver diseases and hemochromatosis, iron overload states are common. It is known that these conditions predispose patients to recurrent infections, septicemia, and high mortality due to pathogens such as *V. vulnificus* (Bullen et al., 1991; Barton and Acton, 2009). This was corroborated by injecting mice with *V. vulnificus* and Fe, resulting in a lower 50% lethal dose and in a reduction in the time to death post-infection (Wright et al., 1981). In addition, elevated serum Fe levels were also produced by liver damage due to injections. Because the infections with *V. vulnificus* are acquired through the consumption of contaminated seafood, the role of iron on infections acquired by the oral route was also studied in mice. On the other hand, the role of iron on the growth of *V. vulnificus* in human and rabbit sera with injections of Fe or Tf were also studied. In the results, lethality and growth of *V. vulnificus* were more efficient in rabbit and human sera; respectively (Wright et al., 1981). Next, an induced peritonitis model was employed in mice to determine whether heme-containing molecules enhance the lethality of infections of *V. vulnificus* (Helms et al., 1984). In this model, the lethality toward mice inoculated intraperitonally with the bacteria and treated with methemoglobin, or hematin but not by myoglobin, was increased compared with those untreated (Helms et al., 1984). These results indicated that *V. vulnificus* has the capacity to produce fatal human infections because of its ability to use human proteins that bind Fe (Helms et al., 1984). Additionally, *V. vulnificus* strains isolated from different sources of Cuddalore coastal waters were tested for their virulence activity based on their LD50 values in mice. The LD50 was in the range of 104– 107 cells in normal mice, but 101–102 cells in iron-injected mice, thus reinforcing the idea that Fe (acquired from human sources *in viv*o) may play a major role in the pathogenesis of *V. vulnificus* (Jayalakshmi and Venugopalan, 1992). Additionally, the virulence mechanisms of *V. vulnificus* biotype 1 and biotype 2 were studied and compared in mice. Both strains presented several properties in common, including capsule expression, the uptake of various Fe sources, and the production of exoproteins (Wright et al., 1981; Amaro et al., 1994). Taken together, data support the importance of iron in the pathogenesis of *Vibrio* sp. and *V. parahaemoluticus in vivo* and *in vitro.*

### Iron regulatory Proteins and Mechanisms in *V. parahaemolyticus*

The study of Fe acquisition systems in *E. coli* led to the discovery of Fur, a DNA binding protein of 16.8 kDa, product of the *fur* (ferric uptake regulation) gene, that represses the transcription of genes involved in Fe uptake systems in iron replete conditions (Litwin and Calderwood, 1993). When the intracellular Fe concentration increases, Fur forms a dimer together with ferrous Fe (Fe2+) and binds to a consensus sequence (Fur-box), which overlaps the promoters of Fur-target genes, resulting in the inhibition of transcription. Although the role of Fur as a repressor is well-documented, emerging evidence demonstrates that Fur can function as an activator (Troxell and Hassan, 2013). Additionally to *E. coli*, Fur has been identified in other Gram-negative and Gram-positive bacteria. An interesting finding was that Fur also participates in functions different to the Fe metabolism for example; defense against oxygen radicals, metabolic pathways, bioluminescence, chemotaxis, swarming and production of toxins, and other virulence factors (Litwin and Calderwood, 1993). We can speculate about the importance of these Fe dependent mechanisms for bacterial virulence inside a host *in vivo*.

*Vibrio parahaemolyticus* contains a Fur protein that is 81% identical with the Fur protein from *E. coli* and over 90% identical with those of the *Vibrio* sp. (Yamamoto et al., 1997). Funahashi et al. (2002) reported that *V. parahaemo*lyticus *psuA* and *pvuA* genes (which encode the TonB-dependent outer membrane receptors for a putative ferric siderophore and ferricvibrioferrin), are regulated by Fur (Funahashi et al., 2002). Additionally, a homolog of the *iutA* gene in *V. parahaemolyticus* (which encodes for the receptor of ferric aerobactin) is apparently regulated by Fur (Funahashi et al., 2003). Furthermore, Fur regulates *V. parahaemolyticus peuA* gene (which encodes for an alternative ferric-enterobactin receptor; Tanabe et al., 2014).

Fur has been involved in the regulation of other virulence factors from *Vibrio* sp. Lee et al. (2013) demonstrated that in *V. vulnificus* Fur regulates hemolysin production at the transcriptional (vvhBA operon) and post-translational (by regulating the expression of two VvhA-degrading exoproteases, VvpE, and VvpM) levels (Lee et al., 2013). In contrast, other transcriptional regulators such as AraC-type family members and LysR-type family members, have been shown to activate transcription initiation of genes involved in the synthesis and utilization of siderophores in bacteria (Balado et al., 2009; Tanabe et al., 2012). In *V. cholerae* Fur regulates the expression of a number of genes in response to changes in the level of available iron. Fur usually acts as a repressor, but it has been shown that Fur positively regulates the expression of ompT, which encodes a major outer membrane porin, involved in the virulence of *V. cholerae* (Craig et al., 2011). It has been reported that Fur also represses the synthesis of RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis (Wyckoff et al., 2007). Mey et al. (2005) reported the effects of iron and Fur on gene expression in *V. cholerae*. According with this work, nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions, and also those genes involved in the transport of iron inside of pathogens (Mey et al., 2005). The iron transport systems regulated negatively by Fur in iron-replete conditions were *feo* and *fbp* genes (involved in the transport of ferrous and ferric iron inside cells; respectively). Both were found to be negatively regulated by iron and Fur (Mey et al., 2005). This is consistent with others genes involved in iron acquisition; in high concentrations of this nutrient the genes for iron acquisition systems are repressed.

### Iron acquisition Systems Used by Pathogenic Microorganisms

An obligate question is how does *V. parahaemolyticus* acquire iron? This theme is complex; however, it has been well established in other pathogens. To acquire Fe from host sources, microorganisms generally use the iron-acquisition systems described below.

### Receptors for Host Iron-Containing Proteins

Transferrins (Tfs) are a family of iron-binding glycoproteins that chelate free ferric Fe in biological fluids (Crichton and Charloteaux-Wauters, 1987). Bacteria such as *Neisseria gonorrhoeae* and *Haemophilus influenzae* are able to use the Fe in these proteins by binding Tf and Lf iron directly through the use of membrane receptors or binding proteins for host ironglycoproteins (Tbp and Lbp, respectively). These receptors have been reported to be induced in some bacteria in the absence of Fe (Gray-Owen and Schryvers, 1996). Some receptors are specific for one iron-containing protein. On the contrary, other receptors that recognizes Tf, also can recognize Lf or other iron proteins, these receptors have been found in *Neisseria* (Morgenthau et al., 2013). Tf binding protein (TbpA) and lactoferrin binding protein (LbpA) are receptors that share 25% amino acid identity and a high degree of similarity. These Tf and Lf receptors can be promiscuous; for example, TbpA can recognizes Lf in some bacteria. Moreover, Tbps also exhibit low homology to other transport proteins and siderophore receptors. The homology among the members of this family of transporters suggests that the ancestral meningococcal Tf and Lf receptors may have been a single-unit transporter, similar to siderophore receptors (Perkins-Balding et al., 2004). The best characterized Tfbps and Lfbps to date belong to *Neisseria* species (Schryvers et al., 1998; Schryvers and Stojiljkovic, 1999).

### Production and Secretion of Siderophores/Hemophores

Siderophores and hemophores are relatively small (*<*1 kDa) compounds produced and secreted by some species of bacteria and fungi to acquire Fe. Once produced and secreted from microorganisms, their function is to chelate ferric Fe with very high affinity (formation constant up to 1050 M; Wandersman and Delepelaire, 2004; Saha et al., 2013). In fact, these compounds can remove ferric Fe from the host proteins Tf, Lf, and ferritin. Siderophores are produced as common products of microbial metabolism under Fe stress conditions and facilitate the solubilization of ferric Fe and transport Fe via a specific receptor expressed in the cell plasma membrane. In addition, *Bacillus subtilis* and *Mycobacterium smegmatis* that are unable to synthesize siderophores resolve this problem by using the Fe from siderophores derived from other microorganisms (xenosiderophores; Schumann and Mollmann, 2001; Miethke et al., 2013). The capacity of these systems to acquire Fe from various environmental or biological sources is an evident advantage for organisms that may exist in several niches. Hemophores, which are less well studied and only found thus far in Gram-negative bacteria, are employed via the following strategy: they are secreted into the extracellular medium where they scavenge heme from various hemoproteins due to their higher affinity for this compound and then return the heme to hemophore-specific outer membrane receptors (Wandersman and Delepelaire, 2012). These proteins are secreted under conditions of iron depletion (Faraldo-Gomez and Sansom, 2003; Wandersman and Delepelaire, 2012).

### Ferrireductases/Proteases

Some microorganisms secrete ferrireductases or produce membrane-associated proteins that reduce the ferric iron in holoTf, holoLf, or ferritin to the more accessible ferrous form. The reduction of ferric iron destabilizes the host iron-containing protein, and the ferrous iron is thus released. It has been reported that some pathogens are able to produce and secrete proteases that cleave host iron-containing proteins, and iron is easily acquired by pathogens in this form, for example, *Entamoeba histoytica* has hemoglobinases (Payne, 1993; Serrano-Luna et al., 1998; Ortiz-Estrada et al., 2012). A scheme for Fe acquisition systems in bacteria is represented in **Figure 2**.

### Human Iron Sources and Iron Acquisition Systems in *V. parahaemolyticus*

*Vibrio parahaemolyticus* and other bacteria from the genus *Vibrio* including *V. cholerae, V. vulnificus*, require Fe for their

growth and have developed systems during evolution to acquire Fe for sustaining metabolism and replication. As mentioned above, the Fe concentration in coastal waters ranges from 1.3– 35.9 to 23.1–573.2 nM, which is sufficient for the growth of *V. parahaemolyticus*. In the human host, *V. parahemolyticus* can utilize host sources, such as the iron-proteins Hb, heme, hemin, and Tf (Yamamoto et al., 1994), and possibly others, such as Lf and Ft (**Figure 1**).

### Siderophores as a Mechanism of Iron Acquisition Used by *V. parahaemolyticus*

One of the strategies of *V. parahaemolyticus* to obtain the iron from the host iron-proteins is the use of siderophores. Under iron-limited conditions *V. parahaemolyticus* secretes Vibrioferrin to facilitate Fe acquisition. Fe-charged Vibrioferrin is recognized by an outer-membrane receptor composed of the proteins PvuA1 and PvuA2, which also recognizes heme and Hb. This receptor is coupled with the ABC transport system PvuBCDE which is located in the inner membrane and whose function is import the ferric-charged vibrioferrin to the inner membrane (Tanabe et al., 2003, 2011, 2012). *V. parahaemolyticus* also contains the TonB system which consists of three proteins designated TonB1, TonB2, TonB3 (Kuehl and Crosa, 2010). The energy for the transport of ferric-vibrioferrin is provided by the TonB2 system for PvuA1 and both the TonB1 and TonB2 systems for PvuA2 (Tanabe et al., 2011, 2012).

The *V. parahaemolyticus* also is able to utilize siderophores produced by other bacteria, for example, exogenous aerobactin, desferri-ferrichrome, and enterobactin (Funahashi et al., 2003, 2009; Tanabe et al., 2012). We have compared and analyzed *in silico* the mechanism used by *V. parahaemolyticus* with *V. cholerae*; the most studied member of the genus *Vibrio. V. cholerae* has multiple strategies for iron acquisition, including the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms (Wyckoff et al., 2007). In general the *Vibrio* species, including *V. cholerae* and *V. anguillarum* can use catecholate-type siderophores as their cognate siderophores (Griffiths et al., 1984; Actis et al., 1986; Balado et al., 2009; Tanabe et al., 2012). Furthermore, some *Vibrio* sp. use the xenosiderophore enterobactin (Ent) that is produced mainly by members of the *Enterobacteriaceae* family (Griffiths et al., 1984; Naka and Crosa, 2012; Tanabe et al., 2012). On the other hand, Tan et al. (2014) described the use of the siderophore vulnibactin, essential in Fe uptake from host proteins. The importance of the vulnibactin in *V. vulnificus* pathogenicity was clinically demonstrated (Ceccarelli et al., 2013; Tan et al., 2014).

Once the iron has been acquired, *V. cholerae* has two systems for the transport of free iron: the Feo system, which transports ferrous iron, and the Fbp system, which transports ferric iron (Occhino et al., 1998; Wyckoff et al., 2007). It has been speculated that *V. cholerae* contains one additional high affinity iron transport system. Apparently, iron transport genes are regulated by Fur (Occhino et al., 1998; Wyckoff et al., 2007).

### Transferrin Receptors as an Iron Acquisition System Used by *V. parahaemolyticus*

Using a basic local alignment search tool (BLAST) analysis of the sequences in *V. parahaemolyticus,* we identified putative genes that could encode for Tf receptors, but not for Lf receptors. The putative *V. parahaemolyticus* Tf receptor gene (*tbpA*) had 88% of identity with for those reported for *N. meningitidis* (not shown; unpublished data). However, apparently *V. parahaemolyticus* also can use LF as in Fe source (Wong et al., 1996). This probably could be due to the siderophore utilization by this bacterium. Because of Lf is one of the main iron transporters at intestinal level, the *V. parahaemolyticus* capacity of to use Lf as an iron source and the mechanism from iron-Lf acquisition must to be determined. Other members of the genus *Vibrio* have receptors for the use of Tf as iron source. For example, recently; Pajuelo et al. (2015) demonstrated that pVvbt2 from *V. vulnificus*, which causes vibriosis in fish (mainly eels), encodes a host-specific Fe acquisition system that depends on an outer membrane receptor called Vep20. This protein recognizes eel Tf and belongs to a new family of plasmid-encoded fish-specific Tf receptors (Pajuelo et al., 2015). Furthermore, it was found that Vep20 is encoded by an iron-regulated gene that is overexpressed in eel blood during artificially induced vibriosis with *V. vulnificus* both *in vitro* and *in vivo* (Pajuelo et al., 2015). The *Vep20* gene homologs have been identified on the transferable plasmids of two species of fish pathogens with broad host ranges: *V. harveyi* (pVh1) and *Photobacterium damselae* subsp. *damselae* (pPHDD1; Pajuelo et al., 2015). It has been hypothesized that *V. cholerae* contains three proteins that could be Tf receptors. These proteins were shown to be involved in the binding of LF, Hemin, Ft, and Hb (Ascencio et al., 1992).

### Binding and Transport of Iron or Iron-Charged compounds in *V. parahaemolyticus*

The expression of two proteolytic proteins of 43 and 90 kDa from *V. parahaemolyticus* were identified. Apparently the protease of 43 kDa is capable of degrading Hb and it has been speculated that this could be one of the strategies of *V. parahaemolyticus* to acquire iron from the human host (Wong and Shyu, 1994). By using the BLAST, we also identified genes that could play a role in Fe acquisition (**Table 1**). Although they have not reported and their biological functions have not been described, these genes likely encode proteins involved in Fe acquisition for *V. parahaemolyticus* in different niches. The probable functions of putative Fe acquisition genes and homologies are described in **Table 1**. Additionally; a schematic view of Fe acquisition systems with putative proteins used by *V. parahaemolyticus* is shown in **Figure 3**.

Apparently, the *V. parahaemolyticus* contain the proteins VctA, VctP, VctG, and VctD (Tanabe et al., 2012). These proteins are involved in the use of the siderophore ferric-enterobactin (Wyckoff et al., 2007, 2009). VctA and IrgA are receptors for

#### TABLE 1 | Genes related to Iron acquisition systems in *Vibrio cholerae* and putative genes in *Vibrio parahaemolyticus*.

*\*indicate proteins that were studied and reported in V. parahaemoyticus. Column 6 contain the V. parahaemolyticus strain with the highest identity. FeoC was not found in the V. parahaemolyticus strains.*

enterobactin in *V. cholerae* and *V. parahaemolyticus* (Goldberg et al., 1990; Tanabe et al., 2012). Also, in *V. parahaemolyticus* were identified the TonB1 and TonB2 system similar to *V. cholerae* (Occhino et al., 1998; Seliger et al., 2001; Wyckoff et al., 2004). The proteins that belong to the TonB systems are involved in the transport of Fe3+ in Gram-negative bacteria. The system includes outer membrane receptors, all of which are connected with a complex of proteins located at the inner membrane such as TonB, ExbB, and ExbD. This system also includes a periplasmic binding protein associated with an ABC transporter

(Larsen et al., 1996; Koster, 2005). The first step is the binding of the ferric-siderophore to the receptor, it has been speculated that this binding induces a conformational change and then the interaction of the receptor with TonB is enhanced. The proteins ExbB and ExbD provide the energy for the transport of the ferric-siderophore to the inner membrane, in this second step a periplasmic binding protein associated with an ABC transporter delivers the ferric-siderophore into the cytoplasm, in the third step the Fe dissociates from the siderophore (Larsen et al., 1996; Occhino et al., 1998; O'Malley et al., 1999). Little is known about the recycling, storage, and modification of the siderophore. The secretion of *E. coli* enterobactin is mediated by the membrane exporter protein EntS (Furrer et al., 2002; Danese et al., 2004).

In *V. parahaemolyticus* the genes encoding both for the TonB1 and TonB2 systems are located on the small chromosome, and the TonB3 system on the large chromosome (O'Malley et al., 1999; Kuehl and Crosa, 2010; Tanabe et al., 2012). In *V. parahaemolyticus* TonB2 is most active than TonB1 in providing the energy necessary for the transport of ferricenterobactin via the receptors IrgA and VctA (Tanabe et al., 2012). However, this is different in *V. cholerae*, during the transport of ferric-enterobactin the energy required for IrgA and VctA receptors is provided by the TonB2 system (Seliger et al., 2001; Tanabe et al., 2012). The TonB3 system is not implicated in the transport of iron either *V. parahaemolyticus* or *V. cholerae* (Kuehl and Crosa, 2010; Tanabe et al., 2012),

has been reported that the TonB3 system from *V. vulnificus* is induced when the bacterium grows in human serum (Alice and Crosa, 2012). The *V. cholerae* has two TonB systems, which are present on small chromosome (this is different from *V. parahaemolyticus*), and those encoding the TonB2 system are located on the large chromosome. They have unique as well as common functions (Seliger et al., 2001). Both mediate the transport of hemin, vibriobactin, and ferrichrome. However, only TonB1 participates in the use of the siderophore schizokinen, but TonB2 is required for the transport of enterobactin (Seliger et al., 2001).

With respect to FhuA, this protein is the receptor for the siderophores desferri-ferrichrome and aerobactin in *V. parahaemolyticus* (Funahashi et al., 2009). In addition, FhuB, FhuC, and FhuD apparently are involved in the transport of the siderophores, and also are present in *V. cholerae* (Rogers et al., 2000; Funahashi et al., 2009). Regarding the hut genes, it has been reported that HutA is the receptor for the uptake of heme. In addition, HutR has significant homology to HutA as well as to other outer membrane heme receptors (Occhino et al., 1998; Mey and Payne, 2001). In the *V. cholerae* the presence of hutBCD stimulated growth when hemin was the iron source, but these genes were not essential for hemin utilization (Occhino et al., 1998; Mey and Payne, 2001; Wyckoff et al., 2004). Other genes found in the *V. parahaemolyticus* genome were the feo system. The feo system consists of genes that encoded proteins involved in the transport of ferrous iron (Fe2+), which is expected to be a major iron source in the intestine (Cartron et al., 2006). This Fe2+ iron transport system feo is widely distributed among bacterial species such as *V. cholerae.* In this bacterium, the feo operon consists of three genes, feoABC. FeoB is an 83-kDa protein involving in the pore formation for iron transport (Weaver et al., 2013). FeoA and FeoC are all required for iron acquisition; however, their functions have not been described in detail. Apparently, in the genome of *V. parahaemolyticus* there are genes that encode for this Fe2+ transport. Moreover, the *V. parahaemolyticus* contains other iron transporters such as FbpA and FbpB. In *Pasteurella haemolytica* the presence of FbpABC family of iron uptake systems has been documented (Kirby et al., 1998). This family of proteins is involved in the utilization and transport of the ferric-xenosiderophore of the bacterium *N. gonorrhoeae,* and is independent of the TonB system (Strange et al., 2011). We speculate that *V. parahaemolyticus* bacteria could have this family of proteins in order to acquire ferric iron from xenosiderophores, in a TonB-independient manner. All of these Fe acquisition systems could be likely involved in the survival of *V. parahaemolyticus* and other *Vibrio* sp. in the different environments that they can colonize, i.e., water, humans, and several other vertebrate hosts (Fouz et al., 1997).

### Conclusion

The element Fe is essential for the growth of pathogenic microorganisms, is fundamental and necessary for establishment and replication inside a host, and is required to cause infection. To this end, microbes that live in hostile environments and extracellular spaces of their host must employ different strategies for Fe acquisition to be successful in these niches. It has been postulated that such strategies were acquired during evolution and are involved in the pathogenesis and virulence of bacteria such as *V. parahaemolyticus*. Based on reported findings, this bacterium can utilize the Fe from the proteins Tf, Hb, and hemin by means of siderophores (vibrioferrin, aerobactin, and desferri-ferrichrome) and likely also receptors to acquire Fe from humans during infection of the gut. In low iron, *V. parahaemolyticus* express two proteins of 78 and 83 kDa (now called PvuA2 and PvuA1; respectively), which are the receptors for the siderophore vibrioferrin, and a protease of 43 kDa, which has been hypothesized is involved in one of the strategies of *V. parahaemolyticus* in order to acquire iron from the host. *V. parahaemolyticus* also encodes for LutA, which is the receptor for the siderophore aerobactin. Additionally, according to **Table 1** and other works, this pathogen possesses genes that encode accessory proteins involved in Fe acquisition, transport and synthesis of molecules implicated in Fe acquisition systems.

The iron *per se* has been involved in increase the virulence of *V. parahaemolyticus* and other bacteria. For example, in recent works it have been demonstrated that iron uptake and Quorum sensing (QS) can act together as global regulators of bacterial virulence factors (Wen et al., 2012). QS is a regulatory mechanism used by several bacteria to regulate or modulate the production of extracellular compounds at high cell densities with the aim of establish bacterial biofilms (nowadays, the main medical problem for the control of infectious diseases). Bacterial QS serves as simple indicator of population density, by means of secreting signaling molecules called autoinducers. The link among iron and QS was reported firstly in *Pseudomonas aeuruginos*a (Bollinger et al., 2001). This bacterium in iron-depleted conditions, retarded biofilm formation and increased the twitching motility and expression of QS-related genes, suggesting a link between iron and QS system during biofilm formation (the most important virulence factor of *P. aeruginosa;* Cai et al., 2010), in contrast; *Staphylococcus aureus* in Fe limitation appeared to stimulate biofilm formation (Johnson et al., 2005). These controversial observations can be explained because biofilm formation QSdependent is nutritionally conditional (Shrout et al., 2006). In other words, in the absence of an acquisition system needed for obtain nutritional iron, or other nutrients such as carbon sources, a bacterium such as *P. aeuruginosa* could establish thin

TABLE 2 | Genes related to quorum sensing in *Vibrio cholerae* and putative genes in *Vibrio parahaemolyticus*.

and weak biofilms instead of mature biofilms (Banin et al., 2005; Shrout et al., 2006). At intracellular level the main regulator is Fur. Therefore, Fur regulates genes that are crucial for the iron acquisition needed for the bacteria replication and consequently; in biofilms development (Banin et al., 2005).

Until now, the association between iron and QS on biofilms formed by *V. parahaemolyticus* has not been studied. The results of a BLAST search indicate that some strains of V*. parahaemolyticus* have genes involved in QS and biofilm formation (luxP, luxQ, cqsA, luxO, hapR, aphA), with high identity for those reported in the *V. cholerae* (Jobling and Holmes, 1997; Skorupski and Taylor, 1999; Miller et al., 2002; Zhu and Mekalanos, 2003; Raychaudhuri et al., 2006; **Table 2**). We speculate that iron and QS could be involved in the virulence of *V. parahaemolyticus*. In the *V. vulnificus* the biosynthesis of the siderophore vulnibactin is regulated by Fur and QS (Wen et al., 2012; Kim et al., 2013). Once vulnibactin sequesters the iron needed for the replication of the bacterium, *V. vulnificus* catalizes the enzyme LuxS, which synthetizes the autoinducer (AI-2), involved in the activation of signals for QS and Biofilm formation. At high cell density, *V. vulnificus* enhances the expression of the gene *vvpE*, which encodes for the virulence factor elastase (Kim et al., 2013). It has been observed that mutations in LuxS reduce not only biofilm formation, also reduce virulence factors such as motility, production of proteases and the secretion of the *V. vulnificus* hemolysin, etc. (Wen et al., 2012).

Although the link between Fur and QS is complex, the siderophores production and the coordinated regulation by the two systems (Fur and QS) probably ensures to bacteria maintain an appropriate iron concentration to optimize its survival and propagation within the human host (Wen et al., 2012). It has been shown that blocking the nutritional support

### References

and the communication pathways of one's adversaries serves as an effective tactic to disrupt cooperative actions among individuals or groups. Removal of iron as a therapeutic approach has been investigated *in vitro* for several infections, with promising results (Gorska et al., 2014). The generation of analogs that block or alternative signals involved in QS have been developed, in order to disrupt biofilm formation and other virulence factors (LaSarre and Federle, 2013). These strategies could be success in bacteria, because Fe limitation and Fe excess affect QS-dependent biofilm formation, therefore understand how these sophisticated and complex regulatory systems are regulated, is vital to predict bacterial behaviors and possibly then, develop drugs that can interfere with the iron acquisitions systems, or with the response of signal molecules involved in iron acquisition systems or QS (Bollinger et al., 2001; Wen et al., 2012; LaSarre and Federle, 2013).

When the host tries to limit infection by lowering iron, pathogens such as *V. parahaemolyticus* triggered increased expression of virulence factors (that are relevant for surface colonization and infection) in order to cause damage to the host. Based in these results, we conclude that unnecessary or excessive iron administration may be harmful, due the possible multiplication of bacterial growth and increase in their virulence. While it is clear that iron levels are important in infection, it is not an easy task to control their levels in the host. The complete detailed mechanism for Fe acquisition and its role in *V. parahaemolyticus* virulence remains to be determined.

### Acknowledgment

This work was supported by a grant from CONACyT México (CB-2014- 236546).

for vanchrobactin analogues. *Appl. Environ. Microbiol.* 75, 2775–2783. doi: 10.1128/AEM.02897-08

in *Vibrio vulnificus*: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases. *Mol. Microbiol.* 88, 813–826. doi: 10.1111/mmi.12224

**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 © 2015 León-Sicairos, Angulo-Zamudio, de la Garza, Velázquez-Román, Flores-Villaseñor and Canizalez-Román. 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.*

# An insight of traditional plasmid curing in *Vibrio* species

*Vengadesh Letchumanan1,2, Kok-Gan Chan2 and Learn-Han Lee1\**

*1 Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Malaysia, 2 Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia*

As the causative agent of foodborne related illness, *Vibrio* species causes a huge impact on the public health and management. *Vibrio* species is often associated with seafood as the latter plays a role as a vehicle to transmit bacterial infections. Hence, antibiotics are used not to promote growth but rather to prevent and treat bacterial infections. The extensive use of antibiotics in the aquaculture industry and environment has led to the emerging of antibiotic resistant strains. This phenomenon has triggered an alarming public health concern due to the increase number of pathogenic *Vibrio* strains that are resistant to clinically used antibiotics and is found in the environment. Antibiotic resistance and the genes location in the strains can be detected through plasmid curing assay. The results derived from plasmid curing assay is fast, cost effective, sufficient in providing insights, and influence the antibiotic management policies in the aquaculture industry. This presentation aims in discussing and providing insights on various curing agents in *Vibrio* species. To our best of knowledge, this is a first review written discussing on plasmid curing in *Vibrio* species.

Keywords: *Vibrio* species, antibiotic, resistance, plasmids, foodborne pathogens

### Introduction

Antibiotic resistant infection has become more challenging to treat with existing antibiotics, leading to infections triggering higher morbidity and mortality, imposing huge costs on our society (Carlet et al., 2011; de Kraker et al., 2011; Finley et al., 2013). This increasing resistance involves many human pathogens including *Vibrio* species. *Vibrio* are Gram-negative bacteria possessing a curved rod shape that naturally inhabits the estuarine and marine environment worldwide (Hazen et al., 2010; Letchumanan et al., 2014; Raghunath, 2015). The presence of this bacterium in the marine environment raises human concern on food safety due to the latter potentially causing disease outbreaks depending on the environmental conditions (Ceccarelli et al., 2013). *Vibrio cholerae*, *V. parahaemolyticus*, *V. vulnificus*, *V. mimicus*, *V. fluvialis*, and *V. alginolyticus* are among the species commonly known to cause human illnesses (Theresa and Kumar, 2014).

The occurrence of multidrug-resistant (MDR) bacteria to clinically used antibiotics is a major health issue and a great challenge to the worldwide drug discovery programs (Alanis, 2005). It is well documented that both clinical and environmental *Vibrio* strains harbors antibiotic resistance traits (Letchumanan et al., 2015; Shrestha et al., 2015; Zavala-Norzagaray et al., 2015). A recent study in Iran has reported multidrug resistance profile towards erythromycin, sulfamethoxazole– trimethoprim and ampicillin in *V. cholerae* isolated from clinical samples (Tabatabaei and Khorashad, 2015). In India, serogroups O1 of *V. cholerae* classical biotype and sub serotype, Ogawa was identified among the *V. cholerae* isolated from clinical strains. All the isolates were

#### *Edited by:*

*Pendru Raghunath, Dr. VRK Women's Medical College, India*

#### *Reviewed by:*

*Weili Liang, National Institute for Communicable Disease Control and Prevention – Chinese Center for Disease Control and Prevention, China Adrian Canizalez-Roman, Autonomous University of Sinaloa, Mexico*

#### *\*Correspondence:*

*Learn-Han Lee, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 27 March 2015 Accepted: 06 July 2015 Published: 17 July 2015*

#### *Citation:*