RESUMEN

Con el objetivo de determinar la diversidad de variantes patogénicas de Vibrio parahaemolyticus en el Perú durante el periodo 1995-2017, se analizaron 102 genomas peruanos (97 clínicos y 5 ambientales) empleando el esquema de tipificación multilocus y BLASTn para la búsqueda de genes de virulencia. Se identificaron 15 tipos de secuencia diferentes, encontrándose que el genotipo ST3, perteneciente al clon pandémico, fue el más abundante, con 52% (n=53); seguido por el ST120, con 23,5% (n=24); y el complejo clonal CC345, con 11,8% (n=12). Un total de 89 cepas analizadas presentaron genes que codifican la isla de patogenicidad VpaI-7 (87,3%), mientras que 96 presentaron el gen tdh (94,1%), y 6, el trh (5,9%). Durante el periodo evaluado, se resalta la predominancia del ST3, causante de un importante brote en el pasado del Perú, además de otros genotipos patógenos que representan un riesgo latente en salud pública asociado al consumo de alimentos marinos.

Palabras clave:
Vibrio parahaemolyticus; Salud Pública; Monitoreo Epidemiológico; Tipificación Molecular; Secuenciación Completa del Genoma

ABSTRACT

During the period from 1995 to 2017, in order to determine the diversity of Vibrio parahaemolyticus pathogenic variants in Peru, 102 Peruvian genomes (97 from a hospital setting and 5 from an out-of-hospital setting) were analyzed using the multilocus typification scheme and BLASTn in the search for virulence genes. Fifteen different sequence types were identified. It was found that the ST3 genotype, which is found in the pandemic clone, was the most abundant, with 52% (n=53); followed by ST120, with 23.5% (n=24); and the CC345 clonal complex, with 11.8% (n=12). A total of 89 analyzed strains presented genes encoding the pathogenicity island VpaI-7 (87.3%), while 96 presented the tdh gene (94.1%), and 6 the trh gene (5.9%). The ST3 genotype was the predominant one during the evaluated period, this genotype was the cause of a major outbreak in Peru’s past history. Other pathogenic genotypes found represent a latent public health risk associated with seafood consumption.

Keywords:
Vibrio parahaemolyticus; Public Health, Epidemiological Monitoring; Molecular Typing; Whole Genome Sequencing

INTRODUCTION

The presence of pathogenic bacteria in the marine environment increases interest in food safety because of their potential to cause outbreaks. Among them, the Vibrio parahaemolyticus stands out, a halophilic gram-negative bacteria widely distributed in coastal ecosystems, whose serotyping depends on somatic (O) and capsular (K) antigens produced under various environmental conditions ¹1. Banerjee SK, Kearney AK, Nadon CA, Peterson C-L, Tyler K, Bakouche L, et al. Phenotypic and Genotypic Characterization of Canadian Clinical Isolates of Vibrio parahaemolyticus Collected from 2000 to 2009. J Clin Microbiol. 2014;52(4):1081-8. doi: 10.1128/JCM.03047-13.
https://doi.org/10.1128/JCM.03047-13... .

Interest in V. parahaemolyticus began many years ago, after it was found to be the causal agent of foodborne infections in an outbreak in Japan. Historically, V. parahaemolyticus has been responsible for 20-30% of cases of foodborne infection in Japan and other Asian countries ²2. Alam MJ, Tomochika KI, Miyoshi SI, Shinoda S. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol Lett. 2002;208(1):83-7. doi: 10.1111/j.1574-6968.2002.tb11064.x.
https://doi.org/10.1111/j.1574-6968.2002... . Peru has recorded significant outbreaks since 1997, which have been associated with climate changes that are part of the El Niño phenomenon. These climate changes alter marine ecological conditions, for example, increasing the rate of plankton abundance ³3. Aliaga R, Miranda J, Zevallos J. Aislamiento e identificación de Vibrio parahaemolyticus O3: K6 en pescados y moluscos bivalvos procedentes de un mercado pesquero de Lima, Perú. Rev Medica Hered. 2010;21(3):139-45. doi: 10.20453/rmh.v21i3.1123.
https://doi.org/10.20453/rmh.v21i3.1123... . Most of these reports associate thermostable direct hemolysin (TDH) with the virulence of V. parahaemolyticus⁴4. Nair GB, Ramamurthy T, Bhattacharya SK, Dutta B, Takeda Y, Sack DA. Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants. Clin Microbiol Rev. 2007;20(1):39-48. doi: 10.1128/CMR.00025-06.
https://doi.org/10.1128/CMR.00025-06... .

Currently, the global prevalence and emergence of V. parahaemolyticus infection is increasing, underlining the need for adequate surveillance of this pathogen. Conventional microbiology is insufficient to determine pathogenic variants and their geographical distribution. In contrast, molecular epidemiology tools, such as the multilocus sequence typing (MLST), are proposed as new alternatives to study infectious diseases based on molecular strain differentiation. This scheme allows the rapid genotypic characterization of microorganisms, due to the development of a centralized database (PubMLST) that allows the comparison of different genetic variants called sequence types (ST) and the delineation of potential dispersion routes⁵5. Gonzalez-Escalona N, Jolley KA, Reed E, Martinez-Urtaza J. Defining a Core Genome Multilocus Sequence Typing Scheme for the Global Epidemiology of Vibrio parahaemolyticus. J Clin Microbiol. 2017;55(6):1682-97. doi: 10.1128/JCM.00227-17.
https://doi.org/10.1128/JCM.00227-17... .

The objective of this study was to determine the genetic variants of pathogenic isolates of V. parahaemolyticus associated with human cases and seafood circulating in Peru during 1995-2017, using the MLST technique and in silico detection of virulence genes.

THE STUDY

A total of 16 strains were submitted as V. parahaemolyticus from the collection of the National Reference Laboratory of Enteropathogens of the Instituto Nacional de Salud (INS) (Bioproject: PRJNA556706) and also 86 Peruvian genomes available in the NCBI database (http://www.ncbi.nlm.nih.gov) were included for MLST analysis (Table 1).

The strains were recovered in alkaline peptone water (Merck, Germany) at 37 °C for 68 hours. Subsequently, they were seeded by striae on bile-esculin citrate thiosulfate agar plates (Merck, Germany), and incubated at 37 °C for 18 to 24 hours. The genus Vibrio was confirmed using conventional biochemical tests, and the species V. parahaemolyticus by PCR for the presence of the toxR gene described by Kim et al. ⁽⁶6. Kim YB, Okuda J, Matsumoto C, Takahashi N, Hashimoto S, Nishibuchi M. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene. J Clin Microbiol. 1999;37(4):1173-7..

DNA extraction was performed using the DNeasy Blood &Tissue kit (Qiagen, Germany). DNA concentration and purity were evaluated by spectrophotometry (Denovix, USA). Sequencing libraries were developed using the Nextera XT kit (Illumina, USA), and genomic sequencing was performed using the MiSeq high throughput sequencer (Illumina, USA) ⁷7. Quino W, Hurtado CV, Escalante-Maldonado O, Flores-León D, Mestanza O, Vences-Rosales F, et al. Multidrogorresistencia de Salmonella infantis en Perú: un estudio mediante secuenciamiento de nueva generación. Rev Peru Med Exp Salud Pública. 2019;36(1):37-45. doi: 10.17843/rpmesp.2019.361.3934.
https://doi.org/10.17843/rpmesp.2019.361... . The quality of the sequences obtained was evaluated using FastQC v0.11.5. The sequences were assembled de novo using the A5-miseq pipeline ⁸8. Coil D, Jospin G, Darling AE. A5-miseq: an updated pipeline to assemble microbial genomes from Illumina MiSeq data. Bioinforma Oxf Engl. 2015;31(4):587-9. doi: 10.1093/bioinformatics/btu661.
https://doi.org/10.1093/bioinformatics/b... . Identification of the genus and detection of contaminated contigs was carried out using the Kraken program ⁹9. Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014;15(3):R46. doi: 10.1186/gb-2014-15-3-r46.
https://doi.org/10.1186/gb-2014-15-3-r46... .

The allelic profiles and the genomes’ STs obtained were assigned according to the information in the MLST data base for V. parahaemolyticus (http://pubmlst.org/vparahaemolyticus), using the MLST v2.10 program, based on the seven-locus scheme described for V. parahaemolyticus⁵5. Gonzalez-Escalona N, Jolley KA, Reed E, Martinez-Urtaza J. Defining a Core Genome Multilocus Sequence Typing Scheme for the Global Epidemiology of Vibrio parahaemolyticus. J Clin Microbiol. 2017;55(6):1682-97. doi: 10.1128/JCM.00227-17.
https://doi.org/10.1128/JCM.00227-17... . Clonal complex assignment was performed using BioNumerics v7.5 (Applied Maths). Inclusion in a clonal complex (CC) was restricted to STs that shared at least 6 of the 7 alleles, while singletons were defined as STs that differed in two or more alleles from the other STs. With the same program a minimum spanning tree (MST) was generated showing the ST and the CC included in this work. In addition, a bar chart was constructed from the isolates studied using Infostat, to visualize genotypes by year of isolation and by number of samples.

The BLASTn tool was used to search for virulence factors of V. parahaemolyticus: the pathogenicity island type 7 (VpI-7) that internally contains the most common variant of TDH obtained from chromosome 2 of the RIMD genome 2210633 (access number: NC_004605.1) and TRH obtained from isolate AQ4299 (access number: LC271586.1), identifying as homologues those with <90% identity and <60% coverage of reference alignment. The code used for the annotation is available at http://github.com/OrsonMM/Blast-score-ratio-for-genomics. The results obtained were ordered in table format indicating the presence or absence of genes. All the sequences obtained during the study have been deposited in GenBank (Bioproject access number: PRJNA556706).

RESULTS

All 16 strains were confirmed as V. parahaemolyticus due to the presence of the ToxR gene. As for the genomic information, an average genomic size of 5.18 bp was obtained, composed by 80 contigs and a GC percentage of 45.2%.

A total of 102 genomes of Peruvian strains of clinical (97) and environmental (5) origin, isolated during 1995-2017, were analyzed (Table 1). The 102 genomes were classified into 15 different STs, which were grouped by year of isolation and origin of the isolate (Figure 1). It is observed that no isolates were obtained in 2004, 2010 and 2012.

The populational structure of the Peruvian V. parahaemolyticus strains (n=102), analyzed by MLST, can be visualized by means of a Minimum spanning tree graph (MST) (Figure 2). All strains belonging to the O3:K6 pandemic complex were grouped in the ST3 (n=53), representing 52% of the strains analyzed. In addition, clonal complex 345 (CC345) was identified, consisting of ST88 (n=3) and ST265 (n=9), both of serotype O4:K8, which differ at a single locus, with an isolation frequency of 11.8%. The remaining strains were included in 12 unrelated STs, while ST120 23.5% (n=24) and ST36 1.9% (n=2) were notable for being related to local or global outbreaks or epidemics (Annex 1).

From the 102 genomes analyzed, 89 isolates had the pathogenicity island, VpaI-7 (87.3%). Genes copies encoding TDH were found in 96 isolates (94.1%), the most frequent STs were ST3, ST36, ST88, ST120 and ST265. Genes for TRH were found only in ST36, ST64, ST65 and ST417, for a total of 6 isolates (5.9%). The results grouped by ST can be seen in Table 2, while the results per gene isolates composing VpaI-7 can be seen in Annex 2.

DISCUSSION

V. parahaemolyticus is a pathogen transmitted by high-demand food, yet little information is available on pathogenic variants and the temporal prevalence of its genotypes in Peru. This under-report of V. parahaemolyticus infections in the hospital setting is due to deficiencies in monitoring and research of foodborne diseases ¹⁰10. Martinez-Urtaza J, Lozano-Leon A, DePaola A, Ishibashi M, Shimada K, Nishibuchi M, et al. Characterization of Pathogenic Vibrio parahaemolyticus Isolates from Clinical Sources in Spain and Comparison with Asian and North American Pandemic Isolates. J Clin Microbiol. 2004;42(10): 4672-78. doi: 10.1128/JCM.42.10.4672-4678.2004.
https://doi.org/10.1128/JCM.42.10.4672-4... .

When analyzing the temporal prevalence of detected genotypes, the oldest isolates are found to correspond to the years 1995-1996, especially serotype O4:K8, but different genotypes: ST88 and ST265. Previous studies report this serotype since 1980, which caused sporadic cases and small outbreaks associated with raw seafood consumption; the highest prevalence was reported in 1983 ⁽¹¹11. Gavilan RG, Zamudio ML, Martinez-Urtaza J. Molecular epidemiology and genetic variation of pathogenic Vibrio parahaemolyticus in Peru. PLoS Negl Trop Dis. 2013;7(5):e2210. doi: 10.1371/journal.pntd.0002210.
https://doi.org/10.1371/journal.pntd.000... . Molecular surveillance studies revealed China as the origin of this serotype, which is composed of several genotypes that have no clonal relationship with the serotype O3:K6, suggesting CC345may be an important clonal complex. In addition, comparative genomics analyses revealed that these isolates presented the secretion system regions type 3 (T3SS) and VpaI-7 ¹²12. Li B, Yang X, Tan H, Ke B, He D, Ke C, et al. Vibrio parahaemolyticus O4:K8 forms a potential predominant clone in southern China as detected by whole-genome sequence analysis. Int J Food Microbiol. 2017;244:90-5. doi: 10.1016/j.ijfoodmicro.2017.01.001.
https://doi.org/10.1016/j.ijfoodmicro.20... , which is consistent with what was found in this study.

In the distribution per year, the presence of ST3 stands out; and MST is the genotype with the highest number of sequenced isolates, from strains isolated between 1997 and 2017. All of them had the complete VpaI-7. The first Peruvian outbreak of ST3 occurred in 1997; however, it is known to have emerged in India in 1996, expanding to the American continent ¹³13. Guerrero A, Lizárraga-Partida ML, Gil BG, Licea-Navarro AF, Revilla-Castellanos VJ, Wong-Chang I, et al. Genetic Analysis of Vibrio parahaemolyticus O3:K6 Strains That Have Been Isolated in Mexico Since 1998. PLoS ONE. 2017;12(1): e0169722. doi: 10.1371/journal.pone.0169722.
https://doi.org/10.1371/journal.pone.016... . This genotype belongs to the pandemic clonal complex CC3, which is distributed worldwide, and still is the dominant clone today ⁵5. Gonzalez-Escalona N, Jolley KA, Reed E, Martinez-Urtaza J. Defining a Core Genome Multilocus Sequence Typing Scheme for the Global Epidemiology of Vibrio parahaemolyticus. J Clin Microbiol. 2017;55(6):1682-97. doi: 10.1128/JCM.00227-17.
https://doi.org/10.1128/JCM.00227-17... . This clonal complex is the most studied genotype because it has the majority of VpaI reported for V. parahaemolyticus, being VpaI-7 the most important one, associated to cytotoxicity and enterotoxicity due to the presence of TDH and T3SS ¹⁴14. Ceccarelli D, Hasan NA, Huq A, Colwell RR. Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors. Front Cell Infect Microbiol . 2013;3:97. doi: 10.3389/fcimb.2013.00097.
https://doi.org/10.3389/fcimb.2013.00097... .

ST120 is the second group with the highest number of isolates in this study, obtained mostly during 2009, due to a serotype O3:K59 outbreak in the northern regions of the country ¹⁵15. Zamudio ML, Meza A, Bailón H, Martinez-Urtaza J, Campos J. Experiences in the epidemiological surveillance of foodborne pathogens by pulsed field gel electrophoresis (PFGE) in Peru. Rev Peru Med Exp Salud Publica. 2011;28(1):128-35.. After the application of molecular surveillance by MLST it was defined to be ST120, which originated from China, and represents the third introduction of pathogenic populations of V. parahaemolyticus¹⁶16. Gonzalez-Escalona N, Gavilan RG, Toro M, Zamudio ML, Martinez-Urtaza J. Outbreak of Vibrio parahaemolyticus Sequence Type 120, Peru, 2009. Emerg Infect Dis. 2016;22(7):1235-7. doi: 10.3201/eid2207.151896.
https://doi.org/10.3201/eid2207.151896... . In the results, the presence of this genotype during 2001, many years before its first report, is noteworthy, which would allow to reconsider when was the introduction of this genotype in Peru. Comparative genomic studies would be necessary to find differences between these isolates and those that caused the 2009 outbreak, which share the presence of VpaI-7.

Additionally, ST36, a small group of epidemiological importance within the Peruvian isolates analyzed, has its origin in the Pacific Northwest region of North America causing outbreaks in the United States and Canada ¹1. Banerjee SK, Kearney AK, Nadon CA, Peterson C-L, Tyler K, Bakouche L, et al. Phenotypic and Genotypic Characterization of Canadian Clinical Isolates of Vibrio parahaemolyticus Collected from 2000 to 2009. J Clin Microbiol. 2014;52(4):1081-8. doi: 10.1128/JCM.03047-13.
https://doi.org/10.1128/JCM.03047-13... . It has expanded since 2012 to rapidly reach other geographical areas such as northwestern Spain ¹⁷17. Martinez-Urtaza J, Aerle RV, Marin MA, Haendiges J, Myers RA, Trinanes J, et al. Genomic variation and evolution of vibrio parahaemolyticus ST36 over the course of a transcontinental epidemic expansion. mBio. 2017;8(6):e01425-17. doi: 10.1128/mBio.01425-17.
https://doi.org/10.1128/mBio.01425-17... . Based on our results, the first strains belonging to this ST appeared in Peru in 2011, as part of the expansion of this clone in the Pacific ¹⁸18. Abanto M, Gavilan RG, Baker-Austin C, Gonzalez-Escalona N, Martinez-Urtaza J. Global Expansion of Pacific Northwest Vibrio parahaemolyticus Sequence Type 36. Emerging Infectious Diseases. 2020;26(2):323-6. doi: 10.3201/eid2602.190362.
https://doi.org/10.3201/eid2602.190362... . Although no outbreak of this genotype has been reported, it represents a latent epidemiological risk due to its pathogenic potential because of the presence of TDH and TRH. In this aspect, molecular surveillance by MLST can be applied for the timely tracking of these isolates and to curb outbreaks.

No information was found about other STs causing outbreaks or epidemics around the world. However, their pathogenic potential is not ruled out, due to the presence of genes encoding TDH or TRH, results that include genotypes such as ST1169 and ST1737.

Virulence factor analysis detected the presence of the genes encoding TDH in many clinical isolates, particularly in the genotypes with the highest number of isolates analyzed. It is known that the genes encoding TDH are mostly located within VpaI-7, so the presence of this region will have an impact on the increase in virulence of V. parahaemolyticus¹⁹19. Raghunath P. Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus. Front Microbiol. 2015;5:805. doi: 10.3389/fmicb.2014.00805.
https://doi.org/10.3389/fmicb.2014.00805... . However, the genetic deletion of the copies of the tdh gene or of the complete VpaI-7 does not determine the absence of virulence ¹⁴14. Ceccarelli D, Hasan NA, Huq A, Colwell RR. Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors. Front Cell Infect Microbiol . 2013;3:97. doi: 10.3389/fcimb.2013.00097.
https://doi.org/10.3389/fcimb.2013.00097... , which explains the detection of clinical strains with absence of TDH. On the other hand, genomes with TDH but without VpaI-7 were detected, which had already been described as TDH variants not associated with VpaI-7 ²⁰20. Okada N, Iida T, Park KS, Goto N, Yasunaga T, Hiyoshi H, et al. Identification and characterization of a novel type III secretion system in trh-positive Vibrio parahaemolyticus strain TH3996 reveal genetic lineage and diversity of pathogenic machinery beyond the species level. Infection and immunity. 2009;77(2):904-13. doi: 10.1128/IAI.01184-08.
https://doi.org/10.1128/IAI.01184-08... . Finally, the gene encoding TRH, which causes a similar effect to TDH, was found in a very small group of isolates.

In conclusion, ST3 is both temporally and quantitatively predominant in Peru, that is why still today, it is a genotype that generates risk in public health associated with the consumption of raw or semi-raw seafood. Notably, its pathogenic potential is due to the presence of VpaI-7, carrier of hemolysins. This, added to the underestimated epidemiological data, as well as the circulation of other pathogenic variants of these bacteria in the country, indicates that bigger efforts in molecular surveillance are needed, because this method is proving to be a powerful tool to detect and control outbreaks and infections.

Acknowledgements:

To Gustavo Bellido Mendoza for his invaluable technical support and to all the team of the National Reference Laboratory of Enteropathogens of the Instituto Nacional de Salud for the support provided during the realization of this work.

References

Publication Dates

History