Studies on prevalence, serotype, antimicrobial resistance and clonal relationship of Salmonella in foods, waters and humans in Ho Chi Minh City, Vietnam

概要

Introduction
Foodborne diseases caused by non-typhoidal Salmonella are a very important public health problem all over the world with 85% of those cases being linked to contaminated foods and waters. Non-typhoidal Salmonella gastroenteritis accounts for 80.3 million global cases of foodborne illness each year. Particularly, Salmonella was the fourth most frequent causative agent of infant diarrhea in southern Vietnam and invasive non-typhoidal Salmonella disease was leading to a high mortality rate (26%) in HIV patients in Vietnam. Notwithstanding, there was no nationwide surveillance program for monitoring Salmonella food contamination. Emergence and spread of multidrug resistant (MDR) bacteria, particularly MDR-Salmonella, have become a major public health concern. This phenomenon occurred by selective pressure due to inappropriate usage of antimicrobials in humans and animals. Along with these reasons, spread of antimicrobial resistant bacteria has contributed to the acquisition of antimicrobial resistance (AMR) determinant through horizontal gene transfer. Currently, extended- spectrum β-lactamase (ESBL) and AmpC-β-lactamase (AmpC)-producing Salmonella, which can hydrolyze third-generation cephalosporins, are also prevalent in many countries. ESBL and ampC genes are often located on large plasmids, which also carry other AMR genes including fluoroquinolones. Nevertheless, cephalosporin and fluoroquinolone are used to treat the Salmonella infections in humans. Therefore, this is a serious threat to global public health because of limited treatment options. Because of emergence of MDR bacteria, colistin, a reused antimicrobial, is a crucial last-resort for the treatment of MDR infections. However, the occurrence of plasmid-mediated colistin resistance (Mcr) has been increasing in many countries. In addition, there is paucity of information about the presence of plasmid-mediated colistin resistant Salmonella in Vietnam. Currently, presence of Salmonella in different foods (meat, fish, vegetable and fruit) and waters as well as in humans (symptomatic and asymptomatic), their antimicrobial resistance profile, and the genetic relatedness of Salmonella serotype, which were concurrently detected in humans and foods/waters, have been lacking in Vietnam.

In this study, we investigated the prevalence of Salmonella and their serotype in symptomatic and asymptomatic humans, raw meats, fishery products, vegetables, fruits and waters in Ho Chi Minh City (HCMC), Vietnam. Furthermore, phenotypic and genotypic characteristics of the isolates were examined particularly focusing on ESBL and Mcr.

Chapter 1. Prevalence, serotype and antimicrobial resistance of non-typhoidal Salmonella isolates in raw meat, fish and shrimp samples in Ho Chi Minh City, Vietnam
Prevalence, serotype and AMR of Salmonella isolates in a total of 409 samples (255 raw meats and 154 seafoods) collected between October 2012 and March 2015 from slaughterhouses, wholesale fish market and retail markets in HCMC, Vietnam were examined. A high rate of Salmonella contamination was detected in pork (69/99, 70%), poultry (47/72, 65%), beef (49/84, 58%), shrimp (26/53, 49%), and farmed freshwater fish samples (37/100, 37%). A total of 53 Salmonella serotypes were found, of which S. Rissen (38) was most prevalent, followed by S. Weltevreden (24), S. London (23), S. Anatum (22), S. Typhimurium (19) and S. Corvallis (18). In addition, 4 monophasic S. Typhimurium strains were identified using a PCR for the detection of a specific IS200 fragment within the fliB-fliA intergenic region. A total of 336 Salmonella isolates were examined for antimicrobial susceprtibility and had a high prevalence (62%) of resistance to antimicrobial agents, particularly tetracycline (TET) (53%), ampicillin (AMP) (44%), chloramphenicol (CHL) (38%), and trimethoprim/sulfamethoxazole (SXT) (31%). Salmonella with MDR being resistance to three or more classes of antimicrobials were commonly found (40%) and, S. Saintpaul and S. Bovismorbificans exhibited resistance to 6 classes of antimicrobials (3.3%). All 7 S. Indiana isolates were resistant against 4 to 6 classes of antimicrobials including ciprofloxacin (CIP), which is used for the treatment of human Salmonella infections. Moreover, plasmid-located blaCMY-2 gene was identified in 4 S. Braenderup and 1 S. Typhimurium isolated from poultry and plasmid-located blaCTX-M-55 and blaTEM-1 genes were detected in 1 S. Bovismorbificans and 1 S. Newport isolated from fish. None of Salmonella isolates were positive for mcr-1~8 genes. This study indicated that MDR Salmonella is widely disseminated not only in meats but also in seafood in the food distribution system of HCMC, Vietnam.

Chapter 2. Prevalence, serotype and antimicrobial resistance of non-typhoidal Salmonella in vegetable, fruit, and water samples in Ho Chi Minh City, Vietnam
Next, prevalence, serotype and AMR of Salmonella in vegetable, fruit and water samples in HCMC, Vietnam were examined. Salmonella was isolated from 75% (30/40), 57% (12/21), 18% (28/160), and 2.5% (1/40) of river water, irrigation water, vegetable and ice water samples, respectively. However, no Salmonella was isolated from 160 fruit and 40 tap water samples examined. A total of 102 isolates, obtained from the 71 samples, belonging to 33 different serotypes, of which S. Rissen (10 isolates) was most prevalent, followed by S. London (9), S. Hvittingfoss (8), S. Newport (7) and S. Weltevreden (7). It should be emphasized that there was the marked similarity in serotypes of Salmonella isolates between vegetables and river or irrigation water. AMR was most prevalent against TET (35%), followed by CHL (34%), AMP (31%), SXT (24%) and nalidixic acid (NAL) (11%). In water samples, S. Agona harboring plasmid containing blaCMY-2 and blaTEM-1 genes was identified. However, mcr-1~8 genes were not detected in all the 102 Salmonella isolates. Of the 102 isolates, 52 (51%) showed resistance to at least 1 antimicrobial class while 28 (28%) showed MDR phenotype. Taken together, these data indicated that environmental water and vegetable but not fruit in HCMC were contaminated with Salmonella including MDR strains, and environmental water might be the source of Salmonella contamination in vegetables.

Chapter 3. Prevalence, serotype and antimicrobial resistance of non-typhoidal Salmonella in fecal samples of diarrheal and healthy humans in Ho Chi Minh City, Vietnam
Fecal specimens were collected from 306 diarrheal and 380 healthy humans, and 30 Salmonella were isolated from 14 patients (15 isolates) and 12 healthy humans (15) by conventional methods, corresponding to 4.6% and 3.2% of positive rate of Salmonella, respectively. Fifteen Salmonella strains of each matrix were grouped into 10 Salmonella serotypes. A total of 18 Salmonella serotypes were identified, of which S. Kentucky (4) was most prevalent, followed by S. Enteritidis (3) and S. Indiana (3). AMR was most prevalent against AMP (73%), followed by TET (70%), CHL (60%), SXT (50%) and NAL (43%). Of the 30 isolates, 24 (80%) showed resistance to at least 1 antimicrobial class while 20 (67%) showed MDR phenotype. Plasmid-mediated blaCTX-M-55 and blaTEM-1 genes were detected in S. Indiana (3) and monophasic S. Typhimurium (1) isolated from healthy and diarrheal humans, respectively. The chromosomally located blaCTX-M-15 and plasmid- located blaCTX-M-65 harboring S. Kentucky (2) and S. Infantis (1) were also isolated from diarrheal patients, respectively. Notably, all ESBL-producing Salmonella were resistant to 6 or 7 antimicrobial classes. Especially, ESBL-producing S. Kentucky (2) and S. Indiana (3) isolates were co-resistant to CIP. Noteworthily, a monophasic S. Typhimurium co-harboring mcr-3 and blaCTX-M-65 genes was identified in a patient sample and mcr-3 gene was located on plasmid. These data indicate that pevalence of Salmonella in diarrheal patient and healthy human was relatively low but Salmonella exhibited MDR, especially against CIP and third generation cephalosporin, are a serious risk to public health.

Chapter 4. Transferability of mcr-3, blaCTX-M-1, blaCTX-M-9 and blaCMY-2 genes, and genetic relatedness between Salmonella serotypes concurrent in human and food/ water samples
As described in Chapter 3, blaCTX-M-1, blaCTX-M-9, blaTEM-1, blaCMY-2 and mcr-3 genes were located on plasmid. Transferability of the R-plasmid was examined by a conjugation assay. All blaCMY- 2, blaCTX-M-65 and mcr-3 gene-harboring plasmids were transferred from Salmonella donor strains to a recipient E. coli. blaCTX-M-55 and blaTEM-1 gene- harboring plasmids were transferred to a recipient E. coli from 3 Salmonella donor strains but not 6. Moreover, transconjugants harboring ESBLs were co- resistant to TET, CHL, kanamycin and SXT and reduced the susceptibility to gentamicin, CIP and NAL. These data indicated that ESBL-, ampC- and mcr-gene harboring Salmonella in food, water and human could be a potential reservoir and source of dissemination of plasmid-mediated ESBL, ampC, mcr and other AMR genes, contribute to spread MDR, and pose a serious concern for public health.

Representational Salmonella serotypes that were concurrently detected in humans and foods/ waters were analyzed for plasmid replicon profile and the genetic relatedness by MLST and PFGE methods. Among 18 plasmid replicons belonging to Enterobacteriacea, the most popular one was IncHI1 (41%), followed by IncFIIAs (12%), IncN (5.8%), IncI1 (5.8%), IncA/C (2.9%) and untypable (41%). Moreover, XbaI-digested PFGE profile and MLST of Salmonella generated 17 major clusters and 10 STs, respectively. S. Typhimurium and monophasic S. Typhimurium were divided into 3 STs: ST19, ST34 and ST36 and corresponding to 3 PFGE patterns. Six S. Enteritidis strains belonging to ST11 and IncFIIAs were comprised of 3 clusters based on the AMR profile and their sources. Twelve S. Kentucky strains were divided into 3 STs: ST198, ST314 and ST2410, and corresponding to 3 clusters, in which ST314 isolate was susceptible to all the tested antimicrobials, while ST198 isolates were MDR including CIP. Ten S. Indiana strains fell the same sequence type, ST17, but divided into 2 PFGE patterns. Taken together, there were strong correlations of STs, PFGE patterns and serotypes and a good association among STs, PFGE patterns and antimicrobial resistance phenotype in S. Kentucky. These data suggest that the genetic characterization of Salmonella strains belonging to the same serotype that was derived from different sources might be closely related, except for isolates from irrigation samples. Impressively, MDR S. Indiana ST17, MDR S. Kentucky ST198, and MDR monophasic S. Typhimurium ST34, a trend of the world, were also isolated in this study. Meats, fishery products, vegetables, river waters might be the source and reservoir of Salmonella as well as antimicrobial resistance genes for humans in HCMC, Vietnam. It is recommended that the Vietnamese government needs to develop the national food safety monitoring and surveillance system, as well as the wastewater treatment system in HCMC.

Conclusions
In this study, Salmonella were detected in various foods, waters, healthy individuals and diarrheal patients in HCMC, Vietnam. In addition, Salmonella in foods, water and humans could be the potential sources of AMR genes including ESBLs, ampC, and mcr genes. Besides, some AMR determinants were successfully transferred among bacteria through plasmid, indicating a challenge of public health. Moreover, the genetic relatedness of representative Salmonella serotypes from human and food/water were closely related. Therefore, meat, fishery product, vegetable and river water might be the sources of Salmonella contamination as well as AMR determinants for humans in HCMC, Vietnam. These data suggest that Vietnamese government needs to develop and improve the national foodborne disease surveillance and AMR surveillance system for restriction of foodborne disease as well as the spread of AMR bacteria.