Dietary supplementation with probiotic Rhodobacter sphaeroides SS15 extract to control acute hepatopancreatic necrosis disease (AHPND)-causing vibrio parahaemolyticus in cultivated white shrimp.

Cultivation of Penaeus vannamei (Pacific white shrimp) is faced with the serious problem of acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus that carries plasmids containing binary toxin genes. The disease is typically moderated by the use of antibiotics. To investigate the control of AHPND and maintenance of water quality without the use of antibiotics, the supplementation of shrimp feed with anti-vibrio compounds from a crude extract of probiotic Rhodobacter sphaeroides SS15 was evaluated. The experimental design comprised four treatments: two that were challenged with AHPND-causing V. parahaemolyticus SR2 at a density of 6.0 × 105 cells mL-1 and two that were not challenged. The unchallenged groups comprised a control group that received commercial feed only (CF) and a group that received CF supplemented with 0.27% (w/w) of the extract of R. sphaeroides SS15 (modified CF: MCF). The treatments challenged with V. parahaemolyticus SR2 comprised a challenge group that received CF only (challenge CF: CF-SR2) and a challenge group that received modified CF (challenge MCF: MCF-SR2). V. parahaemolyticus SR2 was inoculated at the start of cultivation and at day 48 at the same cell density. No significant difference in growth performance was found among all treatments. All water quality parameters were better in the two treatments that received modified CF but excess nitrite, due to overfeeding in low salinity (5-8 ppt), caused shrimp mortality in all treatments. Vibrio populations were much higher in the CF treatments than in the modified CF treatments. After the first challenge, the survival rate was about 67% in both the CF-SR2 and MCF-SR2 treatments, compared with approximately 83% in the unchallenged treatments. One day after the second challenge, mortality in the CF-SR2 treatment was 100%, whereas 16.67% survived in the MCF-SR2 treatment. The survival rate was roughly 27% higher in the MCF treatment than in the CF treatment. The hepatopancreas and gut of both modified CF treatments showed no sign of AHPND. Via better water quality and trained immunity, the anti-vibrio compounds in the modified CF have great potential to increase the survival of cultivated shrimp infected with AHPND-causing strain SR2.

ENTEROAGGREGATIVE ESCHERICHIA COLI O104 FROM THAI AND IMPORTED MALAYSIAN RAW BEEF.

Local Thai and imported Malaysian beef in southern Thailand area carry several Shiga toxin-producing Escherichia coli (STEC) serotypes. STEC O104 is an important pathogen capable of causing outbreaks with considerable morbidity and mortality. This study investigated the presence of E. coli O104 from local Thai and imported Malaysian beef obtained from markets in Hat Yai City, Songkhla Province during August 2015 - February 2016. Thirty-one E. coli O104 strains were isolated from 12 beef samples (16% and 23% Thai and imported Malaysian, respectively). Thirty strains possessed aggA (coding for a major component of AAF/I fimbriae), a gene associated with enteroaggregative E. coli (EAEC) pathotype, and all strains carried fimH (encoding Type 1 fimbriae). Thirty strains belonged to phylogenetic group B1 and one strain (from Malaysian beef) to group A. Agglutination of yeast cells was observed among 29 E. coli O104 strains. Investigation of stx2 phage occupancy loci demonstrated that sbcB was occupied in 12 strains. Antimicrobial susceptibility assay revealed that 7 strains were resistant to at least one antimicrobial agent and two were multi-drug resistant. One strain carried extended spectrum ß-lactamase gene blaCTX-M and three carried blaTEM. PFGE-generated DNA profiling showed identical DNA pattern between that of one EAEC O104 strain from Thai beef and another from Malaysian beef, indicating that these two strains originated from the same clone. This is the first report in Thailand describing the presence of EAEC O104 from both Thai and imported Malaysian beef and their transfer between both countries. Thorough surveillance of this pathogen in fresh meats and vegetables should help to prevent any possible outbreak of E. coli O104.

CHARACTERIZATION OF EXTRAINTESTINAL PATHOGENIC ESCHERICHIA COLI FROM MEAT IN SOUTHERN THAILAND.

Extraintestinal pathogenic Escherichia coli (ExPEC) is an E. coli group, which causes diseases in systems outside human intestinal tract. ExPEC isolates were recovered from fresh chicken (25%) and pork (10%) meats, but not beef and shrimp, from markets in southern Thailand. Among the 14 ExPEC strains isolated, all carried iutA and fimH, coding for aerobactin and type 1 fimbriae, respectively. Two ExPEC strains from chicken meat possessed kpsMTK1 coding for K1 capsular antigen, responsible for neonatal meningitis. Antimicrobial susceptibility assay revealed that all ExPEC were resistant to streptomycin and carried blaTEM, but susceptible to imipenem. Phylogenetic group analysis showed that 4, 4, and 6 ExPEC strains belonged to group A, B1 and D, respectively. ExPEC strains were classified into four serotypes, namely, O8 (2 strains), O15 (2 strains), O25 (1 strain), and O127a (1 strain), with the remaining untypeable. DNA profiling analysis by BOX-PCR revealed clonality of strains with the same serotype. The existence of ExPEC in meat products should cause concern regarding food safety and public health not only in southern Thailand but also throughout the country.

METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS: 1-YEAR COLLECTION AND CHARACTERIZATION FROM PATIENTS IN TWO TERTIARY HOSPITALS, SOUTHERN THAILAND.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing nosocomial and other types of infections worldwide. In a one-year survey of patients in two tertiary hospitals, namely, Maharaj Nakhon Si Thammarat and VachiraPhuket Hospitals, southern Thailand, 64 MRSA strains (prevalence of 8.1%) were obtained mainly from the elderly (> 45 years of age). PCR-based assay revealed high prevalence of virulence genes, femB (30%) and spa (34%), and of SCCmec type II from VachiraPhuket (36%) and Maharaj Nakhon Si Thammarat (38%) Hospitals. The majority of MRSA strains were resistant to clindamycin (98%), erythromycin (95%), gentamicin (84%), and tetracycline (80%), while still sensitive to chloramphenicol, cotrimoxazole, fusidic acid, and vancomycin. These data are important for effective treatment of MRSA-infected patients and for implementing control strategies to prevent spread of MRSA within hospitals.

EX VIVO ADHERENCE TO MURINE ILEAL, BIOFILM FORMATION ABILITY AND PRESENCE OF ADHERENCE-ASSOCIATED OF HUMAN AND ANIMAL DIARRHEAGENIC ESCHERICHIA COLI.

Diarrheagenic Escherichia coli (DEC) are important bacteria causing gastrointestinal infection, which can lead to severe forms of illnesses. This study focused on DEC adherent capabilities using murine intestinal tissue as a model. Ex vivo adherence results showed that enteroaggregative E. coli (EAEC) strain PSU280 exhibited the highest level of adherence, followed by strains from ETEC category. Scanning electron micrographs displayed tight binding and putative bacterial curli fibers, including putative fimbrial structures. The presence of putative curli fibers was confirmed by the presence of csgA, a curli major structural subunit gene. Five and 3 of 15 DEC possessed lpf (encoding long polar fimbriae) and agn43 (encoding antigen43), respectively. Comparable biofilm formation efficiency but variable levels autoaggregation were observed among the DEC strains. In addition, yeast agglutination could be visualized in 11/15 strains. This study demonstrates the adherent ability of DEC strains isolated in southern Thailand as well as a number of crucial adherence-associated genes, information of importance to the understanding of DEC pathogenesis in this region of the country.

REPETITIVE SEQUENCE BASED-PCR PROFILING OF ESCHERICHIA COLI O157 STRAINS FROM BEEF IN SOUTHERN THAILAND.

Beef and its products are potential vehicles of Escherichia coli O157, the most important serotype implicated in many large outbreaks of diarrheal infection in humans worldwide. There is a need for rapid detection of contaminated food in order to implement appropriate and effective control measures. In this study, repetitive sequence (rep)-PCR, using three different primers, BOXA1R, ERIC2 and (GTG)5, singly and in combinations, were employed to compare the genetic relatedness among E. coli O157 group with other diarrheagenic E. coli strains as controls. Although a combination of BOXA1R + ERIC2 + (GTG)5 primers generated a rep-PCR profile containing the highest number of amplicon bands among the DEC strains tested, dendrogram (at 80% similarity) exhibited the lowest DEC classification of 5 clusters, whereas that from BOXA1R or BOXA1R+ (GTG)5 rep-PCR profiling produced 8 clusters. Nevertheless, focusing E. coli O157 strains were grouped into 4 clusters irrespective of the rep-PCR profiles analyzed, and all 14 but two, PSU60 and PSU132, E. coli O157 strains isolated from beef in southern Thailand during 2012 to 2014 fell into a single cluster. Thus, rep-PCR profiling generated with BOXA1R or BOXA1R + (GTG)5 is sufficient for distinguishing among DEC strains, including E. coli O157 in southern Thailand.

HIGH LEVEL OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI AND OCCURRENCE OF STX-NEGATIVE E. COLI O157 FROM RAW MEATS: CHARACTERIZATION OF VIRULENCE PROFILE AND GENETIC RELATEDNESS.

Raw meats, especially beef, are particularly prone to Shiga toxinproducing Escherichia coli (STEC)/enterohemorrhagic E. coli (EHEC) contamination. However, data regarding their quantity in raw meats are seldom reported in Thailand. Among four common meat types, beef possessed highest value of stx1-producing E. coli (STEC1) contamination in February 2015 [> 1,100 most probable number (MPN)/g] and stx2-producing E. coli (STEC2) highest MPN/g (460) in March of the same year. STEC2 was found, for the first time, in shrimp samples in March and April, 2015 with MPN/g value of 6.6 and 9.3, respectively. EHEC at 3 MPN/g was detected in only one (2%) beef sample. Even though stx-negative E. coli O157 from beef has rarely been reported in Thailand, isolation of E. coli O157 using immunomagnetic separation method revealed that four strains (PSVX-1, PSVX-2, PSVX-3, and PSVX-4) from three (8%) beef samples were shown to be stx-negative E. coli O157. These strains were members of phylogenetic group A and were multi-drug resistant. Genetic relatedness as determined by polytrinucleotide (GTG)5-PCR and BOX-PCR showed identical DNA profiles of PSVX-2 and PSVX-4, which by BOX-PCR were 90% to a clinical isolate, O157 strain PSU120, from Hat-Yai Hospital in 2014. The presence of these environment stx-negative E. coli O157 strains with the ability to acquire additional virulence properties could pose a potential public health problem particularly in this region of Thailand.

METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS FROM READY-TO-EAT FOODS IN A HOSPITAL CANTEEN, SOUTHERN THAILAND: VIRULENCE CHARACTERIZATION AND GENETIC RELATIONSHIP.

Due to the existence of sporadic cases caused by methicillin-resistant Staphylococcus aureus (MRSA) in Songklanagarind Hospital, Songkhla, southern Thailand where a canteen is located in close vicinity, this study investigated the presence of MRSA, including mecA-carrying S. aureus (MCSA), contamination in 105 ready-to-eat (RTE) food samples sold in this canteen. Coagulase-negative MRSA (MR-CoN) and coagulase-negative MCSA (MCSA-CoN) contaminations were observed in various RTE foods with unriped-papaya salad having significantly highest incidence of MCSA-CoN contamination (p < 0.05). The majority of MCSA-CoN isolates were resistant to clindamycin and fusidic acid. Two MCSA-CoN strains PSU172 and PSU180 were subsequently shown to be MR-CoN. Staphy- lococcal enterotoxins (SEs)-carrying MCSA-CoN strain PSU109 was isolated from seasoned rice. The SE-carrying MCSA-CoN strain PSU109, MR-CoN strain PSU172 and MCSA-CoN strain PSU173 were able to survive in the acidic unriped-papaya salad sauce (pH 4.0-4.5) for up to 6 hours. Typing by pulsed-field gel electrophoresis revealed 80% genetic relatedness among MCSA-CoN strains from RTE food and clinical strains. This study suggests the plausibility of RTE foods sold in a hospital canteen as vehicles of hospital-acquired MR-CoN and of MCSA-CoN.

IMMUNO-MAGNETIC ISOLATION, CHARACTERIZATION AND GENETIC RELATIONSHIP OF ESCHERICHIA COLI O26 FROM RAW MEATS, HAT YAI CITY, SONGKHLA, THAILAND.

Escherichia coli O26 is the most important serotype in non-O157 group, which plays a significant role in gastrointestinal illnesses. However, information regarding the prevalence and its characteristics are lacking in Thailand. As raw meat is frequently a source of diarrheagenic E. coli, a total of 1,279 E. coli colonies were obtained from 157 raw meat samples obtained from retail markets in Hat Yai City, Songkhla Province, Thailand and E. coli O26 isolated using an immunemagnetic separation technique. Twenty-seven E. coli O26 strains were isolated from 18 samples of raw beef, chicken and pork meats. These E. coli O26 strains could not be classified into the six diarrheagenic E. coli categories and did not harbor virulence genes, except 5 strains carrying escV, encoding type III secretion system component. Phylogenetic group examination demonstrated that 26 strains belonged to phylogenetic group A, and one to group D. Antimicrobial susceptibility test revealed that the E. coli O26 strains were the multi-drug resistant strains. Genetic relatedness employing (GTG)5-PCR and ERIC2-PCR showed that some of O26 which isolated from different samples and different time intervals revealed the identical fingerprint pattern, suggesting that they were derived from the same clone. Examination of five stx2-containing phage integration sites showed that 6 strains had prophage occupancy at sbcB, suggesting that these isolates have the potential in horizontal gene transfer of virulence trait. Moreover, the intactness of yecE and wrbA, the important integration sites in E. coli O26, indicated the possibility of stx2-phage lysogenization in the future.

MOLECULAR CHARACTERIZATION OF VIRULENCE AND ANTIMICROBIAL SUSCEPTIBILITY PROFILES OF UROPATHOGENIC ESCHERICHIA COLI FROM PATIENTS IN A TERTIARY HOSPITAL, SOUTHERN THAILAND.

Among uropathogens, uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI) worldwide, but clinical aspects due to this bacterial species is not fully understood in southern Thailand. Two hundred fifty-four UPEC isolates from patients admitted to Maharaj Nakhon Si Thammarat Hospital, southern Thailand were examined for crucial virulence genes, showing that 33.5% contained at least one of the virulence, genes tested. Genes encoding P fimbria, cytotoxic necrotizing factor-1 and α-hemolysin constituted the majority (15.8%) carried by UPEC isolates. Phylogenetic group classification revealed that 57.5% of UPEC belonged to group D. Antimicrobial susceptibility tests showed that 70.5% and 65.1% of the isolates were resistant to ciprofloxacin and norfloxacin, respectively. Moreover, 50.0% of UPEC were capable of producing extended spectrum beta-lactamases. These findings should be of benefit for more appropriate treatment of UTI patients in this region of Thailand. Keywords: uropathogenic Escherichia coli, antibiotics resistance, cnfl, hlyA, pap, Thailand

First report in Thailand of a stx-negative Escherichia Coli 0157 strain from a patient with diarrhea.

E. coli serotype 0157 is well known to cause serious illnesses in humans. However, there has been no case report to date of this serotype in Thailand. In this study, we report for the first time E. coli 0157 (designated as PSU120) isolated from a stool sample among 228 diarrheal swab samples at Hat Yai Hospital, Songkhla Province, Thailand. This PSU120 was identified as being stx-negative and lacked eae but carried escV, a marker for the locus of enterocyte effacement. Of the five reported integration sites frequently occupied by stx phages, the sbcB and yehV loci were occupied, suggesting that PSU120 is active in horizontal genetic transfer. Antimicrobial susceptibility assay revealed that E. coli 0157 strain PSU120 was resistant to cephalothin, erythromycin, methicillin and vancomycin. Using pulsed- field gel-electrophoresis to compare the genetic relatedness of E. coli 0157 strain PSU120 to two other E. coli 0157 strains, namely, the well-established EHEC strain EDL933 and PSU2, a surrogate of E. coli 0157:H7 whose genotype stx1-, stx2+, eae+ is frequently obtained from the environment in this area during the last decade, revealed 88.6% in similarity. We suggest that PSU120 was originally stx+ but lostthe gene after establishing infection.

ENTEROTOXIGENIC ESCHERICHIA COLI O169:HUT FROM A DIARRHEAL PATIENT: PHYLOGENETIC GROUP AND ANTIMICROBIAL SUSCEPTIBILITY.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common pathogenic E. coli pathotypes causing diarrhea in children worldwide. Its enterotoxins, LT and ST, including colonization factors mainly are responsible for human pathogenesis. From 239 rectal swabs of diarrheal patients at Hat Yai and Pattani Hospitals during August 2013 and May 2014, five isolates from only a single E. coli sample demonstrated the possession of estA1, encoding porcine heat-stable enterotoxin (STp). These isolates all belonged to serotype 0169:H Untypeable (HUT) and carried astA, encoding enteroaggregative heat-stable enterotoxin 1. A PCR-based phylogenetic group investigation classified them as members of the virulent E. coli phylogenetic group D. The isolates were resistant to cephalothin, penicillin G, streptomycin, tetracycline and vancomycin. Confirmation of their clonality was conducted by enterobacterial repetitive intergenic consensus sequence PCR typing, which revealed that these ETEC were derived from the same clone. This is the first report of ETEC O169:HUT in southern Thailand.

MOST PROBABLE NUMBER-POLYMERASE CHAIN REACTION-BASED QUANTIFICATION OF ENTEROTOXIGENIC ESCHERICHIA COLI FROM RAW MEATS IN SOUTHERN THAILAND.

The detection of enterotoxigenic Escherichia coli (ETEC) in food, especially raw meat, has rarely been documented in Thailand, although the presence of this bacterial pathogen is considered of important public health concern. The quantity of ETEC in 150 meat samples collected from fresh food markets in southern Thailand were determined using a most probable number (MPN)-PCR-based quantification approach. ETEC contamination of raw chicken, pork and beef samples was 42%, 25% and 12%, respectively (a significant difference between chicken and beef, p < 0.05). The maximum MPN/g value for enterotoxin gene est-positive ETEC from pork and elt-positive ETEC from chicken were > 1,100 MPN/g, but the range of MPN/g values was greater for ETEC from chicken than from pork or beef. ETEC from raw chicken meat contained significantly more elt- than est-positives (p < 0.05). Thus, a significant proportion of raw meat, in particular chicken, sold in fresh food markets in southern Thailand harbors ETEC and poses a potential threat to consumer health.

Inhibition of shrimp pathogenic vibrios by extracellular compounds from a proteolytic bacterium Pseudomonas sp. W3 / Inhibición de vibrios patógenos del camarón por compuestos extracelulares de una bacterias proteolítica Pseudomonas sp.W3

Pseudomonas sp. W3, a bacterium known to produce an extracellular alkaline protease, secreted secondary metabolites that inhibited pathogenic bacteria responsible for shrimp luminous vibriosis disease. Antivibrio compounds in the culture supernatant or culture filtrates (0.45 um and 0.22 um) of the isolate W3 were tested using an agar well diffusion method on a number of pathogenic vibrios. Vibrio harveyi PSU 2015 a pathogenic isolate was the most sensitive strain. The effectiveness of preparations from the isolate W3 against V. harveyi PSU 2015, and V. cholerae PSSCMI 0062 was in the order of culture supernatant > 0.45 um culture filtrate > 0.22 um culture filtrate. These extracellular antivibrio compounds also lysed both dead and living cells of V. harveyi PSU 2015. Results of the partial characterization tests indicated that there was some particulate antivibrio compound that was destroyed by treatment with enzymes particularly alpha-chymotrypsin, autoclaving at 121ºC for 15 min and was mostly removed by filtration through a 0.22 µm filter. Most of the inhibitory compounds were of small molecular weight able to pass through a 0.22 um filter and were resistant to treatment with various enzymes, pH values between 4-8 and temperatures up to 121ºC for 30 min. The optimum pH for the antivibrio activity in the 0.45 um culture filtrate was between pH 6-7.