Prevalence, virulence characteristics, and antimicrobial resistance of Vibrio parahaemolyticus isolates from raw seafood in a province in Northern Thailand.

Vibrio parahaemolyticus (V. parahaemolyticus) is commonly found in seawater and seafood products, but evidence is limited of its presence in seafood marketed in locations very distant from coastal sources. This study determined the prevalence and characterization of V. parahaemolyticus in seafood from markets in landlocked Phayao province, Northern Thailand. Among 120 samples, 26 (21.7%) were positive for V. parahaemolyticus, being highest in shrimp (43.3%), followed by shellfish (36.7%), and squid (6.7%), but was not found in fish. V. parahaemolyticus comprised 33 isolates that were non-pathogenic and non-pandemic. Almost all isolates from shrimp and shellfish samples were positive for T3SS1. Only five isolates (15.2%) showed two antimicrobial resistance patterns, namely, kanamycin-streptomycin (1) carrying sul2 and ampicillin-kanamycin-streptomycin (4) that carried tetA (2), tetA-sul2 (1), as well as one negative. Antimicrobial susceptible V. parahaemolyticus isolates possessing tetA (67.9%) and sul2 (3.5%) were also found. Six isolates positive for integron class 1 and/or class 2 were detected in 4 antimicrobial susceptible and 2 resistant isolates. While pathogenic V. parahaemolyticus was not detected, contamination of antimicrobial resistance V. parahaemolyticus in seafood in locations distant from coastal areas requires ongoing monitoring to improve food safety in the seafood supply chain.

Prevalence, antimicrobial resistance, and enterotoxin gene profiles of Staphylococcus aureus isolated from mobile phones of the food vendors in Phayao province, Thailand.

BACKGROUND: Mobile phones are widely used and may cause bacterial pathogens to spread among various professionals. Staphylococcus aureus from the mobile phones can contaminate the hands of food vendors and food during the cooking or packaging process. This research aimed to determine the prevalence, enterotoxin genes, and antimicrobial resistance (AMR) profiles of S. aureus contaminating the vendors' mobile phones. METHODS: In this study, 266 mobile phone samples were randomly collected from food vendors selling food on walking streets (n = 139) and in food centers (n = 127) in Phayao province. All samples were identified as S. aureus by the conventional culture method and confirmed species-specific gene by polymerase chain reaction (PCR). Then, all identified S. aureus isolates were tested for antimicrobial susceptibility by broth microdilution method and for the presence of staphylococcal enterotoxin (SE) genes by PCR. RESULTS: The results showed that 12.8% of the mobile phones collected were contaminated with S. aureus. Of 49 S. aureus isolates obtained, 30 (61.2%) were positive for SE genes. The most common SE gene was sea followed by sec, seb, sem, seq, and sel. Moreover, S. aureus was most frequently resistant to penicillin, followed by chloramphenicol and tetracycline, erythromycin, clindamycin, and gentamicin. Methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and multidrug-resistant (MDR) strains were also detected. CONCLUSIONS: This study showed that mobile phones were an intermediate surface for the transmission of S. aureus, including MDR variants. It indicates that hand hygiene and the decontamination of mobile phones are essential to prevent cross-contamination of S. aureus in food settings.

Restoring Ampicillin Sensitivity in Multidrug-Resistant Escherichia coli Following Treatment in Combination with Coffee Pulp Extracts.

Escherichia coli, particularly multidrug-resistant (MDR) strains, is a serious cause of healthcare-associated infections. Development of novel antimicrobial agents or restoration of drug efficiency is required to treat MDR bacteria, and the use of natural products to solve this problem is promising. We investigated the antimicrobial activity of dried green coffee (DGC) beans, coffee pulp (CP), and arabica leaf (AL) crude extracts against 28 isolated MDR E. coli strains and restoration of ampicillin (AMP) efficiency with a combination test. DGC, CP, and AL extracts were effective against all 28 strains, with a minimum inhibitory concentration (MIC) of 12.5-50 mg/ml and minimum bactericidal concentration of 25-100 mg/ml. The CP-AMP combination was more effective than CP or AMP alone, with a fractional inhibitory concentration index value of 0.01. In the combination, the MIC of CP was 0.2 mg/ml (compared to 25 mg/ml of CP alone) and that of AMP was 0.1 mg/ml (compared to 50 mg/ml of AMP alone), or a 125-fold and 500-fold reduction, respectively, against 13-drug resistant MDR E. coli strains. Time-kill kinetics showed that the bactericidal effect of the CP-AMP combination occurred within 3 h through disruption of membrane permeability and biofilm eradication, as verified by scanning electron microscopy. This is the first report indicating that CP-AMP combination therapy could be employed to treat MDR E. coli by repurposing AMP.

Dinactin: A New Antitumor Antibiotic with Cell Cycle Progression and Cancer Stemness Inhibiting Activities in Lung Cancer.

Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the most complex diseases, despite the existence of effective treatments such as chemotherapy and immunotherapy. Since cancer stem cells (CSCs) are responsible for chemo- and radio-resistance, metastasis, and cancer recurrence, finding new therapeutic targets for CSCs is critical. Dinactin is a natural secondary metabolite produced by microorganisms. Recently, dinactin has been revealed as a promising antitumor antibiotic via various mechanisms. However, the evidence relating to cell cycle progression regulation is constrained, and effects on cancer stemness have not been elucidated. Therefore, the aim of this study is to evaluate the new function of dinactin in anti-NSCLC proliferation, focusing on cell cycle progression and cancer stemness properties in Lu99 and A549 cells. Flow cytometry and immunoblotting analyses revealed that 0.1-1 µM of dinactin suppresses cell growth through induction of the G0/G1 phase associated with down-regulation of cyclins A, B, and D3, and cdk2 protein expression. The tumor-sphere forming capacity was used to assess the effect of dinactin on the cancer stemness potential in NSCLC cells. At a concentration of 1 nM, dinactin reduced both the number and size of the tumor-spheres. The quantitative RT-PCR analyses indicated that dinactin suppressed sphere formation by significantly reducing expression of CSC markers (i.e., ALDH1A1, Nanog, Oct4, and Sox2) in Lu99 cells. Consequently, dinactin could be a promising strategy for NSCLC therapy targeting CSCs.

First study on virulence genes, antimicrobial resistance, and integrons in Escherichia coli isolated from cage, free-range, and organic commercial eggs in Phayao Province, Thailand.

Background and Aim: Antimicrobial resistance (AMR) is a global problem that affects human and animal health, and eggs can act as a vehicle for pathogenic and non-pathogenic resistant bacteria in the food chain. Escherichia coli is an indicator of food contamination with fecal materials as well as the occurrence and levels of AMR. This study aimed to investigate the presence of AMR, integrons, and virulence genes in E. coli isolated from eggshell samples of three egg production systems, from supermarkets in Thailand. Materials and Methods: A total of 750 hen's egg samples were purchased from supermarkets in Phayao Province: Cage eggs (250), free-range eggs (250), and organic eggs (250). Each sample was soaked in buffered peptone water (BPW), and the BPW samples were incubated at 37°C for 18-24 h. All samples were tested for E. coli by the standard conventional culture method. Then, all identified E. coli were tested for antimicrobial susceptibility to 15 antimicrobial agents by the agar disk diffusion method. All E. coli strains were subsequently found to have virulence genes and Classes 1 and 2 integrons by polymerase chain reaction. Results: Among the eggshell samples, 91 samples were identified as having E. coli (cage eggs, 24 strains; free-range eggs, 27 strains; and organic eggs, 40 strains). Then, among the E. coli strains, 47 (51.6%) were positive for at least one virulence gene. The proportion of AMR in the eggshell samples was 91.2% (83/91), and streptomycin (STR), ampicillin (AMP), and tetracycline (TET) had a high degree of resistance. Among the E. coli strains, 27 (29.7%) strains were positive for class 1 or 2 integrons, and integron-positive strains were commonly found in STR-, AMP-, and TET-resistant strains. Multidrug resistance (MDR) was detected in 57.1% (52/91) of the E. coli strains, with STR-AMP-TET (5.5%) as the most frequent pattern. The proportion of MDR in cage eggs was 75.0% (18/24), which was higher than in both free-range and organic eggs. On the other hand, 53.2% (25/47) of E. coli carrying virulence genes had MDR, distributed across the production systems as follows: Cage eggs, 76.9% (10/13); free-range eggs, 63.6% (7/11); and organic eggs, 34.8% (8/23). Conclusion: Escherichia coli was detected in eggshell samples from all three egg production systems. The high level of virulence genes, AMR, and integrons indicated the possibility of dissemination of AMR among pathogenic and commensal E. coli through eggshells. These findings could be a major concern to farmers, food handlers, and consumers, especially regarding raw egg consumption.

High prevalence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates: A 5-year retrospective study at a Tertiary Hospital in Northern Thailand.

Background: The global emergence and spread of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales, especially Escherichia coli and Klebsiella pneumoniae, have been recognized as a public health concern as severe infections caused by these microorganisms increase morbidity and mortality. This study aimed to assess the prevalence of ESBL-positive E. coli and K. pneumoniae strains isolated from hospitalized patients in Chiangrai Prachanukroh hospital, Chiangrai province, Thailand. Methods: This retrospective analysis was conducted from January 2016 to December 2020. A total of 384,001 clinical specimens were collected aseptically and further cultivated on an appropriate medium. All clinical isolates (one isolate per patient) were identified based on standard laboratory methods. Antibiotic susceptibility testing was performed by the Kirby Bauer disc diffusion technique following CLSI guidelines. ESBL production was screened with ceftazidime and cefotaxime discs based on the CLSI recommendations. Phenotypic confirmation of ESBL production was carried out using a double-disc synergy technique following the CLSI standard. Results: Of a total of 384,001 clinical samples analyzed for bacterial species identification, 11,065 (2.9%) tested positive for E. coli and 5,617 (1.5%) for K. pneumoniae. Approximately 42.5% (4,706/11,065) of E. coli and 30.2% (1,697/5,617) of K. pneumoniae isolates were classified as ESBL producers. A higher proportion of ESBL producers was found in patients older than 60 years and male groups. The highest infection rates of ESBL-positive pathogens were observed among patients in a medical unit. ESBL-producing E. coli and K. pneumoniae isolates were predominantly found in urine and sputum, respectively. ESBL producers exhibited a high resistance rate to ampicillin (99.8-100%), cefazolin (100%), cefotaxime (100%), fluoroquinolones, and trimethoprim/sulfamethoxazole. Conclusions: This study demonstrated the high prevalence and emerging antibiotic resistance of ESBL-positive E. coli and K. pneumoniae isolates from patients admitted to a provincial hospital in northern Thailand. Most ESBL-producing strains were highly resistant to several antimicrobial agents apart from carbapenems and aminoglycosides. These findings indicated that carbapenems and aminoglycosides should be advised as the first-line drugs of choice for serious infections with ESBL-producing Enterobacterales.

Potential Antimicrobial Properties of Coffee Beans and Coffee By-Products Against Drug-Resistant Vibrio cholerae.

Vibrio cholerae is the causative organism of the cholera epidemic, and it remains a serious global health problem, particularly the multidrug-resistant strain, despite the development of several generic drugs and vaccines over time. Natural products have long been exploited for the treatment of various diseases, and this study aimed to evaluate the in vitro antibacterial activity of coffee beans and coffee by-products against V. cholerae antimicrobial resistant strains. A total of 9 aqueous extracts were investigated, including light coffee (LC), medium coffee (MC), dark coffee (DC), dried green coffee (DGC), dried red coffee (DRC), fresh red coffee (FRC), Arabica leaf (AL), Robusta leaf (RL), and coffee pulp (CP). The influential coffee phytochemicals, i.e., chlorogenic acid (CGA), caffeic acid (CA), and caffeine, were determined using HPLC. The antibacterial properties were tested by agar well-diffusion techniques, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were further determined against 20 V. cholerae isolates. The results revealed that all tested strains were sensitive to coffee extracts, with MIC and MBC values in the range of 3.125-25.0 mg/mL and 12.5-50.0 mg/mL, respectively. With a MIC of 6.25 mg/mL, DGC, DRC, and CP appeared to be the most effective compounds against 65, 60, and 55% of clinical strains, respectively. The checkerboard assay revealed that the combination of coffee extract and tetracycline was greater than either treatment alone, with the fractional inhibitory concentration index (FICI) ranging from 0.005 to 0.258. It is important to note that CP had the lowest FICI (0.005) when combined with tetracycline at 60 ng/mL, which is the most effective dose against V. cholerae six-drug resistance strains (azithromycin, colistin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim), with a MIC of 47.5 µg/mL (MIC alone = 12.5 mg/mL). Time killing kinetics analysis suggested that CA might be the most effective treatment for drug-resistant V. cholerae as it reduced bacterial growth by 3 log10 CFU/mL at a concentration of 8 mg/mL within 1 h, via disrupting membrane permeability, as confirmed by scanning electron microscopy (SEM). This is the first report showing that coffee beans and coffee by-product extracts are an alternative for multidrug-resistant V. cholerae treatment.

Antimicrobial Activity of the Green Tea Polyphenol (-)-Epigallocatechin-3-Gallate (EGCG) against Clinical Isolates of Multidrug-Resistant Vibrio cholerae.

The spread of multidrug-resistant (MDR) Vibrio cholerae necessitates the development of novel prevention and treatment strategies. This study aims to evaluate the in vitro antibacterial activity of green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against MDR V. cholerae. First, MIC and MBC values were evaluated by broth microdilution techniques against 45 V. cholerae strains. The checkerboard assay was then used to determine the synergistic effect of EGCG and tetracycline. The pharmaceutical mode of action of EGCG was clarified by time-killing kinetics and membrane disruption assay. Our results revealed that all of the 45 clinical isolates were susceptible to EGCG, with MIC and MBC values in the range of 62.5-250 µg/mL and 125-500 µg/mL, respectively. Furthermore, the combination of EGCG and tetracycline was greater than either treatment alone, with a fractional inhibitory concentration index (FICI) of 0.009 and 0.018 in the O1 and O139 representative serotypes, respectively. Time-killing kinetics analysis suggested that EGCG had bactericidal activity for MDR V. cholerae after exposure to at least 62.5 µg/mL EGCG within 1 h. The mode of action of EGCG might be associated with membrane disrupting permeability, as confirmed by scanning electron microscopy. This is the first indication that EGCG is a viable anti-MDR V. cholerae treatment.

Prevalence and Multidrug Resistance of Salmonella in Swine Production Chain in a Central Province, Thailand.

ABSTRACT: Salmonella causes foodborne disease outbreaks worldwide and raises concerns about public health and economic losses. To determine prevalence, serovar, antimicrobial resistance patterns, and the presence of extended-spectrum ß-lactamase (ESBL) genes in a cross-sectional study, 418 total samples from feces and carcasses (from three slaughterhouses) and pork and cutting boards (from four markets) were collected in a central Thailand province in 2017 and 2018. Of the 418 samples, 272 (65.1%) were positive for Salmonella. The prevalence of Salmonella-positive samples from markets (158 of 178; 88.8%) was significantly higher than that among samples from slaughterhouses (114 of 240; 47.5%) (P < 0.05). A total of 1,030 isolates were identified; 409 were assigned to 45 serovars, with Salmonella Rissen the most common (82 of 409; 20%). Two serovars, Salmonella Cannstatt and Salmonella Braubach, were identified for the first time in Thailand in market and slaughterhouse samples, respectively. Among 180 isolates representing 19 serovars, 133 (73.9%) exhibited multidrug resistance. Screening for ESBL production revealed that 41 (10.3%) of 399 isolates were ESBL positive. The prevalence of ESBL-producing Salmonella isolates was significantly higher among the market isolates (31 of 41; 75.6%) than among the slaughterhouse isolates in (10 of 41; 24.4%) (P < 0.05). In market samples, 24 (77.4%) of 31 isolates were recovered from pork and 7 (22.6%) were recovered from cutting boards. Nine ESBL-producing isolates carried single ESBL genes, either blaTEM (4 of 41 isolates; 9.8%) or blaCTX-M (5 of 41 isolates; 12.2%), whereas 11 (26.8%) carried both blaTEM and blaCTX-M. No ESBL-producing Salmonella isolate carried the blaSHV gene. These results suggest that pigs, their flesh, and cutting boards used for processing pork could be reservoirs for widespread ESBL-producing Salmonella isolates with multidrug resistance and outbreak potential across the food chain.

Characterization and Genetic Variation of Vibrio cholerae Isolated from Clinical and Environmental Sources in Thailand.

Cholera is still an important public health problem in several countries, including Thailand. In this study, a collection of clinical and environmental V. cholerae serogroup O1, O139, and non-O1/non-O139 strains originating from Thailand (1983 to 2013) was characterized to determine phenotypic and genotypic traits and to investigate the genetic relatedness. Using a combination of conventional methods and whole genome sequencing (WGS), 78 V. cholerae strains were identified. WGS was used to determine the serogroup, biotype, virulence, mobile genetic elements, and antimicrobial resistance genes using online bioinformatics tools. In addition, phenotypic antimicrobial resistance was determined by the minimal inhibitory concentration (MIC) test. The 78 V. cholerae strains belonged to the following serogroups O1: (n = 44), O139 (n = 16) and non-O1/non-O139 (n = 18). Interestingly, we found that the typical El Tor O1 strains were the major cause of clinical cholera during 1983-2000 with two Classical O1 strains detected in 2000. In 2004-2010, the El Tor variant strains revealed genotypes of the Classical biotype possessing either only ctxB or both ctxB and rstR while they harbored tcpA of the El Tor biotype. Thirty O1 and eleven O139 clinical strains carried CTXϕ (Cholera toxin) and tcpA as well four different pathogenic islands (PAIs). Beside non-O1/non-O139, the O1 environmental strains also presented chxA and Type Three Secretion System (TTSS). The in silico MultiLocus Sequence Typing (MLST) discriminated the O1 and O139 clinical strains from other serogroups and environmental strains. ST69 was dominant in the clinical strains belonging to the 7th pandemic clone. Non-O1/non-O139 and environmental strains showed various novel STs indicating genetic variation. Multidrug-resistant (MDR) strains were observed and conferred resistance to ampicillin, azithromycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim and harboured variants of the SXT elements. For the first time since 1986, the presence of V. cholerae O1 Classical was reported causing cholera outbreaks in Thailand. In addition, we found that V. cholerae O1 El Tor variant and O139 were pre-dominating the pathogenic strains in Thailand. Using WGS and bioinformatic tools to analyze both historical and contemporary V. cholerae circulating in Thailand provided a more detailed understanding of the V. cholerae epidemiology, which ultimately could be applied for control measures and management of cholera in Thailand.

The Lake Chad Basin, an Isolated and Persistent Reservoir of Vibrio cholerae O1: A Genomic Insight into the Outbreak in Cameroon, 2010.

The prevalence of reported cholera was relatively low around the Lake Chad basin until 1991. Since then, cholera outbreaks have been reported every couple of years. The objective of this study was to investigate the 2010/2011 Vibrio cholerae outbreak in Cameroon to gain insight into the genomic make-up of the V. cholerae strains responsible for the outbreak. Twenty-four strains were isolated and whole genome sequenced. Known virulence genes, resistance genes and integrating conjugative element (ICE) elements were identified and annotated. A global phylogeny (378 genomes) was inferred using a single nucleotide polymorphism (SNP) analysis. The Cameroon outbreak was found to be clonal and clustered distant from the other African strains. In addition, a subset of the strains contained a deletion that was found in the ICE element causing less resistance. These results suggest that V. cholerae is endemic in the Lake Chad basin and different from other African strains.

O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139.

A novel, sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR), which is based on two-stage amplification pertaining to multiplex PCR and conditional touchdown strategy, was used in detecting and differentiating Vibrio cholerae serogroups. A panel of molecular marker-based TMPCR method generates reproducible profiles of V. cholerae-specific (588 bp) amplicons derived from ompW gene encoding the outer membrane protein and serogroup-specific amplicons, 364 bp for the O1 and 256 bp for the O139, authentically copied from rfb genes responsible for the lipopolysaccharide biosynthesis. The TMPCR amplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588 and 256 bp) or a DNA fragment of non-O1/non-O139 (588 bp) while providing no false positive identifications using the genomic DNA templates of the other vibrios and Enterobacteriaceae. The reciprocal analysis of two-template combinations demonstrated that, using V. cholerae O1, O139, or equally mixed O1 and O139, the TMPCR had a detection limit of as low as 100 pg of the O1, O139, or non-O1/non-O139 in reactions containing unequally or equally mixed gDNAs. In addition, the O serogroup-specific TMPCR method had 100% agreement with the serotyping method when examined for the serotyped V. cholerae reference strains and those recovered from clinical samples. The potential benefit of using this TMPCR tool would augment the serotyping method used in epidemiological surveillance and monitoring of V. cholerae serogroups, O1, O139, and non-O1/non-O139 present in clinical and environmental samples.