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.

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.

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.

Evaluation of loop-mediated isothermal amplification method for detecting enterotoxin A gene of Staphylococcus aureus in pork.

Staphylococcus aureus is an important food-borne pathogen of humans due to ingestion of food containing enterotoxigenic strains. We evaluated the detection of S. aureus enterotoxin A gene (sea) using a loop-mediated isothermal DNA amplification (LAMP) method. Specificity of LAMP method was comparable to that of conventional PCR. Both methods correctly identified sea and did not detect 14 other non-Staphylococcus strains. Limit of detections of LAMP assay for direct detection of S. aureus was 10(4) cfu/ml while that of PCR was 10(3) cfu/ml. Both methods were employed for the detection of S. aureus spiked pork samples to assess their sensitivity. LAMP method was able to detect a minimum of 10(3) cfu/g while PCR 10(4) cfu/g of pork sample following incubation in tryptic soy broth for 6 hours. The LAMP assay allows a one-step identification of gene of interest without any specialized equipment and requires less time than conventional PCR, thus suggesting that the LAMP technique might be an appropriate alternative diagnostic method for detecting enterotoxin A gene of S. aureus in food sample.

Detection of Escherichia coli O157: H7 vt and rfb(O157) by multiplex polymerase chain reaction.

A rapid method for detection of Escherichia coli O157: H7 using multiplex PCR was developed. Two oligonucleotide primer pairs were used for simultaneously detection of vt encoding verotoxin genes for virulence factor and rfb(O157) encoding the O-antigen specific for E. coli O157: H7. Multiplex PCR generated two products of 215 bp and 420 bp for vt and rfb(O157), respectively. Multiplex PCR detected reference strain O157: H7 (NF-7777) with a sensitivity of 10(5) CFU per ml with no amplification of other 15 pathogenic bacteria. After incubation of 10(2) CFU/25 gram raw meat in tryptic soy broth at 37 degrees C for 8 hours, multiplex PCR conducted with the addition of 100 mg bovine serum albumin produced the two specific PCR products for E. coli O157: H7. This modified multiplex PCR is a rapid, sensitive, and specific technique for detecting and differentiating E. coli O157: H7 and has the potential to be used as an alternative to conventional methods for the screening of O157: H7 strains isolated from raw meat.