Piperine, an active ingredient of white pepper, suppresses the growth of multidrug-resistant toxigenic Vibrio cholerae and other pathogenic bacteria.

Emergence and rapid spread of multidrug-resistant (MDR) bacteria including Vibrio cholerae are a global public health issue. Much attention has been paid to natural compounds, such as spices and herbs to find novel antimicrobial compounds as they are considered to be cheaper alternatives to develop as a drug. Here, we show that methanol extract of white pepper could inhibit the growth of V. cholerae O1 El Tor variant, responsible for the recent outbreaks/epidemics. Furthermore, we demonstrate for the first time that piperine, the major component of white pepper, showed a dose-dependent bactericidal effect on V. cholerae growth irrespective of their biotypes and serogroups in the presence of 200 and 300 µg ml-1 of piperine, respectively. Piperine also inhibited the growth of MDR strains of Pseudomonas aeruginosa, Escherichia coli isolated from poultry and enterohemorrhagic/enteroaggregative E. coli O104 in the presence of 200 µg ml-1 . Interestingly, we did not observe any significant inhibitory effect of piperine on E. coli strains isolated from healthy person even up to 200 µg ml-1 . Our data suggest that piperine could be a novel antimicrobial agent in therapeutic and preventive applications against infections caused by pathogenic bacteria including MDR strains.

Extended-spectrum beta-lactamase-producing Escherichia coli harbouring sul and mcr-1 genes isolates from fish gut contents in the Mekong Delta, Vietnam.

This study investigated the existence of sulfonamides and colistin resistance genes among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli recovered from fish gut in Vietnam and evaluated the susceptibility patterns of the ESBL-producing E. coli to relevant antimicrobials. A total of 88 ESBL-producing E. coli isolates were analysed for the presence of the ESBLs, sul (1, 2, 3) and mcr (1-3) genes by PCR. Antimicrobial resistance phenotypes of isolates were determined by disc diffusion. Results showed that: (i) A high prevalence of 94·3% of sulfonamide resistance was observed in 88 isolates. Moreover, the existence of 2·3% of ESBL-producing E. coli harbouring mcr-1 gene were detected; (ii) The phylogenetic types A and B1 were most frequent, and the blaCTX-M group1 and blaTEM genes encoding ESBL were detected in 47·7% of the isolates; (iii) ESBL-producing E. coli harbouring mcr-1 gene exhibited resistance to 11 antibiotics. The existence of mcr-1 and sul1,2,3 genes and the extremely high level of multiple drug resistance in all ESBL-producing E. coli isolates obtained from sampled fish in Vietnam is a major concern. Therefore, it is imperative to monitor ESBL-producing E. coli in the river waters of Vietnam.

Virulence of acute hepatopancreatic necrosis disease PirAB-like relies on secreted proteins not on gene copy number.

AIMS: To investigate the virulence of the Vp_PirAB-like genes in Vibrio parahaemolyticus- acute hepatopancreatic necrosis disease (AHPND)-causing strain and the factors that are associated with the virulence level. METHODS AND RESULTS: The virulence of Vp_PirAB-like was examined using a non-virulent strain FP11 of V. parahaemolyticus transformed with a plasmid harbouring Vp_PirAB-like genes and then it was used to challenge shrimp Litopenaeus vannamei and Marsupenaeus japonicus. Both species experienced 100% mortality at 10 days post infection. Analysis of a mutant strain (E1M), that was originally identified as virulent strain (E1) but lost its virulence to L. vannamei, revealed that it lacked a part of the Vp_PirA-like gene and all of the Vp_PirB-like gene. The copy numbers of Vp_PirA-like and Vp_PirB-like genes varied among virulent strains and were not correlated with their virulence. In Western blotting, Vp_PirA-like and Vp_PirB-like proteins were detected in both the cell lysate and the culture supernatant. The strongest intensity of detecting band in the culture supernatant was observed in the strain that caused the highest mortality. The V. parahaemolyticus AHPND-causing strain, unlike the human tdh-positive strain, did not show any enterotoxicity. CONCLUSION: Vibrio parahaemolyticus AHPND-causing strains secrete the Vp_PirA-like and Vp_PirB-like proteins during the growing phase. The amount of secreted proteins affects the shrimp mortality. SIGNIFICANCE AND IMPACT OF THE STUDY: The secreted proteins of Vp_PirAB-like are key factors of virulence in the V. parahaemolyticus AHPND-causing strain, but not gene copy.

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus.

AIMS: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. METHODS AND RESULTS: Species-specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co-existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. CONCLUSIONS: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple, rapid and cost-effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.

Identification of Vibrio harveyi as a causative bacterium for a tail rot disease of sea bream Sparus aurata from research hatchery in Malta.

A bacterial disease was reported from gilthead sea bream (Sparus aurata) within a hatchery environment in Malta. Symptoms included complete erosion of tail, infection in the eye, mucous secretion and frequent mortality. A total of 540 strains were initially isolated in marine agar from different infected body parts and culture water sources. Subsequently 100 isolates were randomly selected, identified biochemically and all were found to be Vibrio harveyi-related organisms; finally from 100 isolates a total of 13 numbers were randomly selected and accurately identified as V. harveyi by 16S rRNA gene sequencing and species-specific PCR. Ribotyping of these strains with HindIII revealed total of six clusters. In vivo challenge study with representative isolates from each cluster proved two clusters each were highly pathogenic, moderately pathogenic and non-pathogenic. All 13 isolates were positive for hemolysin gene, a potential virulence factor. Further analysis revealed probably a single copy of this gene was encoded in all isolates, although not in the same locus in the genome. Although V. harveyi was reported to be an important pathogen for many aquatic organisms, to our knowledge this might be the first report of disease caused by V. harveyi and their systematic study in the sea bream hatchery from Malta.

Development of simple and rapid PCR-fingerprinting methods for Vibrio cholerae on the basis of genetic diversity of the superintegron.

AIMS: To develop simple and rapid PCR-fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains. METHODS AND RESULTS: PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed targeting region between integrase gene in the SI and its nearby ORF, followed by BglI digestion. Besides, a V. cholerae repeat-amplified fragment length polymorphism (VCR-AFLP) assay was also developed. In the PCR-RFLP, 94 El Tor, 29 classical and 54 O139 strains produced nine, three and six different DNA fingerprints, respectively. On the other hand, VCR-AFLP distinguished these El Tor, classical and O139 strains into five, nine and two DNA fingerprints, respectively. Combining both assays the El Tor, classical and O139 strains could be differentiated into 11, 10 and seven different types, respectively. In a comparative study, pulsed-field gel electrophoresis (PFGE) showed similar differentiation for El Tor (11 types), but lower discrimination for O139 (two types) and classical strains (five types). CONCLUSIONS: The PCR assays based on SI diversity can be used as a useful typing tool for epidemiological studies of V. cholerae. SIGNIFICANCE AND IMPACT OF STUDY: This newly developed method is more discriminatory, simple, rapid and cost-effective in comparison with PFGE, and thus can be widely applicable.

Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus.

AIM: To develop a haemolysin (hly) gene-based species-specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. METHODS AND RESULTS: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species-specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. CONCLUSIONS: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. SIGNIFICANCE AND IMPACT OF THE STUDY: Because there is lack of simple, rapid and cost-effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.