Kanokrat Siripanichgon

Possible horizontal transmission of Salmonella via reusable egg trays in Thailand

Salmonella contamination of eggshells, egg contents, reusable egg trays, and various environmental samples was assessed. Although the overall Salmonella contamination rate from egg farms was low (3.2%), over a quarter (26.7%) of egg trays from farms and more than one third (36.7%) of trays from the market were contaminated. Salmonella strains isolated from reusable egg trays were analyzed by serotyping, antimicrobial susceptibility test and XbaI pulsed-field gel electrophoresis (PFGE) typing. Five serovars (S. Braenderup, S. Emek, S. Weltevreden, S. Stanley, and S. Derby) were isolated, and half of the strains assessed were found to be resistant to one or more of the six antimicrobial agents examined. The overall resistance rates to nalidixic acid, trimethoprim-sulfamethoxazole, tetracycline, and ampicillin were 40.7%, 36.0%, 26.7% and 3.5%, respectively. The PFGE types were matched against sample location and drug resistance. S. Braenderup PFGE type A2 (susceptible to all tested drugs) was isolated from all sample sites; PFGE type A2 (resistant to nalidixic acid) was isolated from Farm C and the market. S. Braenderup PFGE type A1 (resistant to four drugs) was isolated from Farms A and C. S. Weltevreden PFGE type C3 (susceptible to all tested drugs) was isolated from Farms A and B and type C4 (susceptible to all tested drugs) was isolated from Farm A and the market. The distribution of the related genotypes and resistance patterns of Salmonella in egg farms and the market indicate drug-resistant strains of Salmonella may be spread on reusable egg trays.

Characterization of antibiotic resistance in Escherichia coli isolated from shrimps and their environment.

Antimicrobial resistance in bacteria associated with food and water is a global concern. To survey the risk, 312 Escherichia coli isolates from shrimp farms and markets in Thailand were examined for susceptibility to 10 antimicrobials. The results showed that 17.6% of isolates (55 of 312) were resistant to at least one of the tested drugs, and high resistance rates were observed to tetracycline (14.4%; 45 of 312), ampicillin (8.0%; 25 of 312), and trimethroprim (6.7%; 21 of 312); 29.1% (16 of 55) were multidrug resistant. PCR assay of the tet (A), tet (B), tet (C), tet (D), tet (E), and tet (G) genes detected one or more of these genes in 47 of the 55 resistant isolates. Among these genes, tet (A) (69.1%; 38 of 55) was the most common followed by tet (B) (56.4%; 31 of 55) and tet (C) (3.6%; 2 of 55). The resistant isolates were further investigated for class 1 integrons. Of the 55 resistant isolates, 16 carried class 1 integrons and 7 carried gene cassettes encoding trimethoprim resistance (dfrA12 or dfrA17) and aminoglycosides resistance (aadA2 or aadA5). Two class 1 integrons, In54 (dfrA17-aadA5) and In27 (dfrA12-orfF-aadA2), were found in four and three isolates, respectively. These results indicate a risk of drug-resistant E. coli contamination in shrimp farms and selling places. The occurrence of multidrug-resistant E. coli carrying tet genes and class 1 integrons indicates an urgent need to monitor the emergence of drug-resistant E. coli to control the dissemination of drug-resistant strains and the further spread of resistance genes to other pathogenic bacteria.

Enhancement of Legionella pneumophila Culture Isolation from Microenvironments by Macrophage Infectivity Potentiator (mip) Gene‐Specific Nested Polymerase Chain Reaction

The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene‐specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100‐treated water samples, or by using silica‐gel membrane spin column‐eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3′ proximity of the mip‐coding gene of L. pneumophila deposited in genome databases. EcoRI‐ or KpnI‐digested PCR fragments with expected sizes were also confirmed in all 52 PCR‐positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR‐positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.

A novel bacteriocin from Enterococcus faecalis 478 exhibits a potent activity against vancomycin-resistant enterococci

The emergence of multidrug-resistant enterococci (MDRE) and particularly vancomycin-resistant enterococci (VRE) is considered a serious health problem worldwide, causing the need for new antimicrobials. The aim of this study was to discover and characterize bacteriocin against clinical isolates of MDRE and VRE. Over 10,000 bacterial isolates from water, environment and clinical samples were screened. E. faecalis strain 478 isolated from human feces produced the highest antibacterial activity against several MDRE and VRE strains. The optimum condition for bacteriocin production was cultivation in MRS broth at 37°C, pH 5–6 for 16 hours. The bacteriocin-like substance produced from E. faecalis strain EF478 was stable at 60°C for at least 1 hour and retained its antimicrobial activity after storage at -20°C for 1 year, at 4°C for 6 months, and at 25°C for 2 months. A nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) analysis showed that the amino acid sequences of the bacteriocin-like substance was similar to serine protease of E. faecalis, gi|488296663 (NCBI database), which has never been reported as a bacteriocin. This study reported a novel bacteriocin with high antibacterial activity against VRE and MDRE.

New facet of non-O1/non-O139 Vibrio cholerae hemolysin A: a competitive factor in the ecological niche

Different serogroups of Vibrio cholerae may inhabit the same ecological niche. However, serogroup O1/O139 strains are rarely isolated from their ecological sources. Quite plausibly, the non-O1/non-O139 vibrios and other bacterial species suppress growth of O1/O139 strains that share the same niche. Our bacterial inhibition assay data indicated that certain non-O1/non-O139 strains used a contact-dependent type VI secretion system (T6SS) to suppress growth of the O1 El Tor, N16961 pandemic strain. Comparative proteomics of the O1 and the suppressive non-O1/non-O139 strains co-cultured in a simulated natural aquatic microcosm showed that SecB and HlyD were upregulated in the latter. The HlyD-related effective factor was subsequently found to be hemolysin A (HlyA). However, not all hlyA-positive non-O1/non-O139 strains mediated growth suppression of the N16961 V. cholerae; only strains harboring intact cluster I HlyA could exert this activity. The key feature of the HlyA is located in the ricin-like lectin domain (β-trefoil) that plays an important role in target cell binding. In conclusion, the results of this study indicated that non-O1/non-O139 V. cholerae suppressed the growth of the O1 pandemic strain by using contact-dependent T6SS as well as by secreting the O1-detrimental hemolysin A during their co-persistence in the aquatic habitat.