Dongryeoul Bae

Global Gene Expression of Listeria monocytogenes to Salt Stress

Outbreaks of listeriosis caused by the ingestion of Listeria-contaminated ready-to-eat foods have been reported worldwide. Many ready-to-eat foods, such as deli meat products, contain high amounts of salt, which can disrupt the maintenance of osmotic balance within bacterial cells. To understand how Listeria monocytogenes adapts to salt stress, we examined the growth and global gene expression profiles of L. monocytogenes strain F2365 under salt stress using oligonucleotide probe-based DNA array and quantitative real-time PCR (qRT-PCR) analyses. The growth of L. monocytogenes in brain heart infusion (BHI) medium with various concentrations of NaCl (2.5, 5, and 10%) was significantly inhibited (P < 0.01) when compared with growth in BHI with no NaCl supplementation. Microarray data indicated that growth in BHI medium with 1.2% NaCl upregulated 4 genes and down-regulated 24 genes in L. monocytogenes, which was confirmed by qRT-PCR. The transcript levels of genes involved in the uptake of glycine betaine/(L)-proline were increased, whereas genes associated with a putative phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS), metabolic enzymes, and virulence factor were down-regulated. Specifically, the expression levels of PTS transport genes were shown to be dependent on NaCl concentration. To further examine whether the down-regulation of PTS genes is related to decreased cell growth, the transcript levels of genes encoding components of enzyme II, involved in the uptake of various sugars used as the primary carbon source in bacteria, were also measured using qRT-PCR. Our results suggest that the decreased transcript levels of PTS genes may be caused by salt stress or reduced cell growth through salt stress. Here, we report global transcriptional profiles of L. monocytogenes in response to salt stress, contributing to an improved understanding of osmotolerance in this bacterium.

Transcriptome analysis of Listeria monocytogenes grown on a ready-to-eat meat matrix.

The contamination of ready-to-eat (RTE) meat products with Listeria monocytogenes is a major concern for the food industry. For a better understanding of the adaptation and survival ability of L. monocytogenes grown on turkey deli meat, the transcriptome of L. monocytogenes strain F2365 was determined with a microarray. Microarray data were validated with a quantitative real-time reverse transcription PCR assay. Based on the microarray data, 39 and 45 genes from L. monocytogenes were transcriptionally upregulated and down-regulated, respectively. The genes regulated at the transcriptional level were mainly involved in energy metabolism, fatty acid and phospholipid metabolism, biosynthesis of proteins, transport and binding proteins, DNA metabolism, cellular processes, and regulatory functions. No significant change was noted for the expression of genes encoding known virulence factors such as sigB, prfA, inlA, inlB, plcA, plcB, and hly. These results suggest that L. monocytogenes grown on RTE deli meat changes its transcription of proteins involved in its metabolic pathways to obtain an energy source or to adapt to environmental change without increasing the expression of virulence factors. The global transcriptome profiles provide a better understanding of the growth or adaptation of L. monocytogenes in RTE meat products.

Detection and functionality of the CdtB, PltA, and PltB from Salmonella enterica serovar Javiana

Salmonella infection is one of the major foodborne illnesses in the United States. Several Gram-negative bacterial pathogens, including Salmonella Typhi, produce cytolethal distending toxin (CDT), which arrests growth, induces apoptosis of infected host cells and extends persistence of pathogenic bacteria in the host. The aim of this study was to characterize the functionality of CDT (cdtB, pltA and pltB) from nontyphoidal Salmonella isolates. Fifty Salmonella enterica serovar Javiana isolates from food, environmental, and clinical samples were screened for cdtB, pltA, and pltB genes by PCR, and all were positive for all three genes. Nucleotide sequence analysis of all amplified PCR products showed 100% identity to S. Typhi cdtB. To understand the roles of CdtB, PltA, and PltB in S. Javiana, cdtB, pltA, and pltB deletion mutants were constructed using a lambda Red-based recombination system. In vitro-cultured HeLa cell lines were infected with a wild-type strain and its isogenic ∆cdtB, ∆pltA, and ∆pltB to determine whether the strains of S. Javiana are responsible for invasion and cytolethal distending intoxication, including cell cycle arrest, cytoplasmic distension, and nuclear enlargement of host target cells. The results showed that HeLa cells infected with S. Javiana wild type were arrested in G2 /M and had distended cytoplasm and nuclei that were larger than those infected with S. Javiana ∆cdtB and ∆pltA strains. The S. Javiana ∆pltB strain retained the ability to induce cytoplasmic distension and cell cycle arrest, whereas the complemented ∆cdtB and ∆pltA S. Javiana strains showed activity like the wild-type strains. CdtB and pltA from S. Javiana had apparent effects on the distension of both cytoplasm and nucleus as well as cell cycle arrest of HeLa cell lines after 72 h of infection. Our data show a significant difference between the wild-type cdtB strain and its isogenic ∆cdtB for invasion of the cell lines. Therefore, CdtB produced from S. Javiana strains may play an important role in pathogenesis in host cells.

Characterization of extended-spectrum β-lactamase (ESBL) producing non-typhoidal Salmonella (NTS) from imported food products

Food contaminated with extended-spectrum β-lactamase (ESBL)-producing Salmonella enterica has emerged as an important global issue due to the international food-product trade. Therefore, the purpose of this study was to investigate whether imported food products can serve as a reservoir for non-typhoidal Salmonella (NTS) that can transmit β-lactam-resistance to humans through ingestion of the contaminated food. NTS isolates (n=110) were collected from various imported food products (n=3480) from 2011 to 2013. The NTS isolates were analyzed by serotyping, antimicrobial susceptibility tests, and plasmid profiling. Salmonella ser. Weltevreden, Salmonella ser. Newport, Salmonella ser. Senftenberg, Salmonella ser. Virchow, Salmonella ser. Enteritidis, Salmonella ser. Typhimurium, and Salmonella ser. Bareilly were the most prevalent serovars. Nine NTS strains were resistant to ampicillin and/or one or more cephalosporins (MIC>32 μg/mL). Polymerase chain reaction (PCR) detection revealed that all nine isolates carried the bla(TEM-1) β-lactamase gene, with or without the bla(CTX-M-9) or bla(OXA-1) genes. Two isolates, PSS_913 and PSS_988, exhibited decreased susceptibility to extended-spectrum cephalosporins and ampicillin. Plasmids ranging in size from less than 8 to over 165 kbp, from all of the 9 resistant isolates, belonged to the IncHI1, IncI1, IncN, or IncX groups. Conjugation experiments and Southern hybridization, using bla(TEM-1), confirmed the plasmid-mediated transfer of ESBL genes, which resulted in increased MICs of β-lactams for Escherichia coli transconjugants. The contamination of imported food products by NTS with conjugative plasmid-borne ESBL genes may contribute to the spread of ESBL-producing NTS and compromise the therapeutic activity of extended-spectrum β-lactam antibiotics.

The sub-species characterization and antimicrobial resistance of Listeria monocytogenes isolated from domestic and imported food products from 2004 to 2011

Thirty-five Listeria monocytogenes strains isolated from domestic and imported food products including seafood, vegetables, and dairy foods were characterized by serotyping, molecular sub-typing, and antimicrobial susceptibility. L. monocytogenes serovars 1/2a and 1/2b strains were dominant as compared to other two serovars 4b and 1/2c strains. The dendrogram of AscI or ApaI-digested PFGE profiles of L. monocytogenes strains was classified into 23 (with 8 groups) or 3 (with 2 groups) different PFGE types, respectively. The AscI-digested groups consisted of the same serovar or food-source. Antimicrobials such as ampicillin, gentamicin, and trimethoprim-sulfamethoxazole are widely used in the treatment of listeriosis. Of the isolates used in this study, NCTR_LM14 and NCTR_LM57 were resistant to several classes of antimicrobials including aminoglycosides, penicillin, tetracycline, glycopeptide and fluoroquinolones. The multi-antimicrobial resistant isolates also showed higher efflux pump activity as compared to the antimicrobial sensitive strains NCTR_LM06 and L. monocytogenes EGD-e. This study demonstrates that L. monocytogenes isolates from various food products are genetically diverse with some isolates being resistant to more than 3 different antibiotic classes. This study also indicates that the efflux pump activity of the antibiotic resistant strains was higher than antimicrobial susceptible strains. Therefore, we propose that the antibiotic resistance observed in these strains may be conferred by the results of a highly active efflux pump system.

Isolation and Characterization of Antimicrobial-Resistant Nontyphoidal Salmonella enterica Serovars from Imported Food Products.

The objective of this study was to determine antimicrobial resistance and elucidate the resistance mechanism in nontyphoidal Salmonella enterica serovars isolated from food products imported into the United States from 2011 to 2013. Food products contaminated with antimicrobial-resistant nontyphoidal S. enterica were mainly imported from Taiwan, Indonesia, Vietnam, and China. PCR, DNA sequencing, and plasmid analyses were used to characterize antimicrobial resistance determinants. Twentythree of 110 S. enterica isolates were resistant to various antimicrobial classes, including β-lactam, aminoglycoside, phenicol, glycopeptide, sulfonamide, trimethoprim, and/or fluoroquinolone antimicrobial agents. Twelve of the isolates were multidrug resistant strains. Antimicrobial resistance determinants blaTEM-1, blaCTX-M-9, blaOXA-1, tetA, tetB, tetD, dfrA1, dfrV, dhfrI, dhfrXII, drf17, aadA1, aadA2, aadA5, orfC, qnrS, and mutations of gyrA and parC were detected in one or more antimicrobial-resistant nontyphoidal S. enterica strains. Plasmid profiles revealed that 12 of the 23 antimicrobial-resistant strains harbored plasmids with incompatibility groups IncFIB, IncHI1, IncI1, IncN, IncW, and IncX. Epidemiologic and antimicrobial resistance monitoring data combined with molecular characterization of antimicrobial resistance determinants in Salmonella strains isolated from imported food products may provide information that can be used to establish or implement food safety programs to improve public health.

Prevalence, toxin gene profile, antibiotic resistance, and molecular characterization of Clostridium perfringens from diarrheic and non-diarrheic dogs in Korea

Clostridium perfringens causes diarrhea and other diseases in animals and humans. We investigated the prevalence, toxin gene profiles, and antibiotic resistance of C. perfringens isolated from diarrheic dogs (DD) and non-diarrheic dogs (ND) in two animal hospitals in Seoul, Korea. Fecal samples were collected from clinically DD (n = 49) and ND (n = 34). C. perfringens was isolated from 31 of 49 DD (63.3%) and 21 of 34 ND dogs (61.8%). All C. perfringens strains were positive for the α toxin gene, but not for the β, ε, or ι toxin genes; therefore, all strains were identified as type A C. perfringens. All isolates were cpe-negative, whereas the β2 toxin gene was identified in 83.9% and 61.9% of isolates from DD and ND, respectively. Most isolates were susceptible to ampicillin (94%), chloramphenicol (92%), metronidazole (100%), moxifloxacin (96%), and imipenem (100%). However, 25.0% and 21.2% of isolates were resistant to tetracycline and clindamycin, respectively. Molecular subtyping of the isolated strains was performed by using pulsed-field gel electrophoresis. Fifty-two isolates were classified into 48 pulsotypes based on more than 90% similarity of banding patterns. No notable differences were observed among the isolates from DD and ND.

Addition of Rifampicin to Bolton Broth to Inhibit Extended‐Spectrum β‐Lactamase‐Producing Escherichia coli for the Detection of Campylobacter

Exponential growth of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Campylobacter media has become a common problem for the detection of Campylobacter in chicken meats. We investigated the minimum inhibitory concentration of 40 ESBL-producing E. coli isolates from meats obtained from various countries against antibacterial agents in Bolton broth (cefoperazone, vancomycin, and trimethoprim). All ESBL-producing E. coli strains were resistant to cefoperazone and vancomycin, whereas 50% of them were resistant to trimethoprim and grew in Bolton broth. We found that 20 μg/mL of rifampicin inhibited the growth of trimethoprim-resistant E. coli strains. Hence, we added 20 μg/mL of rifampicin to Bolton broth to improve the isolation of Campylobacter from chicken carcass rinses. The isolation rate of Campylobacter was significantly higher in the modified broth (44 out of 58, 75.9%, P < 0.05) than in the normal broth (0 out of 58, 0%). Furthermore, the number of agar plates with non-Campylobacter spp. was much lower after enrichment in the modified broth (4 out of 58, 6.9%, P < 0.05) than in the normal broth (58 out of 58, 100%).