The characteristics of volatile compounds from five different bacterial species, Escherichia coli O157:H7, Salmonella Enteritidis, Shigella flexneri, Staphylococcus aureus, and Listeria monocytogenes, growing, respectively, in trypticase soy broth were monitored by headspace solid-phase micro-extraction/gas chromatography-mass spectrometry. The results showed that most volatile organic compounds (VOCs) of five pathogens started to increase after the sixth to tenth hour. Methyl ketones and long chain alcohols were representative volatiles for three Gram-negative bacteria. The especially high production of indole was characterized to E. coli O157:H7. The production of 3-hydroxy-2-butanone was indicative of the presence of two Gram-positive bacteria. Both 3-methyl-butanoic acid and 3-methyl-butanal were unique biomarkers for S. aureus. The population dynamics of individual pathogen could be monitored using the accumulation of VOCs correlated with its growth. And these five pathogens could be distinguishable though principle component analysis of 18 volatile metabolites. Moreover, the mixed culture of S. aureus and E. coli O157:H7 was also investigated. The levels of 3-methyl-butanal and 3-methyl-butanoic acid were largely reduced; while the level of indole almost unchanged and correlated with E. coli O157:H7 growth very well. The characteristics of volatiles from the five foodborne pathogens could lay a fundamental basis for further research into pathogen contamination control by detecting volatile signatures of pathogens.

The aim of the present study was to investigate the antibiotic resistance profiles and the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from two production swine operations in Sichuan Province, China, between August 2002 and February 2007. The prevalence of ESBL-producing E. coli increased dramatically from 2.2% to 10.7% during this period. This increase appeared mostly related to dissemination of CTX-M-type ESBLs among E. coli isolates. Of 212 E. coli isolates studied, 14 harbored ESBL genes. Among them, 13 harbored bla(CTX-M-15/22) and one harbored bla(SHV-2). To our knowledge, this is the first study to identify bla(CTX-M-22) from production animals. One isolate in 2002 harbored bla(SHV-2), indicating that ESBL genes have been present in farm animals in China since at least 2002. Molecular characterization and pulsed-field gel electrophoresis of the ESBL-producing isolates suggested that different mechanisms may be involved in the dissemination of the CTX-M genes and revealed that additional resistance determinants for non-beta-lactam antibiotics were carried by plasmids encoding certain ESBL genes. Results of this study provide an example of how ESBL genes, particularly those of CTX-M lineages, are rapidly spreading among E. coli isolates from commercial pig farms in Sichuan province of China.

Aims:  To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results:  Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions:  This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study:  This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same genetic origin. However, two ORFs in S. aureus genomes were predicted to encode nucleases. One encoded an unnamed nuclease A (SNase) (termed nuc1), and the other encoded a thermonuclease (TNase) named nuc (termed nuc2). In order to verify whether the two thermostable nuclease proteins are coexpressed in S. aureus, the nuc1 and nuc2 genes were cloned and expressed in Escherichia coli, and both of the recombinant proteins showed thermostable nuclease activity in a toluidine blue-DNA assay. Furthermore, a nuc1-deleted mutant of S. aureus strain RN4220 (termed RNDeltanuc1) was successfully constructed by homologous recombination. Selection and characterization of this mutant strain revealed that it still exhibited thermostable nuclease activity, but at a relative lower level than that of the parent strain. The nucleases secreted by the parent strain and nuc1-deleted strain still showed functional activity after 30 min at 121 degrees C. The findings indicated that two types of thermostable nucleases, encoded by two different genes, coexisted in S. aureus.