This paper presents a handheld device that is capable of simplifying multistep assays to perform sensitive detection of foodborne pathogens. The device is capable of multiplexed detection of Escherichia coli (E. coli) O157:H7, Salmonella Typhimurium (S. Typhimurium), Staphylococcus aureus, and Bacillus cereus. The limit of detection for each bacterium was characterized, and then, the detection of bacteria from contaminated fresh lettuces was demonstrated for two representative foodborne pathogens. We employed a sample pretreatment protocol to recover and concentrate target bacteria from contaminated lettuces, which can detect 1.87 × 10 CFU of E. coli O157:H7 and 1.47 × 10 CFU of S. Typhimurium/1 g of lettuce without an enrichment process. Lastly, we demonstrated that the limit of detection can be reduced to 1 CFU of E. coli O157:H7 and 1 CFU of S. Typhimurium/1 g of lettuce by including a 6 h enrichment of contaminated lettuces in growth media before pretreatment.

Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.Electronic supplementary materialThe online version of this article (doi:10.1007/s00203-017-1415-9) contains supplementary material, which is available to authorized users.

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Staphylococcus aureus is an important foodborne pathogen on global basis. The current study investigated the genetic patterns in S. aureus isolates from leaf vegetables (n = 53). Additional isolates from livestock (n = 31) and humans (n = 27) were compared with the leaf vegetable isolates. Genes associated with toxins, antibiotic resistance, and pulsed-field gel electrophoresis (PFGE) patterns were analyzed. At least 1 enterotoxin-encoding gene (sea, seb, sec, sed, and see) was detected in 11 of 53 (20.75%) leaf vegetable isolates. When the agr (accessory gene regulator) grouping was analyzed, agr II was the major group, whereas agr IV was not present in leaf vegetable isolates. All S. aureus isolates from leaf vegetables were resistant to more than one of the antibiotics tested. Nineteen of 53 (35.85%) isolates from leaf vegetables exhibited multidrug-resistance, and 11 of these were MRSA (methicillin-resistant S. aureus). A dendrogram displaying the composite types of S. aureus isolates from 3 origins was generated based on the combination of the toxin genes, agr genes, antibiotic resistance, and PFGE patterns. The isolates could be clustered into 8 major composite types. The genetic patterns of S. aureus isolates from leaf vegetables and humans were similar, whereas those from livestock had unique patterns. This suggests some S. aureus isolates from leaf vegetables to be of human origin.