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Staphylococcus aureus, S. epidermidis, S. haemolyticus, and S. hominis are important pathogenic microorganisms that cause food contamination and clinical infections. This study aimed to develop a rapid, accurate, and monitoring technique for simultaneous detection of these Staphylococcus spp. in food. A total of 19 novel candidate species-specific targets for these four Staphylococcus spp. were mined based on pangenome analysis. Four novel targets (comFA for S. aureus, group_14348 for S. epidermidis, group_26190 for S. haemolyticus, and group_26478 for S. hominis) were selected for quantitative polymerase chain reaction (qPCR). The limit of detection in pure culture without pre-enrichment were 102 colony-forming units (CFU)/mL for S. aureus, 103 CFU/mL for S. epidermidis and S. haemolyticus, and 100 CFU/mL for S. hominis, respectively. This method allowed fast, sensitive, and accurate detection of the target Staphylococcus spp. in actual samples and the presence of nontarget bacteria. The feasibility of this method was satisfying in terms of sensitivity, specificity and efficiency after evaluating 100 samples, as well as being consistent with the national standard detection method. Thus, this qPCR assay, based on novel species-specific targets, allows efficient screening for pathogenic Staphylococcus spp., and may help prevent staphylococcal contamination of food.

Cronobacter species are opportunistic pathogens associated with severe infections in immunocompromised individuals. Species differentiation is essential as the sensitivity of different species to chemical reagents, antibiotics, and virulence factors may be diverse. This study aimed to mine new molecular targets for the detection of Cronobacter strains at the species level. Using a bioinformatics approach and the online BLAST software, we obtained serial Cronobacter species-specific target genes. Primers for the selected genes were constructed, and the PCR products were evaluated using various Cronobacter species and non-Cronobacter strains. As a result, 2, 5, 7, and 5 specific target genes were found for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis, respectively. Thereafter, 4- and 5-multiplex PCR (mPCR) was successfully developed to identify these four species based on the validated species-specific primers. Overall, our data indicated that PCR and mPCR assays have excellent specificity and sensitivity for the detection of Cronobacter species, suggesting the potential of their use in food inspection and clinical diagnosis.

Salmonella serotyping is a critical step for the identification of pathological isolates in humans and animals. Whole-genome analysis is a proven method for Salmonella serovars identification. In this study, we analysed 1668 genomic sequences of 23 serogroup B serovars and 129 genomic sequences of 80 non-target serovars to detect serovar-specific molecular targets for serogroup B (O:4 epitopes). Serogroup B is the most common serogroup found in China, whose presence is increasing in recent years. We identified five, eight, one, and seven new serovar-specific genes for Indiana, Derby, Typhimurium, and Agona serovars, respectively. We then designed specific PCR primer sets for these genes to validate our results. All target Salmonella serovars could be detected successfully by PCR in different types of food samples. The analytical sensitivity of PCR for 19 out of 21 pairs of primers was 101–103 CFU/mL and 102–105 CFU/g in pure cultures of the bacteria and artificially contaminated fresh pork samples, respectively. Furthermore, we performed a functional analysis of the newly-identified serovar-specific genes. We found that the function was unknown for most of the genes, although for some of them we were able to identify their generic functional categories (membrane protein genes and secreted proteins genes). Our findings revealed that whole-genome analysis is a valuable tool to identify new serovar-specific targets, which could be used to develop rapid and accurate molecular detection methods and provide additional information about different Salmonella serogroup B serovars.

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 101 CFU/mL in pure culture and 102 CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.

Bacillus cereus is a major foodborne pathogen that can cause a type of diarrhea. Diarrheal syndrome results from the ingestion of food products contaminated with enterotoxin-producing B. cereus. This study investigated the presence of four enterotoxins genes in 130 B. cereus isolated from various food products from Jiangxi, China. The expression levels of the enterotoxin genes in three B. cereus strains of different origins were subsequently analyzed. All of the B. cereus strains harbor at least 1 enterotoxin gene, whereas 34 strains harbor 2 enterotoxin genes, 44 strains harbor 3 enterotoxin genes, and 47 strains carry all of the four enterotoxin genes. The detection rates of the cytK, hblD, nheA, and entFM enterotoxin genes in all B. cereus isolates were 71%, 43%, 92%, and 99%, respectively. Moreover, the food matrix significantly affected the expression of these enterotoxin genes. The majority of the enterotoxin genes were also downregulated in four food matrices, indicating that the relative expression of certain enterotoxin genes, especially the entFM gene, was lower in a real food matrix than in laboratory broths. Hence, these data revealed that B. cereus is a serious health hazard and that the food matrix may influence the virulence expression of B. cereus. Our results provide better insights into the physiology of the microorganism grown in food products.