Molecular epidemiology and antibiotic resistance phenotypes and genotypes of salmonellae from food supply chains in China

Salmonella is an important zoonotic agent and a vehicle for antibiotic resistance genes. Here, 294 isolates from humans and food-producing animals were subjected to serotyping, multilocus sequence typing, and assessment of phenotypic (15 antibiotics) and genotypic (32 resistance genes) antimicrobial resistance. Twenty-two serotypes and 35 sequence types (STs) were identified, the most common STs being ST11, including S. Enteritidis from chickens and humans; ST17, including S. Indiana from chickens; and ST40, including S. Derby from pigs and humans. Antimicrobial resistance phenotypes and genotypes exhibited ST- and serovar-specific features. ST11, clonal complex (CC) 19, ST40, and ST155 were moderately multidrug-resistant (MDR) clones, most of the isolates of which were resistant to between 3 and 6 antibiotics. Isolates of a super-MDR clone, ST17, demonstrated resistance against 9 to 14 antimicrobials, in particular, ampicillin, amoxicillin-clavulanic acid, cefotaxime, cefepime, cefoperazone, ceftriaxone, and ciprofloxacin. Consistent with this, ST17 (S. Indiana) was associated with a gene cluster comprising bla_CTX-M (and/or bla_OXA-1-like together with bla_TEM-1-like), sul1, aacC4, aac(6)-1b, floR, and dfrA17, while the moderately-MDR clones (ST11, S. Enteritidis) were more closely linked to the bla_TEM-1-like gene. The similar genetic clones isolated from animals and humans indicate a common ancestor, and implicate animals as a major salmonellae source. Antibiotic abuse in animal production appears to be the origin of MDR and super-MDR isolates, the latter being closely associated with the β-lactamase genes bla_CTX-M, bla_OXA-1-like, and bla_TEM-1-like. Carried by chickens, ST17 (S. Indiana) is an emerging super-MDR clone whose associated resistance genes are expanding to other ST clones and serotypes being transmitted to animals and humans.     

Salmonella isolated from the slaughterhouses and correlation with pork contamination in free market

This study surveyed the distribution, antimicrobial susceptibility profiles and serotype of Salmonella isolated from three slaughterhouses, and performed molecular typing on these isolates, to understand the relationship between strains of Salmonella obtained from the pork production chain in Yangzhou, China. Samples from slaughtered pigs and the slaughtering environment were collected from three slaughterhouses in Yangzhou, Jiangsu province, from October 2012 to July 2013. The positive identification rates of Salmonella in slaughtered pigs and the environmental samples were 46.6% and 48.8%, respectively. The prevalence of Salmonella in slaughterhouses were affected by seasonal factors and reached the peak in summer. Among the Salmonella serovars identified, S. Derby was most prevalent in slaughterhouses, but other serovars like S. Typhimurium, S. Meleagridis and S. Anatum were also widespread. Antimicrobial susceptibility testing revealed that 32 and 131 different MDR patterns were found among the strains from the environment and slaughtered pig samples, respectively. Fifty-six isolates of S. Derby and 16 strains of S. Typhimurium were characterized by the technique of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme Xba I. 35 and 11 PFGE patterns were generated among the selected isolates. Four isolates of S. Derby isolates with the same pattern (PF26) were isolated from cooling water, evisceration and carcass, suggesting that cross contamination occurred between the environment and the slaughtered pigs. Six S. Typhimurium in cluster 1 with the same ST type (ST19) came from different parts of the slaughtered pig, which could have occurred because of horizontal transmissions along the slaughtering process. The same PFGE patterns of Salmonella were found in both samples from carcasses in the slaughterhouse and in the Yangzhou pork market, proving that Salmonella had spread from the slaughterhouse to the pork market. In conclusion, our study demonstrate that serious cross contamination occurred in Yangzhou slaughterhouses and can contribute Salmonella contamination in pork sold in the local public market.     

Phylogenetic and toxinogenic characteristics of Bacillus cereus group members isolated from spices and herbs

The foodborne pathogen Bacillus (B.) cereus is a common contaminant in spices and herbs. To further characterise B. cereus and its closely related group members present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with view to B. cereus group species, phylogenetic affiliation and toxinogenic potential.Of a total of 59 isolates 44 were identified as B. cereus sensu stricto (s.s.), four as B. toyonensis-like, five as B. thuringiensis, one as B. weihenstephanensis, two as B. pseudomycoides/B. mycoides and three as undefined B. cereus group species. A maximum of three different species occurred simultaneously in the same spice sample. The isolates comprised 33 multilocus (ML) sequence types (STs), which can be assigned to three different phylogenetic groups. Except two B. pseudomycoides/B. mycoides strains, all isolates were able to produce enterotoxins and one strain the emetic toxin cereulide as detected by an immunoassay and LC-MS, respectively. The prevalence of toxin genes was 96.6% for nheA, 94.9% for hblD, 50.8% for cytK-2 and 1.7% for ces. The emetic strain was characterised by ST 869, which for the first time was assigned to an emetic B. cereus (s.s.) strain and is not part of the previously known two emetic MLST clusters.Our results demonstrate that not only B. cereus (s.s.) but also toxin producing B. thuringiensis, B. weihenstephanensis and B. toyonensis-like strains could be detected in condiments. For some isolates MLST revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described species markers.     

Substratum attachment location and biofilm formation by Bacillus cereus strains isolated from different sources: Effect on total biomass production and sporulation in different growth conditions

Bacillus cereus, an endospore forming human pathogen associated with foodborne diseases, can form biofilms and attach to surfaces of processing equipment in the food industry. It is a consistent source of contamination and/or cross contamination of processed food products. The objective of this study was to understand substratum attachment location and biofilm formation behavior of B. cereus strains under different growth conditions. A total of 60 strains isolated from food, human, or farm and a number of reference strains were used in this study. Substratum attachment locations of these strains in 96-well microtiter plates were highly diversified among these strains. Strains isolated from food showed higher preference to attach at the air-liquid interface during early stage of biofilm formation. To the best of our knowledge, this is the first report showing the level of substratum attachment location and biofilm formation of B. cereus strains isolated from different sources. Substratum properties did not affect biofilm formation location when a number of selected strains were grown on stainless steel coupon, indicating that biofilm formation location might be independent of the type of substratum. Substratum attachment location and biofilm formation related phenotypes such as total biomass production, number of sessile cells, and sporulation were closely correlated. Substratum attachment location and sporulation behavior were strongly affected during biofilm formation under nutrient stress condition. The number of spores was significantly increased in biofilms grown under nutrient stress condition even though total biomass formation was lower. Our results on substratum attachment location and related biofilm formation behavior are substantially important for food industries where different surfaces are prone to B. cereus attachment, particularly for setting up and implementing clean in place (CIP) protocols.     

Prevalence and antimicrobial resistance of Staphylococcus aureus isolated from raw milk and dairy products

The objectives of this study were to determine the prevalence and antimicrobial resistance of Staphylococcus aureus isolated from raw milk (cow and sheep) and dairy products (traditional cheese and kashk) in Mazandaran Province, Iran. A total of 2650 samples, including 1930 raw milk and 720 dairy products were purchased from retail stores. Out of 2650 samples, S. aureus was detected in 328 samples (12.4%) in which 53 (16.2%) were positive for methicillin-resistant S. aureus. The S. aureus isolates showed resistance to tetracycline (56.1%), followed by penicillin G (47.3%), oxacillin (16.2%), lincomycin (11.9%), clindamycin (11.3%), erythromycin (7.9%), streptomycin (5.8%), cefoxitin (5.5%), kanamycin (4%), chloramphenicol (3.7%), and gentamicin (2.1%). A high frequency of blaZ (46%) and tetM (34.8%) resistance genes was found in S. aureus isolates. The findings of this study revealed consumption of raw milk and dairy products as a potential risk of foodborne infection in this region.     

The effects of high pressure processing and slightly acidic electrolysed water on the structure of Bacillus cereus spores

The aim of this study was to investigate the effects of high pressure processing (HPP) and slightly acidic electrolysed water (SAEW) on the structure of B. cereus spores in order to understand the inactivation mechanism. The physiological response of spores was studied by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), dipicolinic acid (DPA) release, su­per-res­o­lu­tion mul­ti­pho­ton con­fo­cal mi­cro­scope (SMCM), and flow cytometry (FCM). The morphology of B. cereus spores treated with HPP-SAEW exhibited a large fraction damage, and also an incomplete exosporium. Furthermore, spores treated with HPP-SAEW had an irregular surface bumps, and even a critical distortion. Approximately 90% of DPA was released in the groups of treated by HPP-SAEW, and 80% of DPA was released in single HPP treatment groups. The SMCM and FCM results indicated that HPP-SAEW methods was not mainly depended on germination, but directly killed. The mechanism of HPP-SAEW possibly was that one of the proteins was particularly susceptible to SAEW under HPP conditions.     

Distribution and expression of the enterotoxin genes of Bacillus cereus in food products from Jiangxi Province,China

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.