Prevalence and profile of Salmonella from samples along the production line in Chinese beef processing plants

This work investigated the prevalence of Salmonella, the serotypes and antibiotic resistance of the isolated strains from four beef processing plants of China. The prevalence of Salmonella in hide (n = 70), feces (n = 70), pre-evisceration carcass (n = 70), post-evisceration carcass (n = 70), post-washing carcass (n = 70), chilled carcass (n = 80), and raw meat (n = 80) samples was 20.0%, 18.6%, 1.4%, 1.4%, 2.9%, 1.3%, and 1.3%, respectively. Among the four plants, there were significant differences in the prevalence of Salmonella on hides and in feces. During the processing, Salmonella was significantly reduced after hide removal. Seven serotypes of Salmonella were identified among the eighty-three isolates. Salmonella Agona was the dominant serotype (p < 0.05, 53.0%), followed by Salmonella Senftenberg (16.9%), Salmonella Meleagridis (10.8%), and Salmonella Derby (9.6%). None of the isolated strains were found to be resistant to sixteen commonly used antimicrobial agents. The results of this study indicate that Salmonella contamination is common in samples along the production line, with S. Agona as dominant serotype. Specific measures should be taken to prevent and/or treat Salmonella contamination in corresponding products in Chinese beef processing plants. Furthermore, the current research might provide baseline information of Salmonella prevalence profile in Chinese beef processing plant, which could be used for the future study.     

Prevalence and characterization of Yersinia enterocolitica isolated from retail foods in China

Yersinia enterocolitica is an important food-borne enteropathogen that causes gastrointestinal syndromes. The aims of this study were to identify Y. enterocolitica in food samples in China, and to assess the pathogenic potential and antimicrobial resistance, and to characterize the genotypes of the isolates. From July 2011 to May 2014, a total of 2320 food samples were obtained, and 47 (2.03%) were found positive for Y. enterocolitica, while 706 retail-level ready-to-eat products and 249 vegetable samples were negative. A total of 58 Y. enterocolitica strains were isolated. All isolates belonged to biotype 1A, and the primary serotype was O:8. All strains lacked the ail, virF, ystA, and ystC virulence genes, but harbored the ystB, fepD, ymoA, fes, and sat genes. All 58 strains were sensitive to kanamycin and sulfonamide, but were resistant to two or more antibiotics. Most of the strains expressed the β-lactamase genes; the presence of blaA and blaB was detected in 97% and 100% of isolates, respectively. Many strains were resistant to trimethoprim/sulfamethoxazole (79.3%), ampicillin (91.4%), and cephalothin (91.4%). The 58 strains were grouped into three clusters and one singleton by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) at a similarity coefficient of 70%, and each cluster was largely organized by geographical region. This study provides a valuable accounting of the prevalence of Y. enterocolitica from a nationwide survey of foods in China, and highlights the seasonal effects of Y. enterocolitica prevalence in foods in China for the first time.     

Isolation and characterization of Listeria monocytogenes in Chinese food obtained from the central area of China

The objective of this study was to investigate the prevalence and characteristics of Listeria monocytogenes isolated from Chinese food, including frozen dumplings, flavored raw meat, roasted meat, braised meat, and a cold vegetable dish with sauce. A total of 900 food samples were collected from supermarkets, open-air markets, and delicatessens in three large cities in the central area of China to examine the presence of L. monocytogenes; 21 (2.3%) of the samples were positive for this pathogen. Among the different samples, braised meat showed the highest L. monocytogenes detection rate (4.4%). Samples obtained from delicatessens showed a much higher L. monocytogenes contamination rate (8.3%) than those from open-air markets (6.7%) or supermarkets (0%). Multilocus sequence typing (MLST) analysis indicated that the 21 bacterial isolates belonged to 12 ST subgroups. ST5 was the largest and contained 7 isolates (33.3%); it was followed by ST474, ST121 and ST9 (each containing 2 isolates [10.5%]). Antibiotic susceptibility analysis showed that the 21 L. monocytogenes isolates were thoroughly resistant to cefoxitin but highly susceptible to doxycycline and ciprofloxacin. The presence of 10 virulence genes was evaluated by PCR, which showed that inlA, inlC, inlJ, prfA, hlyA, and plcB were present in all isolates and that inlB, actA, plcA and iap were present in 71.4–90.5% of the isolates. This study provides a useful reference for risk assessment and control of L. monocytogenes contamination in Chinese food and for the treatment of clinical listeriosis.     

Evaluation of a novel antimicrobial solution and its potential for control Escherichia coli O157:H7, non-O157:H7 shiga toxin-producing E. coli,Salmonella spp., and Listeria monocytogenes on beef

The goal of this study was to evaluate the efficacy of a novel antimicrobial solution made with chitosan, lauric arginate ester, and organic acids on Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and non-O157 shiga toxin-producing E. coli cocktails and to test its potential to be used as a marinade for raw beef. Fresh beef top round steaks were surface-inoculated with the pathogen cocktails at approximately 2.5 or 4.5 Log CFU/cm², marinated with the antimicrobial solution (AMS), and then stored at 4 °C for 6, 24, and 48 h. Three commercially available marinades were used for comparison. Results revealed that AMS had the most antimicrobial effect regardless of the type or inoculation level of pathogens (P < 0.05). After 6 h, the AMS marination reduced all pathogens to levels below the limit of detection (<1 Log CFU/cm²), resulting in a 3.5 Log CFU/cm² reduction. When AMS was diluted with autoclaved distilled water by 5 times (AMS 1:5) or 10 times (AMS 1:10), its antimicrobial efficacy was impacted by marination time, the inoculated pathogens, and the inoculation levels. This study demonstrates that the developed antimicrobial solution has a great potential to be used during marination by consumers to ensure better food safety.     

Genotypic characterization of Shiga toxin-producing Escherichia coli O157:H7 isolates in food products from china between 2005 and 2010

Shiga toxin-producing Escherichia coli (STEC) O157 is an important foodborne pathogen. The aims of this study were to determine genetic relatedness of STEC O157 isolated from foods in China. STEC O157 isolates from food were characterized by virulence gene typing, antibiotyping, Multi-locus sequence typing (MLST), pulsed-field gel electrophoresis analysis (PFGE), Multi-locus variable number tandem repeat analysis (MLVA), and the single nucleotide polymorphism (SNP) clade typing. Of the 30 STEC O157 isolates analyzed, all isolates harbored eae, exhA, stx1 and/or stx2 genes with stx2c subtype predominating. By MLST, they were relatively homogenous with only 4 STs. PFGE and MLVA generated 22 pulsotyples and 23 patterns, respectively, which showed considerable diversity. Only one clade 8 isolate was detected. These results indicate that STEC O157 isolates from foods in China were heterogeneous. There was no correlation between genotypic characteristics and sources of isolates. Since different subtyping methods often give different discriminatory powers, the use of more than one subtyping approach is necessary in providing a more accurate picture of the genetic diversity of STEC O157. Four isolates were from ready-to-eat meat or salads, underscores the risk of infections. There is a need for surveillance of STEC O157 in foods and clinically and implementation of prevention strategies to prevent outbreaks of STEC O157 infections in China.     

Trends in the occurrence and characteristics of Campylobacter jejuni and Campylobacter coli isolates from poultry meat in Northern Poland

The aim of this study was to investigate the occurrence of Campylobacter spp. in poultry meat available in retail stores in the northern part of Poland during a five-year period (2009–2013). A total of 742 poultry meat samples were collected from butcher shops and supermarkets including the following types of samples: chicken breast filets (n = 133), turkey breast filets (n = 112), chicken wings (n = 135), chicken leg quarters (n = 128), chicken drumsticks (n = 115), and chicken giblets (n = 119).The results indicated that 41.6% of the samples were positive for Campylobacter spp., and Campylobacter jejuni was predominant in this study. The prevalence of Campylobacter spp. changed during the study period, decreasing from 60.2% in 2009 to 32% in 2013.The characterization of the isolates revealed a high prevalence of Campylobacter virulence genes. All Campylobacter spp. isolates from poultry meat contained the cadF gene, which is responsible for adherence. The flaA gene, which is involved in motility, was present in all C. jejuni and Campylobacter coli strains. The cdtB, which is associated with toxin production, was present in 93.3% of C. jejuni strains and 89.6% of C. coli strains. The iam gene, which is associated with the invasiveness of Campylobacter spp., was predominant in C. coli strains (95.6%) compared to C. jejuni strains (84.5%).Resistance to four antimicrobials was also examined. The prevalence of resistance among the obtained C. jejuni and C. coli isolates was as follows: ciprofloxacin (62.8% and 72.2%, respectively), tetracycline (42.3% and 42.6%, respectively), erythromycin (3% and 1.7%, respectively) and azithromycin (1%). Multidrug resistance was more frequent among C. jejuni isolates (29.8%) than among C. coli isolates (18.2%).In conclusion, the results of this study demonstrated the importance of poultry meat as a source of Campylobacter spp., especially macrolide-resistant strains. The trend of decreasing Campylobacter spp. occurrence in retail poultry meat in this region of Poland requires further investigation, and monitoring.     

Cloth-based hybridization array system for the identification of Escherichia coli O157:H7

A simple cloth-based hybridization array system (CHAS) utilizing polyester cloth as the solid phase for DNA hybridizations has been adapted as a tool for the identification of presumptive verotoxigenic Escherichia coli O157:H7 colonies isolated from foods by standard culture techniques. Key indicator genes for this organism, rfbO157, fliCH7, vt1 and vt2 (encoding the O and flagellar antigenic determinants and verotoxin genes, respectively) were amplified in a multiplex PCR incorporating digoxigenin-dUTP, followed by hybridization of the amplicons with an array of specific oligonucleotide probes immobilized on polyester cloth, and subsequent immunoenzymatic assay of the bound digoxigenin label. This system includes a simple internal amplification control (IAC) to gauge PCR inhibition based on the incorporation of a primer pair with complementary 3′ ends, resulting in the generation of a unique “primer-dimer” detectable by hybridization with a specific capture probe immobilized on polyester cloth. The CHAS provided sensitive and specific detection of the E. coli O157:H7gene markers, exhibiting the expected patterns of reactivity with a panel of various target and non-target organisms.     

Development of a controlling method for Escherichia coli O157:H7 and Salmonella spp. in fresh market beef by using polylysine and modified atmosphere packaging

Escherichia coli O157:H7 and Salmonella spp. often contaminate fresh beef. In Japan, an E coli outbreak caused by raw beef made 181 people ill and 5 individuals dead in 2011. Responding to this outbreak, an effective sterilization method for fresh beef is expected to be developed. In this study, ε-polylysine combined with CO₂-packaging method was examined for controlling these pathogens in fresh beef. At an incubation temperature of 4 °C, approximately 4.3 log and 2.4 log reduction in bacterial numbers were observed after 7-day incubation for E. coli O157:H7 and Salmonella, respectively, in ε-polylysine-added beef. When effectiveness of CO₂-packaging combined with ε-polylysine was investigated, CO₂ did not have additional inhibiting effect on bacterial growth compared to only-ε-polylysine-treated samples when incubated at 4 °C. However, effectiveness of CO₂ was observed when incubated at 10 °C where approximately 2.9 log and 4.4 log reduction in E. coli cell numbers were observed in only-ε-polylysine-treated samples and polylysine- and CO₂-treated group, respectively, and approximately 1.7 log and 3.5 log reduction in Salmonella cell numbers were observed in only-ε-polylysine-treated samples and polylysine and CO₂-treated group, respectively. This study confirmed that ε-polylysine or ε-polylysine combined with CO₂ packaging are effective in preventing foodborne diseases caused by raw beef.     

Detection of viable but non-culturable Escherichia coli O157:H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays

Quantitative differentiation of the live fraction of pathogens in raw food samples is highly critical from a public health risk perspective, as many studies have shown that under stress conditions major foodborne pathogens enter a viable but non-culturable (VBNC) state in which bacteria can remain for long periods of time and maintain the potential for virulence. The objective of this study was to evaluate the applicability of propidium monoazide (PMA) quantitative PCR (qPCR) and immunological methods for detection of Escherichia coli O157:H7 VBNC populations induced by low temperature on the surface of lettuce and spinach plants. The primer/probe set selected influenced the qPCR signal in mixtures with a defined ratio of viable and non-viable cells. The PMA qPCR used in a background of added dead pathogens and epiphytic bacteria gave a detection limit of 10³ CFU/g leaf and a linear quantitative detection range of 5 log. During quantification of VBNC cells from lettuce and spinach samples there was a good correlation between the PMA qPCR results and viable counts detected by microscopy, showing that PMA qPCR gives an accurate indication of the VBNC population. However, the commercially available immunoassay methods used to detect Shiga-like toxin production and the O157 antibody in vegetable samples with no detectable culturable pathogen underestimated the number of samples contaminated with E. coli O157:H7 VBNC cells. Results indicate that PMA qPCR is a suitable technique for the detection and quantification of VBNC cells of foodborne pathogens in contaminated raw lettuce and spinach.     

Adherence of cold-adapted Escherichia coli O157:H7 to stainless steel and glass surfaces

The effects of previous cold-induced cell elongation on adherence of Escherichia coli O157:H7 to glass slides and stainless steel surfaces was evaluated at 4 °C for ≤48 h. Planktonic E. coli O157:H7 with and without cold adaptation were prepared at 15 and 37 °C, respectively, and planktonic E. coli O157:H7 containing elongated (>4 ≤ 10 μm) and filamentous (>10 μm) cells were prepared at 6 °C. Despite morphological differences in planktonic E. coli O157:H7 preparations, all three cell types attached to a greater extent to glass than to the stainless steel surfaces. E. coli O157:H7 cells adapted to growth at 15 °C attached better to both glass and stainless steel surfaces (3.2 and 2.6 log cfu/cm², respectively) than cells of the other treatments at ≥24 h. Cells adapted at 6 °C attached to glass slides and stainless steel coupons at levels of 3.0 and 1.8 log cfu/cm², respectively, while E. coli O157:H7 cells grown at 37 °C attached to these surfaces at levels of 2.0 and 1.7 log cfu/cm², respectively. No further attachment of cells from any of the treatments was noted between 24 and 48 h at 4 °C. These results suggest that E. coli O157:H7 cells adapted at 6 °C–15 °C have greater potential to attach to food contact surfaces than those grown at higher temperature. The enhanced biofilm-forming ability of 6 °C or 15 °C-adapted, elongated and filamentous E. coli O157:H7 cells did not appear to be related to the greater entanglement of longer cells within biofilm matrices.     

Antimicrobial activity of essential oils against Escherichia coli O157:H7 in organic soil

Soil can be a significant source of preharvest contamination of produce by pathogens. Demand for natural pesticides such as essential oils for organic farming continues to increase. We examined the antimicrobial activity of several essential oils against Escherichia coli O157:H7 in soil. Two essential oils (cinnamaldehyde and eugenol), two bio-pesticides (Ecotrol and Sporan) containing essential oils, and an organic acid (acetic acid) at 0.5%, 1.0%, 1.5% and 2.0%, were mixed with organic sandy soil and inoculated with five different strains of E. coli O157:H7. Soils were incubated at room temperature (22 °C) and samples obtained at 1, 7 and 28 days were enumerated to determine survival. E. coli O157:H7 populations in soil were reduced by up to 5 log cfu/g after 24 h incubation at room temperature with 2% cinnamanaldehyde, Ecotrol, Sporan or vinegar. Reduction in E. coli O157:H7 by eugenol was not significantly different from control. Overall, E. coli O157:H7 strain 4406 was the most sensitive of all the five strains tested and cinnamaldehyde was superior to other treatments in reducing E. coli O157:H7 in soil. In general, increases in essential oil concentrations corresponded to reduced survival of E. coli O157:H7 with all oils used in this study. The results suggest that oils can reduce potential contamination of fresh organic produce inadvertently contaminated by soil.     

Antimicrobial efficacy of grape seed extract against Escherichia coli O157:H7 growth,motility and Shiga toxin production

Escherichia coli O157:H7 produces Shiga toxin (Stx) which is heat stable and causes Hemolytic Uremic Syndrome (HUS), a serious disease associated with bloody diarrhea and even death. To ensure food safety, both live E. coli O157:H7 and its toxin production in food products need to be controlled. Natural ingredients with inhibitory effects on E. coli O157:H7 growth and toxin production are top choices of antimicrobials for the food industry. The objectives of this study were to evaluate efficacy of grape seed extract (GSE) against the growth, swimming motility and Stx production of E. coli O157:H7. The disc diffusion assay indicated that 3.2 mg GSE per disc resulted in an inhibition zone of 14.8 ± 0.21 mm. The minimal inhibitory concentration of GSE against E. coli O157: H7 was 4.0 mg/ml. At high inoculation level (1 × 10⁷ CFU/ml), including GSE at 0.25–2.0 mg/ml reduced Stx production without inhibiting E. coli O157:H7 growth. At 5 × 10⁵ CFU/ml inoculation level, 2.0 and 4.0 mg/ml GSE effectively inhibited the growth of E. coli O157:H7 for at least 72 h, however, a low level of GSE (0.125–1.0 mg/ml) enhanced E. coli O157:H7 growth and Stx2 production. At 4 mg/ml, GSE completely abolished Stx2 production in addition to it bactericidal effect against E. coli O157:H7. In addition, GSE at concentration as low as 0.125% blocked the swimming motility, which is important for E. coli O157:H7 surface adherence. In conclusion, GSE is effective in inhibiting the motility of E. coli O157:H7, GSE shows potential to be used as a natural antimicrobial to control E. coli O157:H7.     

Sublethal injury and recovery of Escherichia coli O157:H7 by high pressure carbon dioxide

A study was carried out to investigate possible sublethal injury caused by high pressure carbon dioxide (HPCD) to Escherichia coli O157:H7 suspended in phosphate-buffered saline (PBS, pH 7.00). The treatment pressure was 5 MPa, treatment temperatures were 25–37 °C and treatment time was 5–70 min. Only 1.21 log₁₀ cycles reduction was obtained in the nonselective medium, while a higher reduction of 5.18 log₁₀ cycles was obtained in the selective medium after treatment at 5 MPa and 25 °C for 50 min, indicating HPCD could induce sublethal injury of E. coli cells and these cells could not recover in the selective medium. The proportion of the sublethally injured E. coli cells in the survivors increased dramatically up to 72.4% with increasing the treatment time from 20 min to 30 min by HPCD at 5 MPa and 25 °C, and this proportion increased gradually with increasing the treatment time, up to 100% at 5 MPa and 25 °C for 60 min. The effect of recovery media, inhibitors of specific metabolic processes, Mg and Ca cations on the recovery of the sublethally injured cells was also investigated. When PBS, peptone water, tryptic soy broth (TSB) and carrot juice were used as recovery media, the maximum repair rate of the sublethally injured cells was found in TSB and the minimum was in PBS. The addition of inhibitors such as sodium azide, chloramphenicol, rifampicin and penicillin G showed that repair of most sublethally injured cells required energy, protein and RNA synthesis, but was not dependent on peptidoglycan synthesis. Moreover, Mg and Ca cations were also needed in the repair.     

Prevalence,enumeration, and characterization of Salmonella isolated from aquatic food products from retail markets in China

This study aimed to estimate the extent and level of Salmonella contamination of aquatic food products in China, and to determine serotype, virulotype, and the antimicrobial resistance profiles of recovered Salmonella isolates. Out of 554 samples collected from July 2011 to May 2014, 86 (15.5%) tested positive for Salmonella. The highest contamination rate occurred in oysters (23.1%, 6/26), followed by freshwater fish (18.6%, 43/231), shrimp (13.0%, 13/100), and saltwater fish (12.2%, 24/197). The contamination levels generally corresponded to a most probable number (MPN)/g of 0.3–10, although one sample exceeded 110 MPN/g. Among the 103 isolates, S. Typhimurium, S. Wandsworth, S. Thompson, and S. Derby were the most prevalent serovars. Sixty-eight isolates (66.0%) were resistant to at least one antimicrobial, and 35 (34.0%) were resistant to more than three. High rates of resistance were observed for tetracycline (35.9%), ampicillin (28.2%), nalidixic acid (26.2%), trimethoprim-sulfamethoxazole (25.2%), chloramphenicol (20.4%) and streptomycin (18.4%). Of note, S. Thompson isolates exhibited resistance to multiple extended-spectrum cephalosporins, ciprofloxacin, and other antimicrobials. PCR analysis of 15 virulence genes showed that ssaQ, mgtC, siiD, sopB, and bcfC were present in all 103 isolates, whereas the remaining loci were variably distributed. S. Typhimurium, S. Enteritidis, and S. Weltevreden isolates exhibited a wider range of pathogenicity determinants compared with the other strains. Our study provides a comprehensive surveillance on prevalence of Salmonella in aquatic food products from China and indicates its potential risk to public health. These data are valuable for epidemiological studies, risk management, and public health strategies.     

Antimicrobial activity of mustard essential oil against Escherichia coli O157:H7 and Salmonella typhi

The aim of this study was to investigate how mustard essential oil (EO) affected the cell membrane of Escherichia coli O157:H7 and Salmonella typhi. Intracellular pH and ATP concentration and the release of cell constituents were measured when mustard EO was in contact with E. coli and S. typhi at its minimal inhibitory concentration (MIC) and maximal tolerated concentration (MTC). The treatment with mustard EO affected the membrane integrity of bacteria and induced a decrease of the intracellular ATP concentration. Also, the extracellular ATP concentration increased and a reduction of the intracellular pH was observed in both bacteria. A significantly (P ? 0.05) higher release of cell constituent was observed when both bacteria cells were treated with mustard EO. Electronic microscopy observations showed that the cell membranes of both bacteria were apparently damaged by mustard EO. In conclusion, mustard EO affects the concentration of intracellular component, such as ATP in both bacteria and affects the pH suggesting that cytoplasmic membrane is involved in the antimicrobial action of mustard EO. Mustard EO can be used as an effective antimicrobial agent. We have demonstrated that mustard EO affected the cell membrane integrity, resulting in a loss of cell homeostasis.     

Incidence of Listeria monocytogenes and Escherichia coli O157:H7 in two Kasar Cheese processing environments

This study was designed to determine the presences of two environmental pathogens in two dairy factories in Sakarya, Turkey. A total of 264 environmental samples, raw milk and cheese samples were taken at four different seasons. According to the results, Listeria monocytogenes or Escherichia coli O157:H7 was isolated from 26 or 2.7% of the samples collected from both factories, respectively. None of the cheese or curd samples were found to be positive for Listeria or E. coli O157:H7. However, 50% of raw milk samples contained Listeria innocua. Listeria was mostly isolated from the swap samples taken from the drains or the floors in processing or packaging areas. However, E. coli was also isolated from the swap samples taken from the workers’ hands and gloves as well as the drains and the floor. Only one raw milk sample contained E. coli O157:H7. A higher prevalence of both pathogens was observed in the summer months than in the other months.     

Detection of Escherichia coli O157:H7 in ground beef using immunomagnetic separation and multiplex PCR

In the study 251 fresh ground beef samples sold in Ankara were analyzed to evaluate the prevalence of Escherichia coli O157:H7 by immunomagnetic separation (IMS) based cultivation technique. Virulence factors of the isolates were determined by multiplex PCR. Nineteen (7.6%) of 251 ground beef samples were found as contaminated with E. coli O157. By PCR analyse in two of the isolates fliC_h7 gene was detected and identified as E. coli O157:H7. According to the multiplex PCR one of the isolate has all stx₁, stx₂, eaeA, hly and fliC_h7 genes and the other has stx₁, eaeA, hly and fliC_h7 genes.     

Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7

Immunomagnetic separation uses antibody-coated paramagnetic particles to selectively bind and purify the target organisms from a comprehensive range of complex food matrices. Aim of this study was to develop and optimize an immunomagnetic separation method based on monoclonal antibody for efficient isolation of Escherichia coli O157:H7 in food samples. The key parameters for preparing the immunomagnetic beads; the coupling rate between monoclonal antibody and magnetic beads, additive amount of immunomagnetic beads, and magnetic separation times were optimized with different concentrations of E. coli O157:H7. Under optimized conditions, the capture efficiency (CE) was greater than 98% against 10¹–10⁶ cells of E. coli O157:H7 with 0.05 mg immunomagnetic beads. The immunomagnetic beads exhibited high specific binding with E. coli O157:H7 strains (CE > 98%), and low binding with non-target bacteria (CE < 2%) except for S. aureus (CE = 23.6%). The capture efficiency of immunomagnetic beads against E. coli O157:H7 in ground beef and milk samples were 94.4% and 99.8%, respectively.