Total coliforms, microbial diversity and multiple characteristics of Salmonella in soil-irrigation water-fresh vegetable system in Shaanxi, China.

Global occurrences of foodborne disease outbreaks have been documented, involving fresh agricultural produce contaminated by various pathogens. This contamination can occur at any point in the supply chain. However, studies on the prevalence of total coliforms, Salmonella and microbial diversity in vegetable and associated environments are limited. This study aimed to assess 1) the number of total coliforms (n = 299) and diversity of microbial communities (n = 52); 2) the prevalence, antibiotic susceptibility, genomic characteristics, and potential transmission relationships of Salmonella in soil-irrigation water-vegetable system (n = 506). Overall, 84.28 % samples were positive to total coliforms, with most frequently detected in soil (100 %), followed by irrigation water (79.26 %) and vegetables (62.00 %). A seasonal trend in coliform prevalence was observed, with significantly higher levels in summer (P < 0.05). Detection rates of Salmonella in soil, vegetable and irrigation water were 2.21 %, 4.74 % and 9.40 %. Fourteen serotypes and sequence types (STs) were respectively annotated in 56 Salmonella isolates, ST13 S. Agona (30.36 %, 17/56), ST469 S. Rissen (25.00 %, 14/56), and ST36 S. Typhimurium (12.50 %, 7/56) were dominant serotypes and STs. Thirty-one (55.36 %) isolates were multi-drug resistant, and the resistance was most frequently found to ampicillin (55.36 %, 31/56), followed by to sulfamethoxazole (51.79 %, 29/56) and tetracycline (50.00 %, 28/56). The genomic characteristics and antibiotic resistance patterns of Salmonella isolates from soil, vegetables, and irrigation water within a coherent geographical locale exhibited remarkable similarities, indicating Salmonella may be transmitted among these environments or have a common source of contamination. Microbial alpha diversity indices in soil were significantly higher (P < 0.05) than that in vegetable and irrigation water. The microbial phylum in irrigation water covered that in the vegetable, demonstrating a significant overlap in the microbial communities between the vegetables and the irrigation water.

Prevalence, antibiotic susceptibility and genomic analysis of Salmonella from retail meats in Shaanxi, China.

Salmonella is a major foodborne pathogen that poses a substantial risk to food safety and public health. This study aimed to assess the prevalence, antibiotic susceptibility, and genomic features of Salmonella isolates recovered from 600 retail meat samples (300 pork, 150 chicken and 150 beef) from August 2018 to October 2019 in Shaanxi, China. Overall, 40 (6.67 %) of 600 samples were positive to Salmonella, with the highest prevalence in chicken (21.33 %, 32/150), followed in pork (2.67 %, 8/300), while no Salmonella was detected in beef. A total of 10 serotypes and 11 sequence types (STs) were detected in 40 Salmonella isolates, with the most common being ST198 S. Kentucky (n = 15), ST13 S. Agona (n = 6), and ST17 S. Indiana (n = 5). Resistance was most commonly found to tetracycline (82.50 %), followed by to ampicillin (77.50 %), nalidixic acid (70.00 %), kanamycin (57.50 %), ceftriaxone (55.00 %), cefotaxime (52.50 %), cefoperazone (52.50 %), chloramphenicol (50.00 %), levofloxacin (57.50 %), cefotaxime (52.50 %), kanamycin (52.50 %), chloramphenicol (50.00 %), ciprofloxacin (50.00 %), and levofloxacin (50.00 %). All ST198 S. Kentucky isolates showed multi-drug resistance (MDR; ≥3 antimicrobial categories) pattern. Genomic analysis showed 56 distinct antibiotic resistance genes (ARGs) and 6 target gene mutations of quinolone resistance determining regions (QRDRs) in 40 Salmonella isolates, among which, the most prevalent ARG types were related to aminoglycosides and ß-lactams resistance, and the most frequent mutation in QRDRs was GyrA (S83F) (47.5 %). The number of ARGs in Salmonella isolates showed a significant positive correlation with the numbers of insert sequences (ISs) and plasmid replicons. Taken together, our findings indicated retail chickens were seriously contaminated, while pork and beef are rarely contaminated by Salmonella. Antibiotic resistance determinants and genetic relationships of the isolates provide crucial data for food safety and public health safeguarding.

Conjugative transfer of mcr-1-bearing plasmid from Salmonella to Escherichia coli in vitro on chicken meat and in mouse gut.

Since mcr-1 was first discovered in 2015, this gene has shown excellent transmission ability and evolutionary characteristics worldwide, leading to major public health and food safety concerns. In this study, chicken meat was used as a food vehicle for the conjugation of mcr-1-bearing Salmonella at different storage temperatures (4 °C, 25 °C, and 37 °C) to simulate mcr-1 transmission during food transportation and storage and determine its efficiency and mechanism. In addition, conjugation experiments were performed in mouse gut to further confirm that mcr-1 is horizontally transferred in vivo during food consumption. 16S rDNA sequencing of mouse stool samples was performed to understand the effect of horizontal transfer of mcr-1 on mouse gut bacteria. mcr-1-bearing plasmids were characterized using pulsed-field gel electrophoresis (PFGE) and S1 nuclease-PFGE and sequenced by Illumina sequencing. Our results showed that mcr-1-bearing plasmids in donors are successfully transferred to recipients on chicken meat at not only 25 °C and 37 °C but also 4 °C with conjugation frequencies between 1.32 × 10-6 and 3.85 × 10-4 per recipient cell. In mouse gut, mcr-1 was transferred not only to the recipient bacteria introduced by intragastric administration but also to the intestinal bacteria (E. coli strain named as E6353). Horizontal transfer of mcr-1-bearing plasmid in mouse gut negatively affected the mouse intestinal microbiota. In a constant conjugative environment, plasmid replicon type is the most decisive factor affecting the conjugation frequency. The peak number of transconjugants in group D6-E. coli C600 with an IncHI2-type mcr-1-bearing plasmid (1.43 × 102 colony-forming units [CFU]/g feces) was significantly higher than that of transconjugants in group D7-E. coli C600 with an IncX4-type mcr-1-bearing plasmid (0.3 × 102 CFU/g feces). The upstream and downstream genetic environment of mcr-1 in different plasmid replicon types in Salmonella varied during conjugation in different horizontal transfer environments. An IncI2 plasmid (p25-D4R7S1_mcr-1) lost the insertion sequence ISApl1, which originally existed upstream of mcr-1, when this plasmid transferred from donor to recipient cells on chicken meat at 25 °C. An IncHI2 plasmid was more active than IncI2 and IncX4 plasmids during bacterial reproduction and evolution; an IncFIB-IncHI2 hybrid plasmid (p6176253_mcr-1) was formed in mouse gut during conjugation from pD6_tet(M) and pD6_mcr-1. mcr-1 is captured by mobile genetic elements IS26 in IncX4 plasmids and ISApl1 in IncI2, IncHI2, and IncFIB-IncHI2 hybrid plasmids and is disseminated among bacteria.

Cunning plasmid fusion mediates antibiotic resistance genes represented by ESBLs encoding genes transfer in foodborne Salmonella.

Foodborne disease caused by antibiotic resistant Salmonella is quite difficult to deal with. In order to further explore the antibiotic resistance associated with gene transfer among foodborne Salmonella, several wild-type Salmonella strains were used as donors and recipients, respectively, to investigate how extended spectrum ß-lactamases (ESBLs) encoding genes co-transfer with transposable elements to transmit antibiotic resistance. Antibiotic susceptibility was determined by agar dilution method, the transposase encoding gene was detected via PCR combined with DNA sequencing, S1 nuclease and pulsed field gel electrophoresis (S1-PFGE), and southern-blot. Illumina HiSeq 4000 platform and Nanopore MinION long-read sequencing technology were used to determine the antibiotic resistance encoding genes (ARGs) and their surrounding gene environment. The results indicated that the conjugation frequency was from ×10-4 to ×10-5 per recipient cell. A 185,608-bp-long DNA fragment and two short backbone protein encoding regions in pG19 in the donor fused with part genes in pS3 in the recipient during conjugation, the size of this fusion plasmid is as same as that of pG19. Cefoxitin resistance of the transconjugant was mediated by a tnpA21-related blaDHA-1 transfer. Resistance of Salmonella to ceftriaxone, cefoperazone and ceftiofur was mediated by a tnpU1548 related blaTEM-1B and blaCTX-M-3 transfer. The study indicated that transposase synergy and plasmid selective fusion act as important roles for foodborne Salmonella gathering ARGs. The consistent size of the plasmid before and after fusion suggested the invisibility and complexity of bacterial conjugation without DNA sequencing, the fact reminded us that the rampant transmission of antibiotic-resistance encoding genes would pose tremendous threat to food safety.

Epidemiology and Characterization of CTX-M-55-Type Extended-Spectrum ß-Lactamase-Producing Salmonella enterica Serovar Enteritidis Isolated from Patients in Shanghai, China.

The emergence of extended-spectrum ß-lactamase-producing Salmonella enterica serovar Enteritidis (ESBL-SE) in humans and foods has gained global attention. In particular, CTX-M-type ESBL-SE are increasingly being detected from various sample types. The aim of this study was to comprehensively analyze the epidemiology and characteristics of blaCTX-M-55-carrying ESBL-SE isolates of clinical origin in Shanghai, China. A total of 292 S. Enteritidis isolates were recovered from the feces and blood of outpatients and inpatients between 2006 and 2014. Overall, there was a high frequency of cefotaxime-resistant isolates (97.3%), which was significantly higher (p < 0.01) than that of isolates resistant to the other tested antibiotics. All S. Enteritidis isolates exhibited resistance to ≥1 antibiotic, and 98.0% were multidrug resistant. A total of 233 isolates were identified as ESBL-SE, 166 of which were CTX-M type. Six subtypes of CTX-M-encoding genes were detected, among which blaCTX-M-55 (91.6%, 152/166) was the most prevalent genotype. There was high genetic similarity among blaCTX-M-55-positive ESBL-SE. The blaCTX-M-55 gene in the ESBL-SE donor strains could be easily transferred into Enterobacteriaceae recipient strains. This study highlights that CTX-M-55 should be considered an important surveillance target in Shanghai, China. Cephalosporins, especially cefotaxime, must be used with caution in empirical treatment for Salmonella infections.

Prevalence, Serotype, Antibiotic Susceptibility, and Genotype of Salmonella in Eggs From Poultry Farms and Marketplaces in Yangling, Shaanxi Province, China.

Poultry products such as eggs provide essential nutrients to the human body and thus play vital roles in the human food network. Salmonella is one of the most notorious foodborne pathogens and has been found to be prevalent in eggs. To better understand the characteristics of Salmonella in eggs, we investigated the prevalence of Salmonella spp. in 814 fresh eggs collected from poultry farms and retail marketplaces in Yangling, Shaanxi Province, China. The serotype, genotype, and antibiotic susceptibilities of 61 Salmonella isolates recovered from the eggs were analyzed. The average detection rate of Salmonella-positive eggs was 5.6%, with 6.6% of the eggs collected from poultry farms and 5.1% from marketplaces. Thirteen serotypes were identified from the 61 isolates, among which Salmonella Typhimurium (24.5%) and Salmonella Indiana (22.9%) were the most prevalent serotypes. Other dominant serotypes included Salmonella Thompson (13.1%) and Salmonella Enteritidis (11.4%), with the remaining nine serotypes detected at low rates (1.6-4.9%). All the Salmonella isolates tested were resistant to sulfisoxazole (100.0%). The majority (77.1%) of the isolates were resistant to nalidixic acid, amoxicillin-clavulanate, and ampicillin, while nearly two-thirds (63.9-68.9%) were resistant to trimethoprim-sulfamethoxazole, kanamycin, tetracyclines, and chloramphenicol. The rate of resistance to ciprofloxacin was 40.1%; the resistance rates to streptomycin, ceftiofur, and ceftriaxone ranged from 21.3 to 26.2%; and those to gentamicin, amikacin, and cefoxitin were relatively low (3.3-16.4%). Forty-nine (80.3%) Salmonella isolates exhibited resistance to multiple antibiotics, 20 (32.8%) of which were resistant to at least 10 antibiotics. Subtyping by pulse-field gel electrophoresis revealed a close genetic relatedness of Salmonella isolates from poultry farms, in striking contrast to the high diversity of the isolates obtained from marketplaces. Isolates of the same serotype always shared identical genotype and antibiotic resistance profiles, even the ones that were recovered from eggs sampled at different locations and times. These findings indicate that diverse Salmonella spp. with high rates of multidrug resistance are prevalent in fresh eggs in the study area. More attention should be paid to egg production, transportation, and storage to prevent foodborne outbreaks caused by Salmonella.

Genes and Proteomes Associated With Increased Mutation Frequency and Multidrug Resistance of Naturally Occurring Mismatch Repair-Deficient Salmonella Hypermutators.

The emergence of antibiotic-resistant Salmonella through mutations led to mismatch repair (MMR) deficiency that represents a potential hazard to public health. Here, four representative MMR-deficient Salmonella hypermutator strains and Salmonella Typhimurium LT2 were used to comprehensively reveal the influence of MMR deficiency on antibiotic resistance among Salmonella. Our results indicated that the mutation frequency ranged from 3.39 × 10-4 to 5.46 × 10-2 in the hypermutator. Mutation sites in MutS, MutL, MutT, and UvrD of the four hypermutators were all located in the essential and core functional regions. Mutation frequency of the hypermutator was most highly correlated with the extent of mutation in MutS. Mutations in MMR genes (mutS, mutT, mutL, and uvrD) were correlated with increased mutation in antibiotic resistance genes, and the extent of antibiotic resistance was significantly correlated with the number of mutation sites in MutL and in ParC. The number of mutation sites in MMR genes and antibiotic resistance genes exhibited a significant positive correlation with the number of antibiotics resisted and with expression levels of mutS, mutT, and mutL. Compared to Salmonella Typhimurium LT2, a total of 137 differentially expressed and 110 specifically expressed proteins were identified in the four hypermutators. Functional enrichment analysis indicated that the proteins significantly overexpressed in the hypermutators primarily associated with translation and stress response. Interaction network analysis revealed that the ribosome pathway might be a critical factor for high mutation frequency and multidrug resistance in MMR-deficient Salmonella hypermutators. These results help elucidate the mutational dynamics that lead to hypermutation, antibiotic resistance, and activation of stress response pathways in Salmonella.

Diversity of Serotype, Genotype, and Antibiotic Susceptibility of Salmonella Prevalent in Pickled Ready-to-Eat Meat.

Pickled ready-to-eat meat (PRTEM) is a meat product that is treated with various seasonings and then cooked. PRTEM is a popular food consumed mostly in China and some Asian countries. Since this food is considered 'ready to eat', once it is contaminated by foodborne pathogens such as Salmonella, the prospect for significant morbidity, mortality, and immeasurable economic losses can occur. Here we investigated the prevalence and concentration of Salmonella in 107 PRTEM samples collected from Shaanxi, China during 2015-2016. Furthermore, we analyzed the serotype, antibiotic susceptibility, and presence of antibiotic resistance genes and amino acid mutations in 219 Salmonella isolates, followed by subtyping of 115 representative isolates. The average detection rate of Salmonella-positive PRTEM was 58.9%, and the average most probable number (MPN) of Salmonella in positive samples was 2.27 logMPN per gram of sample (range: 2.10-2.43). Ten serotypes were identified from the 219 Salmonella isolates, with S. Thompson (37.9%) and S. Indiana (20.5%) being predominant. The remaining serotypes were S. Typhi (7.8%), S. Typhimurium (7.3%), S. Mbandaka (6.9%), S. Albany (6.4%), S. Blockley (5.5%), S. Infantis (4.1%), S. Escanaba (3.2%), and S. Dusseldorf (0.5%). All isolates were resistant to ceftiofur (100%), while most of them were resistant to ciprofloxacin (99.1%), amoxicillin-clavulanic acid (97.7%), trimethoprim-sulfamethoxazole (96.4%), ampicillin (92.3%), sulfisoxazole (92.2%), tetracyclines (90.4%), and nalidixic acid (90.4%), respectively. A single mutation of Ser83Phe (27.1%) and double mutations of Ser83Phe-Asp87Gly (25.9%) in GyrA were detected in 85 isolates, whereas mutations of Thr57Ser (63.9%) and Ser80Arg (36.1%) in ParC were detected in 122 isolates. qnrB, oqxAB, aac(6')-Ib, and qnrA were present in 50 (22.8%), 48 (21.9%), 26 (11.9%), and 1 (0.5%) isolate(s), respectively. Pulse field gel electrophoresis results revealed that those isolates recovered from the same type of PRTEM or the same sampling place shared identical or similar DNA profiles, antibiotic resistance phenotypes, and even plasmid-mediated quinolone resistance encoding genes. The findings indicate that Salmonella is commonly prevalent in PRTEMs at high concentrations in Shaanxi, China. More attention should be paid to the processing and storage of this ready-to-eat food to prevent bacterial contamination and foodborne outbreaks.

Influence of initial protein structures and xanthan gum on the oxidative stability of O/W emulsions stabilized by whey protein.

In this article, oil-in-water (O/W) emulsions stabilized by natural whey protein concentrate (WPC) and microparticulated whey protein (MWP) and their mixtures with xanthan gum (XG) were prepared to investigate the lipid oxidative stability of O/W emulsions with the same interfacial composition but different interfacial structures. High-performance size exclusion chromatography, Fourier transform infrared spectrometry, X-ray diffraction analysis and steady-state fluorescence spectroscopy were used to reveal the differences in the structures of natural whey protein and the microparticulated whey proteins (MWP, pH 3.5-8.5). Dispersions of the proteins (70% w/w) and XG (30% w/w) were mixed to prepare the mixtures (protein-XG). Emulsions of 60% peanut oil that were stabilized by the proteins and the protein-XG mixtures were subjected to oxidation. In addition, the peroxide values (PVs) were measured to evaluate the oxidative stability of each emulsion. The MWP(pH 4.5)-XG and MWP(pH 6.5)-XG-stabilized emulsions showed high oxidative stabilities that were not significantly different from each other. The results indicated that a single complex layer formed by the spherical protein microparticles and XG can better inhibit the lipid oxidation of O/W emulsions than a double layer. This study has significant implications for the development of novel structures containing lipid phases that are susceptible to oxidation.

Effect of microparticulation and xanthan gum on the stability and lipid digestion of oil-in-water emulsions stabilized by whey protein.

Since lipid digestion is an interfacial process, food emulsions are increasingly being seen as a mechanism for controlling lipid uptake. Oil-in-water emulsions stabilized by whey protein (WP) and protein-xanthan gum (XG) mixtures were designed to investigate the influence of interfacial structures on lipid digestion using an in vitro digestion model. The interfacial layers with different structures were designed using microparticulated whey protein (MWP) and MWP-XG mixtures. The increase in the volume average diameter of proteins indicated that the WPs aggregated to form micro-particles during microparticulation. The increase in the protein surface hydrophobicity index and the measurement results from the Magnetic Resonance Imaging System indicated that the protein hydrophilic groups were embedded and that the protein hydrophobic groups were exposed. Under in vitro conditions, the emulsions stabilized by microparticulated whey proteins and protein-XG mixtures were more stable than the WP emulsions, and the microparticulated whey proteins and protein-XG mixtures were more effective for decreasing the digestion rate, as shown by the stability analysis and free fatty acid release rates. These results help elucidate the influence of the interfacial structure on lipid digestion. The control of lipid digestibility within the gastrointestinal tract might be important for the design and development of reduced-fat foods and novel functional foods for controlling bioactive release.