Cold-tolerance mechanisms in foodborne pathogens: Escherichia coli and Listeria monocytogenes as examples.

The cold chain is an integral part of the modern food industry. Low temperatures can effectively alleviate food loss and the transmission of foodborne diseases caused by microbial reproduction. However, recent reports have highlighted shortcomings in the current cold chain technology's ability to prevent and control cold-tolerant foodborne pathogens. Furthermore, it has been observed that certain cold-chain foods have emerged as new sources of infection for foodborne disease outbreaks. Consequently, there is a pressing need to enhance control measures targeting cold-tolerant pathogens within the existing cold chain system. This paper aims to review the recent advancements in understanding the cold tolerance mechanisms of key model organisms, identify key issues in current research, and explore the potential of utilizing big data and omics technology in future studies.

Dual-recognition colorimetric platform based on porous Au@Pt nanozymes for highly sensitive washing-free detection of Staphylococcus aureus.

A dual-recognition strategy is reported to construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and are robust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus. In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.

Semiquantitative Fingerprinting Based on Pseudotargeted Metabolomics and Deep Learning for the Identification of Listeria monocytogenes and Its Major Serotypes.

The rapid identification of pathogenic microorganism serotypes is still a bottleneck problem to be solved urgently. Compared with proteomics technology, metabolomics technology is directly related to phenotypes and has higher specificity in identifying pathogenic microorganism serotypes. Our study combines pseudotargeted metabolomics with deep learning techniques to obtain a new deep semiquantitative fingerprinting method for Listeria monocytogenes identification at the serotype levels. We prescreened 396 features with orthogonal partial least-squares discrimination analysis (OPLS-DA), and 200 features were selected for deep learning model building. A residual learning framework for L. monocytogenes identification was established. There were 256 convolutional filters in the initial convolution layer, and each hidden layer contained 128 filters. The total depth included seven layers, consisting of an initial convolution layer, a residual layer, and two final fully connected classification layers, with each residual layer containing four convolutional layers. In addition, transfer learning was used to predict new isolates that did not participate in model training to verify the method's feasibility. Finally, we achieved prediction accuracies of L. monocytogenes at the serotype level exceeding 99%. The prediction accuracy of the new strain validation set was greater than 97%, further demonstrating the feasibility of this method. Therefore, this technology will be a powerful tool for the rapid and accurate identification of pathogens.

Prevalence of antimicrobial resistance in a full-scale drinking water treatment plant.

Antibiotic resistance in drinking water has received increasing attention in recent years. In this study, the occurrence and abundance of antibiotic resistance genes (ARGs) in a drinking water treatment plant (DWTP) was comprehensively investigated using metagenomics. Bioinformatics analysis showed that 381 ARG subtypes belonging to 15 ARG types were detected, and bacitracin had the highest abundance (from 0.26 × 10-2 to 0.86 copies/cell), followed by multidrug (from 0.57 × 10-1 to 0.47 copies/cell) and sulfonamide (from 0.83 × 10-2 to 0.35 copies/cell). Additionally, 933 ARG-carrying contigs (ACCs) were obtained from the metagenomic data, among which 153 contigs were annotated as pathogens. The most abundant putative ARG host was Staphylococcus (7.9%), which most frequently carried multidrug ARGs (43.2%). Additionally, 38 high-quality metagenome-assembled genomes (MAGs) were recovered, one of which was identified as Staphylococcus aureus (Bin.624) and harboured the largest number of ARGs (n = 16). Using the cultivation technique, 60 isolates were obtained from DWTP samples, and Staphylococcus spp. (n = 11) were found to be dominant in all isolates, followed by Bacillus spp. (n = 17). Antimicrobial susceptibility testing showed that most Staphylococcus spp. were multidrug resistant (MDR). These results deepen our understanding of the distribution profiles of ARGs and antibiotic resistant bacteria (ARB) in DWTPs for potential health risk evaluation. Our study also highlights the need for new and efficient water purification technologies that can be introduced and applied in DWTPs.

An Investigation on the Occurrence and Molecular Characterization of Bacillus cereus in Meat and Meat Products in China.

Bacillus cereus is a common foodborne pathogen that can cause both gastrointestinal and nongastrointestinal diseases. In this study, we collected 603 meat and meat products from 39 major cities in China. The positive contamination rate of B. cereus in the collected samples was 26.37% (159/603), and the contamination level in 5.03% (8/159) positive samples exceeded 1100 most probable number/g. The detection rates of virulence genes were 89.7% for the nheABC gene group, 37.1% for the hblACD gene cluster, 82.3% for cytK-2, and 2.9% for cesB. Notably, all isolates presented with multiple antibiotic resistance, and 99.43% of isolates were resistant to five classes of antibiotics. In addition, the multilocus sequence typing results indicated that all isolates were rich in genetic diversity. Collectively, we conducted a systematic investigation on the prevalence and characterization of B. cereus in meat and meat products in China, providing crucial information for assessing the risk of B. cereus occurrence in meat and meat products.

Amplified electrochemical antibiotic aptasensing based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework.

A facile and versatile competitive electrochemical aptasensor for tobramycin (TOB) detection is described using electrochemical-deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework (AuNPs/P-MOF) as signal-amplification platform and a DNA probe labeled with methylene blue (MB) at the 3'-end (MB-Probe) as a signal producer. First, F-Probe (short complementary DNA strands of both the aptamer and the MB-Probe label with a sulfhydryl group at the 5'-end) was immobilized on the AuNPs/P-MOF modified electrode as detection probes, which competed with TOB in binding to the aptamer. TOB-aptamer binding resulted in F-Probe remaining unhybridized on the electrode surface, so that a significant current response was generated by hybridizing with MB-Probe instead. The developed strategy showed favorable repeatability, with a relative standard deviation (RSD) of 4.3% computed over five independent assays, and high stability, with only 6.8% degradation after 15 days of storage. Under optimal conditions, the proposed aptamer strategy exhibited a linear detection range from 100 pM to 500 nM with a limit of detection (LOD) of 56 pM (S/N = 3). The electrochemical aptasensor demonstrated remarkable selectivity, and its feasibility for accurate and quantitative detection of TOB in milk samples was confirmed (RSD < 4.5%). Due to its simple design, easy operation, and high sensitivity and selectivity, the proposed method could expect to detect other antibiotics by replacing the aptamers. In summary, this study provides a simple and effective new strategy for electrochemical aptasening based on MOF-based sensing interface. Scheme illustration of label-free competitive electrochemical aptamer-based detection of tobramycin based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework as signal-amplification platform.

Assessment and molecular characterization of Bacillus cereus isolated from edible fungi in China.

BACKGROUND: Bacillus cereus is a foodborne pathogen commonly found in nature and food and can cause food spoilage and health issues. Although the prevalence of B. cereus in foods has been reported worldwide, the extent of contamination in edible fungi, which has become increasingly popular as traditional or functional food, is largely unknown. Here we investigated the prevalence, toxin genes' distribution, antibiotic resistance, and genetic diversity of B. cereus isolated from edible fungi in China. RESULTS: Six hundred and ninety-nine edible fungi samples were collected across China, with 198 (28.3%) samples found to be contaminated by B. cereus, with an average contamination level of 55.4 most probable number (MPN)/g. Two hundred and forty-seven B. cereus strains were isolated from the contaminated samples. Seven enterotoxin genes and one cereulide synthetase gene were detected. The detection frequencies of all enterotoxin genes were ≥ 80%, whereas the positive rate of the cesB gene in B. cereus was 3%. Most isolates were resistant to penicillins, ß-lactam/ß-lactamase inhibitor combinations, cephems, and ansamycins, but were susceptible to penems, aminoglycosides, macrolides, ketolide, glycopeptides, quinolones, phenylpropanol, tetracyclines, lincosamides, streptogramins, and nitrofurans. Meanwhile, 99.6% of all isolates displayed multiple antimicrobial resistance to three or more classes of antimicrobials. Using genetic diversity analysis, all isolates were defined in 171 sequence types (STs), of which 83 isolates were assigned to 78 new STs. CONCLUSIONS: This study provides large-scale insight into the prevalence and potential risk of B. cereus in edible fungi in China. Approximately one-third of the samples were contaminated with B. cereus, and almost all isolates showed multiple antimicrobial resistance. Detection frequencies of all seven enterotoxin genes were equal to or more than 80%. These new findings may indicate a need for proper pre-/post-processing of edible fungi to eliminate B. cereus, thereby preventing the potential risk to public health.

Staphylococcus argenteus isolated from retail foods in China: Incidence, antibiotic resistance, biofilm formation and toxin gene profile.

Staphylococcus argenteus is a novel species of coagulase-positive staphylococci which was separated from Staphylococcus aureus in 2014. It can threaten human health like S. aureus but can not identify with conventional biochemical or other phenotypic testing. From 2011 to 2016, 1581 S. aureus strains were isolated from 4300 samples from retail foods covering most provincial capitals in China. According to multilocus sequence typing (MLST) and PCR confirmation, 7.2% of isolates (114/1581) were confirmed as S. argenteus. The pathogen was distributed in 22 of 39 sampled cities and all food types. Interestingly, most S. argenteus positive samples were collected from coastal cities in South China. MLST detected 8 different sequence types (STs), including five new STs. CC2250 was the predominant lineage of S. argenteus, followed by CC1223. To further characterize the isolates, their antibiotic resistance, virulence genes, biofilm formation and biofilm-related genes were examined. The pvl gene was not detected in S. argenteus, and only 1 isolate (0.9%) was positive for the tsst-1 gene. For 18 enterotoxin genes, 16.7% (19/114) of isolates harboured more than three genes, whereas 70.2% (80/114) of isolates had none of the investigated genes. Penicillin and ampicillin were the major antibiotics to which the S. argenteus isolates were resistant, followed by tetracycline, kanamycin and fusidic acid. A total of 94.7% of isolates had the ability to produce biofilms and all isolates harboured icaA, fnbA, and fib genes. Other biofilm-related genes, such as eno, clfB, fnbB, and icaC, were also found in 99.1%, 92.1%, 88.6%, and 74.6% of isolates, respectively. This study is the first systematic investigation of the prevalence of S. argenteus in retail foods in China and shows their ubiquity in food. We also provide comprehensive surveillance of the incidence of S. argenteus in retail foods and information to enable more accurate and effective treatment of infections of this new species.

Genetic characteristics and virulence of Listeria monocytogenes isolated from fresh vegetables in China.

BACKGROUND: Ready-to-eat (RTE) vegetables have become increasingly popular along with the trend of moving towards a healthy lifestyle. However, RTE vegetables are at a higher risk of containing pathogens, maybe owing to lack of rigorous sanitization procedures. To understand the prevalence and potential risk of Listeria monocytogenes in RTE vegetables, we investigated the contamination level and characteristics of L. monocytogenes isolated from fresh vegetables. RESULTS: Twenty-three (5.49%) of the 419 vegetables samples were positive for L. monocytogenes. Phylogenetic group I.1 (1/2a-3a) and II.2 (1/2b-3b-7) strains were predominant in 30 isolates, which accounted for 33.3 and 50.0%, respectively. Multilocus sequence typing of the 30 isolates grouped them into nine sequence types (STs). The most common STs were ST87 (36.7%) and ST8 (26.7%). Virulence analysis showed that all 30 isolates harbored eight classical virulence genes, 10.0% isolates harbored the llsX gene (ST3 and ST1 strains), and 36.7% carried the ptsA gene and belonged to ST87. Approximately 83.3% isolates carried full-length inlA, whereas five isolates had premature stop codons in inlA, three of which belonged to ST9 and two to ST8. Antibiotic susceptibility showed the isolates were varyingly resistant to 13 antibiotics, 26.7% of the isolates were multi-drug resistant. CONCLUSIONS: The fresh vegetables contain some potential hypervirulent L. monocytogenes (ST1 and ST87) in the Chinese markets. In addition, the high rate of L. monocytogenes isolates was multi-drug resistant. Fresh raw vegetables may be a possible transmission route for L. monocytogenes infection in consumers. Therefore, sanitization of raw fresh vegetables should be strengthened to ensure their microbiological safety when used as RTE vegetables.

High-throughput sequencing analysis of bacterial community composition and quality characteristics in refrigerated pork during storage.

Spoilage bacteria seriously influence meat quality. In this study, the bacterial community, sensory scores, pH, and total volatile basic nitrogen (TVB-N) in refrigerated (4 °C) pork, the most commonly consumed meat in China, were investigated. In a high-throughput sequencing analysis of the V3-V4 region of the 16S rDNA gene, 259 bacterial genera were belonging to 21 phyla were identified. With the passage of time, the bacterial community diversity decreased. After 5 days, Pseudomonas, Acinetobacter and Photobacterium were dominant in refrigerated pork, especially Photobacterium, which rarely associated with meat spoilage. Our results suggest that these taxa contribute to refrigerated pork spoilage. During storage, pH and TVB-N showed similar trends. Additionally, total viable counts (TVC) increased steadily and sensory score decreased. On day 5, TVC, pH, TVB-N and sensory scores changed dramatically, and sensory scores indicating that the shelf life of refrigerated pork was less than 5 days. The predicted metabolic pathways, based o the data of 16S rDNA, indicated an abundant carbohydrate metabolism and amino metabolism in refrigerated pork. This study provides insight into the determinants of shelf life. Furthermore, it provides insight into the process involved in refrigerated pork spoilage.

An ultrasensitive smartphone-assisted bicolor-ratiometric fluorescence sensing platform based on a "noise purifier" for point-of-care testing of pathogenic bacteria in food.

Non-specific binding in fluorescence resonance energy transfer (FRET) remains a challenge in foodborne pathogen detection, resulting in interference of high background signals. Herein, we innovatively reported a dual-mode FRET sensor based on a "noise purifier" for the ultrasensitive quantification of Escherichia coli O157:H7 in food. An efficient FRET system was constructed with polymyxin B-modified nitrogen-sulfur co-doped graphene quantum dots (N, S-GQDs@PMB) as donors and aptamer-modified yellow carbon dots (Y-CDs@Apt) as acceptors. Magnetic multi-walled carbon nanotubes (Fe@MWCNTs) were employed as a "noise purifier" to reduce the interference of the fluorescence background. Under the background purification mode, the sensitivity of the dual-mode signals of the FRET sensor has increased by an order of magnitude. Additionally, smartphone-assisted colorimetric analysis enabled point-of-care detection of E. coli O157:H7 in real samples. The developed sensing platform based on a "noise purifier" provides a promising method for ultrasensitive on-site testing of trace pathogenic bacteria in various foodstuffs.

A systematic review and meta-analysis indicates a high risk of human noroviruses contamination in vegetable worldwide, with GI being the predominant genogroup.

Human noroviruses (HuNoVs) are the most predominant viral agents of acute gastroenteritis. Vegetables are important vehicles of HuNoVs transmission. This study aimed to assess the HuNoVs prevalence in vegetables. We searched the Web of Science, Excerpta Medica Database, PubMed, and Cochrane databases until June 1, 2023. A total of 27 studies were included for the meta-analysis. Statistical analysis was conducted using Stata 14.0 software. This analysis showed that the pooled HuNoVs prevalence in vegetables was 7 % (95 % confidence interval (CI): 3-13) worldwide. The continent with largest number of studies was Europe, and the highest number of samples was lettuce. As revealed by the results of the subgroup meta-analysis, the prevalence of GI genogroup was the highest (3 %, 95 % CI: 1-7). A higher prevalence was seen in vegetables from farms (18 %, 95 % CI: 5-37), while only 4 % (95 % CI: 1-8) in retail. The HuNoVs prevalence of ready-to-eat vegetables and non-ready-to-eat vegetables was 2 % (95 % CI: 0-8) and 9 % (95 % CI: 3-16), respectively. The prevalence by quantitative real time RT-PCR was 8 % (95 % CI: 3-15) compared to 3 % (95 % CI: 0-13) by conventional RT-PCR. Furthermore, the HuNoVs prevalence in vegetables was 6 % (95 % CI: 1-14) in ISO pretreatment method and 8 % (95 % CI: 1-19) in non-ISO method, respectively. This study is helpful in comprehensively understanding the prevalence of HuNoVs contamination in vegetables worldwide.

Occurrence, molecular characterization and antibiotic resistance of Cronobacter spp. isolated from wet rice and flour products in Guangdong, China.

This study explored the prevalence of Cronobacter spp. in wet rice and flour products from Guangdong province, China, the molecular characteristics and antimicrobial susceptibility profiles of the isolates were identified. Among 249 samples, 100 (40.16%) were positive for Cronobacter spp., including 77 wet rice and 23 wet flour products. Eleven serotypes were characterized among 136 isolates with C. sakazakii O2 (n = 32) predominating. Forty-nine MLST patterns were assigned, 15 of which were new. C. sakazakii ST4 (n = 17) was the dominant ST, which is previously reported to have caused three deaths; followed by C. malonaticus ST7 (n = 15), which is connected to adult infections. All strains presented susceptibility to ampicillin/sulbactam, imipenem, aztreonam and trimethoprim/sulfamethoxazole. The isolates showed maximum resistance to cephalothin, and the resistance and intermediate rates were 91.91% and 3.68%, each. Two strains, croM234A1 and croM283-1, displayed resistance to three antibiotics. High contamination level and predominant number of pathogenic STs of Cronobacter in wet rice and flour products implied a potential risk to public healthiness. This survey could provide comprehensive information for establishing more targeted control methods for Cronobacter spp.

Nitrogen-metabolising microorganism analysis in rapid sand filters from drinking water treatment plant.

Sand filters (SFs) are common treatment processes for nitrogen pollutant removal in drinking water treatment plants (DWTPs). However, the mechanisms on the nitrogen-cycling role of SFs are still unclear. In this study, 16S rRNA gene amplicon sequencing was used to characterise the diversity and composition of the bacterial community in SFs from DWTPs. Additionally, metagenomics approach was used to determine the functional microorganisms involved in nitrogen cycle in SFs. Our results showed that Pseudomonadota, Acidobacteria, Nitrospirae and Chloroflexi dominated in SFs. Subsequently, 85 high-quality metagenome-assembled genomes (MAGs) were retrieved from metagenome datasets of selected SFs involving nitrification, assimilatory nitrogen reduction, denitrification and anaerobic ammonia oxidation (anammox) processes. Read mapping to reference genomes of Nitrospira and the phylogenetic tree of the ammonia monooxygenase subunit A gene, amoA, suggested that Nitrospira is abundantly found in SFs. Furthermore, according to their genetic content, a nitrogen metabolic model in SFs was proposed using representative MAGs and pure culture isolate. Quantitative real-time polymerase chain reaction (qPCR) showed that ammonia-oxidising bacteria (AOB) and archaea (AOA), and complete ammonia oxidisers (comammox) were ubiquitous in the SFs, with the abundance of comammox being higher than that of AOA and AOB. Moreover, we identified a bacterial strain with a high NO3-N removal rate as Pseudomonas sp. DW-5, which could be applied in the bioremediation of micro-polluted drinking water sources. Our study provides insights into functional nitrogen-metabolising microbes in SFs of DWTPs.

Occurrence, Antibiotic Susceptibility, Biofilm Formation and Molecular Characterization of Staphylococcus aureus Isolated from Raw Shrimp in China.

The aim of this study was to determine the prevalence and characterization of Staphylococcus aureus isolated from 145 shrimp samples from 39 cities in China. The results show that 41 samples (28%) from 24 cities were positive, and most of the positive samples (39/41, 95.1%) were less than 110 MPN/g. Antimicrobial susceptibility testing showed that only seven isolates were susceptible to all 24 antibiotics, whereas 65.1% were multidrug-resistant. Antibiotic resistance genes that confer resistance to ß-lactams, aminoglycosides, tetracycline, macrolides, lincosamides and streptogramin B (MLSB), trimethoprim, fosfomycin and streptothricin antibiotics were detected. All S. aureus isolates had the ability to produce biofilm and harbored most of the biofilm-related genes. Genes encoding one or more of the important virulence factors staphylococcal enterotoxins (sea, seb and sec), toxic shock syndrome toxin 1 (tsst-1) and Panton-Valentine leukocidin (PVL) were detected in 47.6% (30/63) of the S. aureus isolates. Molecular typing showed that ST15-t085 (27.0%, 17/63), ST1-t127 (14.3%, 9/63) and ST188-t189 (11.1%, 7/63) were the dominant genetic types. The finding of this study provides the first comprehensive surveillance on the incidence of S. aureus in raw shrimp in China. Some retained genotypes found in this food have been linked to human infections around the world.

Cronobacter spp. Isolated from Quick-Frozen Foods in China: Incidence, Genetic Characteristics, and Antibiotic Resistance.

Cronobacter spp. are emerging foodborne pathogens that cause severe diseases. However, information on Cronobacter contamination in quick-frozen foods in China is limited. Therefore, we studied the prevalence, molecular characterization, and antimicrobial susceptibility of Cronobacter in 576 quick-frozen food samples collected from 39 cities in China. Cronobacter spp. were found in 18.75% (108/576) of the samples, and the contamination degree of the total positive samples was 5.82 MPN/g. The contamination level of frozen flour product samples was high (44.34%). Among 154 isolates, 109 were C. sakazakii, and the main serotype was C. sakazakii O1 (44/154). Additionally, 11 serotypes existed among four species. Eighty-five sequence types (STs), including 22 novel ones, were assigned, indicating a relatively high genetic diversity of the Cronobacter in this food type. Pathogenic ST148, ST7, and ST1 were the main STs in this study. ST4, epidemiologically related to neonatal meningitis, was also identified. All strains were sensitive to cefepime, tobramycin, ciprofloxacin, and imipenem, in which the resistance to cephalothin was the highest (64.94%).Two isolates exhibited multidrug resistance to five and seven antimicrobial agents, respectively. In conclusion, these findings suggest that the comparatively high contamination level of Cronobacter spp. in quick-frozen foods is a potential risk warranting public attention.

Sensitive and Selective Detection of Enterococcus faecalis Using a New Turn-on Fluorogenic ß-glucosidase Substrate Combined with a Modified Selective Broth.

A new, simple-to-synthesize and sensitive turn-on fluorogenic substrate (CFMU-Glu) for ß-glucosidase activity was developed. This probe was based on a 7-hydroxycoumarin derivative (CFMU) that could emit green fluorescence and had the low pKa value of 5.61 ± 0.01. CFMU-Glu could be used for sensitive monitoring of the almond ßGLU and Enterococcus faecalis (E. faecalis) at the optimal pHs of 6.50 and 7.00, respectively. Moreover, a new sensitive and selective fluorogenic broth (PBF-B) for E. faecalis, utilizing CFMU-Glu and polymyxin B, was also developed. Polymyxin B was discovered to can significantly improve the detection selectivity and signal intensity. The proposed 4-four method using PBF-B and a microcentrifuge tube could provide fluorogenic detection limits of 5.01 × 104 and 1.0 × 105 CFU mL-1 by fluorescence microplate reader and naked eye, respectively; it could also provide a turn-on chromogenic detection limit of 1.0 × 106 CFU mL-1 by naked eye. The proposed method could detect 8 CFU mL-1 of E. faecalis in drinking water, Liangcha (herbal tea) and milk samples within 10 h, without pre-enrichment.

High prevalence of multidrug-resistant Escherichia coli in retail aquatic products in China and the first report of mcr-1-positive extended-spectrum ß-lactamase-producing E. coli ST2705 and ST10 in fish.

Contamination of food by multidrug-resistant (MDR) bacteria is a potential threat to consumers. Aquatic products are increasingly consumed due to their high value and rich nutrient. Nevertheless, the prevalence of multi-drug resistant (MDR) E. coli in retail aquatic products has not been systematically investigated in China. In this study, we conducted a national investigation on the prevalence of E. coli and MDR E. coli in retail aquatic products and the characteristics of the MDR E. coli isolates. A total of 849 samples consisting of 680 fish, 143 shrimp, and 26 shellfish were purchased from markets in 39 cities in China and investigated for the presence of E. coli. Overall, 340 (40.0 %) and 169 (19.9 %) samples were contaminated with E. coli and MDR E. coli, indicating poor hygiene conditions of retail aquatic products. A total of 190 MDR E. coli were isolated, which were recovered from 38.5 % shellfish, 20.1 % shrimp, and 19.0 % fish. Thirty-two ESBL-producing E. coli were identified from 3.5 % fish, 3.5 % shrimp, and 3.8 % shellfish. The MDR E. coli isolates showed a high prevalence of resistance to tetracycline (93.7 %), trimethoprim-sulfamethoxazole (78.9 %), ampicillin (78.4 %), chloramphenicol (72.1 %), nalidixic acid (73.2 %), cephalothin (65.3 %), and streptomycin (65.8 %). Resistances to kanamycin (42.1 %), gentamicin (37.9 %), ciprofloxacin (42.6 %), and norfloxacin (45.8 %) were also common. Further, 15.3 % and 8.4 % of the isolates were resistant to cefotaxime and ceftazidime, respectively. Four isolates were resistant to colistin. More than 85.0 % of the isolates were resistant to 5-15 antibiotics. Of the antibiotic resistance genes, TEM-1, tetA, strA/B, sul2, aadA, floR, and qnrS were the most prevalent, followed by sul1, aac(6')-Ib, oqxA/B, cmlA and aphA1. Six CTX-M-types were found, among which CTX-M-55 and CTX-M-14 were predominant. All 4 colistin-resistant isolates carried the mcr-1 gene, of which three were ESBL strains. WGS indicated that the mcr-1 gene was located on two types of plasmids (IncHI2 and IncX4). Conjugation experiments showed the mcr-1 gene could be transferred to E. coli C600. To our knowledge, this is the first report of mcr-1-positive ESBL E. coli (ST10 and ST2705) in retail aquatic products. The emergence of such strains poses a serious threat to consumers, with the potential to spread resistance genes to humans. Our results indicate that retail aquatic products are important reservoirs of MDR E. coli and facilitate the dissemination of the resistance genes. Continuous surveillance and interventions of restricting antibiotic use in aquatic environments should also be applied to reduce the prevalence of MDR bacteria.

Presence and characterization of methicillin-resistant Staphylococcus aureus co-carrying the multidrug resistance genes cfr and lsa(E) in retail food in China.

Staphylococcus aureus is an important food-related pathogen associated with bacterial poisoning that is difficult to treat due to its multidrug resistance. The cfr and lsa(E) genes both cause multiple drug resistance and have been identified in numerous Staphylococcus species, respectively. In this study, we found that a methicillin-resistant S. aureus (MRSA) strain, 2868B2, which was isolated from a sample of frozen dumplings in Hangzhou in 2015, co-carried these two different multidrug resistance genes. Further analysis showed that this strain was resistant to more than 18 antibiotics and expressed high-level resistance to florfenicol, chloramphenicol, clindamycin, tiamulin, erythromycin, ampicillin, cefepime, ceftazidime, kanamycin, streptomycin, tetracycline, trimethoprim-sulfamethoxazole and linezolid (MIC = 8 µg/mL). Whole genome sequencing was performed to characterize the genetic environment of these resistance genes and other genomic features. The cfr gene was located on the single plasmid p2868B2 (39,159 bp), which demonstrated considerable similarity to many plasmids previously identified in humans and animals. p2868B2 contained the insertion sequence (IS) element IS21-558, which allowed the insertion of cfr into Tn558 and played an important role in the mobility of cfr. Additionally, a novel multidrug resistance region (36.9 kb) harbouring lsa(E) along with nine additional antibiotic resistance genes (ARGs) (aadD, aadE, aacA-aphD, spc, lnu(B), lsa(E), tetL, ermC and blaZ) was identified. The multidrug resistance region harboured four copies of IS257 that were active and can mediate the formation of four circular structures containing ARGs and ISs. In addition, genes encoding various virulence factors and affecting multiple cell adhesion properties were identified in the genome of MRSA 2868B2. This study confirmed that the cfr and lsa(E) genes coexist in one MRSA strain and the presence of plasmid and IS257 in the multi-ARG cluster can promote both ARG transfer and dissemination. Furthermore, the presence of so many ARGs and virulence genes in food-related pathogens may seriously compromise the effectiveness of clinical therapy and threaten public health, its occurrence should pay public attention and the traceability of these genes in food-related samples needs further surveillance.

Characterization of Escherichia coli O157:non-H7 isolated from retail food in China and first report of mcr-1/IncI2-carrying colistin-resistant E. coli O157:H26 and E. coli O157:H4.

Escherichia coli O157 belongs to a diverse serogroup including different H serotypes. E. coli O157: H7 is the most common serotype that can cause acute gastroenteritis, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (HUS) in humans. In recent years, some E. coli O157:non-H7 strains have been reported to cause sporadic cases and outbreaks of diarrheal diseases. However, the phenotypic and genotypic characteristics of E. coli O157:non-H7 are poorly understood. In this study, E. coli O157:non-H7 strains were isolated from retail food sold on markets in 13 cities in China during 2012-2016 and characterized systematically in terms of their H serotypes, virulence genes, antibiotic resistance, and genotypes. Six H serotypes (H26, H42, H11, H38, H4, and H5) were identified, of which, O157:H42 (31.4 %) and O157:H26 (28.6 %) were the most prevalent. Most of the isolates (82.9 %) carried virulence genes. Ten isolates (28.6 %) carried the eae gene and were identified as atypical enteropathogenic E. coli. Multilocus sequence typing showed that the E. coli O157:non-H7 strains demonstrated diverse sequence types with different evolutionary trends than E. coli O157:H7. All the isolates exhibited multidrug resistance. The isolates showed a high prevalence of resistance to AMC, AMP, CTX, CIP, T/S, TE, and FFC. The predominant antibiotic-resistance genes were TEM-1 (40.0 %), CTX-M-55 (34.3 %), aadA (74.3 %), sul2 (62.9 %), floR (91.4 %), tetA (85.7 %), qnrS (37.1 %), oqxA (62.9 %), and oqxB (62.9 %). For the first time, we identified IncI2 plasmid-mediated colistin-resistant strains (six O157:H26 and one O157:H4). These strains co-harbored plasmid-mediated mcr-1 gene, CTX-M-55, OXA-4, PMQR, and other resistant genes, which is of great concern. Colistin and cefotaxime are generally used as the last defense to treat complicated infections. The emergence of virulent and multidrug resistant strains in food poses a serious threat to human health. The strict monitoring and surveillance of multiple-drug resistant strains in food are needed to prevent their dissemination to humans.