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.

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.

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.

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.

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.

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.

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.

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.

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.

Pseudotargeted Metabolomic Fingerprinting and Deep Learning for Identification and Visualization of Common Pathogens.

Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF) spectrometry fingerprinting has reduced turnaround times, costs, and labor as conventional procedures in various laboratories. However, some species strains with high genetic correlation have not been directly distinguished using conventional standard procedures. Metabolomes can identify these strains by amplifying the minor differences because they are directly related to the phenotype. The pseudotargeted metabolomics method has the advantages of both non-targeted and targeted metabolomics. It can provide a new semi-quantitative fingerprinting with high coverage. We combined this pseudotargeted metabolomic fingerprinting with deep learning technology for the identification and visualization of the pathogen. A variational autoencoder framework was performed to identify and classify pathogenic bacteria and achieve their visualization, with prediction accuracy exceeding 99%. Therefore, this technology will be a powerful tool for rapidly and accurately identifying pathogens.

Controlled PAH-mediated method with enhanced optical properties for simple, stable immunochromatographic assays.

Despite their potential for signal amplification in immunochromatographic assays (ICAs) with Au nanoparticles (AuNPs) as probes, metal growth methods are of limited practical applicability given their complex non-specificity and lack of robust growth schemes. Here, we propose a novel method of polyallylamine hydrochloride (PAH)-mediated metal growth for the detection of Escherichia coli O157:H7 by AuNP-ICA. The developed method relies on the highly controlled growth of Cu shells on the AuNP core and allows one to achieve highly enhanced colorimetric signals by controlling PAH as the growth framework. The introduction of PAH eliminates the non-specific adsorption of Cu ions on the nitrocellulose membrane and thus provides maximized and effective signal-to-noise ratios to achieve a detection limit of 9.8 CFU/mL for E. coli O157:H7. Moreover, the newly developed detection method exhibits good reproducibility (coefficient of variation <13%), remarkable stability, and practical applicability. The PAH-mediated signal enhancement system paves the way to the realization of stable metal growth methods based on Au, Ag, and other metals and is well suited for the rapid, stable, and sensitive detection of food-borne pathogens using the AuNP-ICA platform.

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.

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.

First report of the optrA-carrying multidrug resistance genomic island in Campylobacter jejuni isolated from pigeon meat.

Campylobacter spp., such as Campylobacter jejuni and Campylobacter coli, are important zoonotic Gram-negative pathogens that cause acute intestinal diseases in humans. The optrA gene, encoding an ATP-binding cassette F (ABC-F) protein that confers resistance to oxazolidinones and phenicols, has been found in C. coli in China. In this study, the optrA gene was first identified in C. jejuni collected from retail meat in China from 2013 to 2016. Nine strains, isolated from a pigeon meat sample, carry the optrA gene. The molecular characteristics of the optrA-positive strains were determined by whole genome sequencing. Pulsed-field gel electrophoresis, multilocus sequence typing, and single nucleotide polymorphism analyses demonstrated that the nine optrA-positive isolates were genetically homogeneous. Phylogenetic characteristics and sequence comparison revealed that optrA was located on a chromosome-borne multidrug resistance genomic island. The optrA gene along with the tet(O) gene formed two different translocatable units (TUs), thereby supporting the transmission of TU-associated resistance genes. The emergence and spread of such TUs and strains are of great concern in terms of food safety, and measures must be implemented to avoid their dissemination in other Gram-negative bacteria and food chains.

Genomic Analysis and Stability Evaluation of the Phenol-Degrading Bacterium Acinetobacter sp. DW-1 During Water Treatment.

Phenol is a toxic organic molecule that is widely detected in the natural environment, even in drinking water sources. Biological methods were considered to be a good tool for phenol removal, especially microbial immobilized technology. However, research on the "seed" bacteria along with microbial community analysis in oligotrophic environment such as drinking water system has not been addressed. In this study, Acinetobacter sp. DW-1 with high phenol degradation ability had been isolated from a drinking water biofilter was used as seeded bacteria to treat phenol micro-polluted drinking water source. Meanwhile, the whole genome of strain DW-1 was sequenced using nanopore technology. The genomic analysis suggests that Acinetobacter sp. DW-1 could utilize phenol via the ß-ketoadipate pathway, including the catechol and protocatechuate branches. Subsequently, a bio-enhanced polyhedral hollow polypropylene sphere (BEPHPS) filter was constructed to investigate the stability of the seeded bacteria during the water treatment process. The denatured gradient gel electrophoresis (DGGE) profile and the quantification of phenol hydroxylase gene results indicate that when the BEPHPS filter was operated for 56 days, Acinetobacter sp. was still a persistent and competitive bacterium in the treatment group. In addition, 16S rRNA gene amplicon sequencing results indicate that Acinetobacter sp., as well as Pseudomonas sp., Nitrospira sp., Rubrivivax sp. were the predominant bacteria in the treatment group, which were different from that in the CK group. This study provides a better understanding of the mechanisms of phenol degradation by Acinetobacter sp. DW-1 at the gene level, and provides new insights into the stability of seeded bacteria and its effects on microbial ecology during drinking water treatment.

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.

An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection.

Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high patient mortality. Accordingly, it is necessary to develop a L. monocytogenes detection platform with high specificity, sensitivity, and exploitability. CRISPR/Cas systems have shown great potential in the development of next-generation biosensors for nucleic acid detection, owing to the trans-cleavage capabilities of the Cas effector proteins. Herein, we introduce the trans-cleavage activity of CRISPR/Cas12a into an electrochemical biosensor (E-CRISPR), combined with recombinase-assisted amplification (RAA), to establish a cost-effective, specific and ultrasensitive method; namely RAA-based E-CRISPR. The concept behind this approach is that the target will induce the number change of the surface signaling probe (containing an electrochemical tag), which leads to a variation in the electron transfer of the electrochemical tag. The introduction of an RAA-based Cas12a system into the E-CRISPR sensor achieves a more prominent signal change between the presence and absence of the target. Under optimized conditions, RAA-based E-CRISPR can detect as low as 0.68 aM of genomic DNA and 26 cfu/mL of L. monocytogenes in pure cultures. More importantly, the RAA-based E-CRISPR enables rapid and ultrasensitive detection of L. monocytogenes in spiked and natural Flammulina velutipes samples. Moreover, no cross-reactivity with other non-target bacteria was observed. This system thus demonstrates to be a simple, high-sensitivity, and high-accuracy platform for L. monocytogenes detection.

Characteristics of Antibiotic Resistance Genes and Antibiotic-Resistant Bacteria in Full-Scale Drinking Water Treatment System Using Metagenomics and Culturing.

The contamination of antibiotic resistance genes (ARGs) may directly threaten human health. This study used a metagenomic approach to investigate the ARG profile in a drinking water treatment system (DWTS) in south China. In total, 317 ARG subtypes were detected; specifically, genes encoding bacitracin, multidrug, and sulfonamide were widely detected in the DWTS. Putative ARG hosts included Acidovorax (6.0%), Polynucleobacter (4.3%), Pseudomonas (3.4%), Escherichia (1.7%), and Klebsiella (1.5%) as the enriched biomarkers in the DWTS, which mainly carried bacitracin, beta-lactam, and aminoglycoside ARGs. From a further analysis of ARG-carrying contigs (ACCs), Stenotrophomonas maltophilia and Pseudomonas aeruginosa were the most common pathogens among the 49 ACC pathogens in the DWTS. The metagenomic binning results demonstrated that 33 high-quality metagenome-assembled genomes (MAGs) were discovered in the DWTS; particularly, the MAG identified as S. maltophilia-like (bin.195) harbored the greatest number of ARG subtypes (n = 8), namely, multidrug (n = 6; smeD, semE, multidrug_transporter, mexE, semB, and smeC), beta-lactam (n = 1; metallo-beta-lactamase), and aminoglycoside [n = 1; aph(3')-IIb]. The strong positive correlation between MGEs and ARG subtypes revealed a high ARG dissemination risk in the DWTS. Based on the pure-culture method, 93 isolates that belong to 30 genera were recovered from the DWTS. Specifically, multidrug-resistant pathogens and opportunistic pathogens such as P. aeruginosa, Bacillus cereus, and S. maltophilia were detected in the DWTS. These insights into the DWTS's antibiotic resistome indicated the need for more comprehensive ARG monitoring and management in the DWTS. Furthermore, more effective disinfection methods need to be developed to remove ARGs in DWTSs, and these findings could assist governing bodies in the surveillance of antibiotic resistance in DWTSs.