Sequence Polymorphisms in Vibrio cholerae HapR Affect Biofilm Formation under Aerobic and Anaerobic Conditions.

We investigated the influence of hapR sequence mutations on the biofilm formation of Vibrio cholerae. In this study, hapR sequences from 85 V. cholerae strains belonging to both pandemic and nonpandemic serogroup were investigated through phylogenetic and sequence analyses. Biofilm formation assays under aerobic and anaerobic conditions were also performed. Sequence variations include single point mutations and insertions/deletions (indels) leading to either truncated or frameshifted HapR. Population structure analysis revealed two major hapR haplogroups, hapR1 and hapR2. Phylogenetic reconstruction displayed a hypothetical ancestral hapR sequence located within the hapR1 haplogroup. Higher numbers of single nucleotide polymorphisms and genetic diversity indices were observed in hapR1, while indels occurred dominantly in hapR2. Aerobic conditions supported more robust biofilms compared to anaerobic conditions. Strains with frameshifted HapR produced the largest amount of biofilm under both oxygen conditions. Quantitative real-time PCR assay confirmed that strains with truncated and frameshifted HapR resulted in a nonfunctional regulator as exhibited by the significantly low hapA gene expression. The present study shows that HapR mutations had a strong influence on biofilm formation and that sequence polymorphisms leading to the disruption of DNA-binding sites or dimerization of the HapR will result in more-robust V. cholerae biofilms. IMPORTANCE Our study revealed an ancestral hapR sequence from a phylogenetic reconstruction that displayed the evolutionary lineage of the nonpandemic to the pandemic strains. Here, we established hapR1 and hapR2 as major hapR haplogroups. The association of the O1 and O139 serogroups with the hapR2 haplogroup demonstrated the distinction of hapR2 in causing cholera infection. Moreover, mutations in this regulator that could lead to the disruption of transcription factor-binding sites or dimerization of the HapR can significantly affect the biofilm formation of V. cholerae. These observations on the relationship of the hapR polymorphism and V. cholerae biofilm formation will provide additional considerations for future biofilm studies and insights into the epidemiology of the pathogen that could ultimately help in the surveillance and mitigation of future cholera disease outbreaks.

Occurrence, Phenotypic and Molecular Characteristics of Vancomycin-Resistant Enterococci Isolated from Retail Raw Milk in Egypt.

The aim of this study was to determine the occurrence, phenotypic and molecular characteristics of vancomycin-resistant enterococci (VRE), isolated from retail raw cow's milk. One hundred milk samples collected from retail shops in Egypt were examined for the occurrence of VRE by using kanamycin aesculin azide agar supplemented with 4 µg/mL vancomycin. PCR was conducted to determine enterococcal species and to screen the isolated strains for the presence of antibiotic resistance and virulence genes. All isolated strains were characterized by antimicrobial susceptibility testing for 12 antibiotics. From 24 samples (24%), we recovered 22 isolates (91.6%) classified as VRE (minimum inhibitory concentration ≥32) and 2 isolates (8.3%) classified as intermediate resistant to vancomycin (≤16). Enterococcus faecium (29.1%), Enterococcus faecalis (12.5%), Enterococcus casseliflavus (16.6%), and Enterococcus gallinarum (4.1%) were identified by using multiplex PCR. The genus Enterococcus was resistant to clindamycin (100%), linezolid (91.6%), teicoplanin (91.6%), erythromycin (87.5%), and tetracycline (29.1%). Co-resistance to vancomycin, teicoplanin, and linezolid was detected in 83.3% of isolates. Antibiotic resistance genes vanB, tet(M), tet(L), and erm(B) were identified in 29.1%, 16.6%, 8.3%, and 4.1% of isolates, respectively. Virulence genes gelE and esp were detected in 16.6% and 12.5% of isolates, respectively. In conclusion, the high occurrence of co-resistance to vancomycin, teicoplanin, and linezolid reported in this study is alarming. The high frequency of linezolid resistance prompts increased the attention of researchers to routinely perform linezolid susceptibility in food isolates. This study declares potential food safety risks from consumption and improper handling of raw milk regarding clinically important bacteria and promotes necessary legislation for forbidding the selling and consumption of retail raw milk.

Coproduction of Tet(X7) Conferring High-Level Tigecycline Resistance, Fosfomycin FosA4, and Colistin Mcr-1.1 in Escherichia coli Strains from Chickens in Egypt.

The plasmid-mediated tet(X7) conferring high-level tigecycline resistance was identified in five mcr-1.1-positive Escherichia coli strains (ST10 [n = 3] and ST155 [n = 2]) isolated from chickens in Egypt. Two fosfomycin-resistant fosA4-carrying IncFII plasmids (∼79 kb in size) were detected. Transposase ISCR3 (IS91 family) is syntenic with tet(X7) in all isolates, suggesting its role in the mobilization of tet(X7). To our knowledge, this is the first global report of ST4-IncHI2 plasmids cocarrying tet(X7) and mcr-1.1 from chickens.

Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of blaNDM-1, blaOXA-66, blaOXA-72, and an AbaR4-Like Resistance Island in the Same Sample.

This study was conducted to characterize carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii isolated from fresh vegetables in Japan. Two K. pneumoniae isolates (AO15 and AO22) and one A. baumannii isolate (AO22) were collected from vegetables in the city of Higashihiroshima, Japan, and subjected to antimicrobial susceptibility testing, conjugation experiments, and complete genome sequencing using Illumina MiniSeq and Oxford Nanopore MinION sequencing platforms. The two K. pneumoniae isolates were clonal, belonging to sequence type 15 (ST15), and were determined to carry 19 different antimicrobial resistance genes, including blaNDM-1 Both the isolates carried blaNDM-1 on a self-transmissible IncFII(K):IncR plasmid of 122,804 bp with other genes conferring resistance to aminoglycosides [aac(6')-Ib, aadA1, and aph(3')-VI], ß-lactams (blaCTX-M-15, blaOXA-9, and blaTEM-1A), fluoroquinolones [aac(6')-Ib-cr], and quinolones (qnrS1). A. baumannii AO22 carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid of 10,880 bp. Interestingly, A. baumannii AO22 harbored an AbaR4-like genomic resistance island (GI) of 41,665 bp carrying genes conferring resistance to tetracycline [tet(B)], sulfonamides (sul2), and streptomycin (strAB). Here, we identified Japanese carbapenemase-producing Gram-negative bacteria isolated from vegetables, posing a food safety issue and a public health concern. Additionally, we reported a GR2-type plasmid carrying two copies of blaOXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii isolate.IMPORTANCE Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases. The NDM carbapenemase, rarely described in Japan, was detected in two K. pneumoniae isolates. The A. baumannii isolate identified in this study carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid. This study indicates that even one fresh ready-to-eat vegetable sample might serve as a significant source of genes (blaNDM-1, blaOXA-72, blaCTX-M-14b, and blaCTX-M-15) encoding resistance to frontline and clinically important antibiotics (carbapenems and cephalosporins). Furthermore, the detection of these organisms in fresh vegetables in Japan is alarming and poses a food safety issue and a public health concern.

Toxigenic Potential of Bacillus cereus Strains Isolated from Retail Dairy Products in Egypt.

Little is known about the virulence in Bacillus cereus strains isolated from retail dairy products in the Middle East and particularly from Egypt. In this study, the occurrence of B. cereus in 290 samples of dairy products (raw milk, Ras cheese, pasteurized extended shelf life [ESL] milk) collected from retail shops was investigated. The potential of 126 selected isolates of B. cereus to possess genes encoding nonhemolytic enterotoxin, hemolysin BL, and cytotoxin K (cytK), and to grow at 7°C was verified. The highest occurrence of B. cereus was found in raw milk (85%, 85/100) followed by Ras cheese (10%, 10/100) and ESL milk samples (8.8%, 8/90). A large proportion of the B. cereus isolates from raw milk (48.9%, 48/99) and Ras cheese (71.4%, 10/14) had at least one complete set of toxin genes (nhe or hbl). Enterotoxin genes, nheA, nheB, nheC, hblA, hblD, and hblC, were detected in 38.4% (5/13), 53.8% (7/13), 61.5% (8/13), 46.1% (6/13), 46.1% (6/13), and 23.1% (3/13) of ESL milk isolates, respectively. cytK was identified in 42.4% (42/99), 50% (7/14), and 46.2% (6/13) of raw milk, Ras cheese, and ESL milk isolates, respectively. The psychrotrophic ability was observed in 22.2% and 15.3% of isolates recovered from raw milk and ESL milk, respectively. The toxigenic potential of B. cereus strains described in this study may pose a health risk to the consumer and, therefore, the presence of these bacteria in retail dairy products should be monitored to ensure consumers' safety.

Correction to: Full genome sequence of a polyvalent bacteriophage infecting strains of Shigella, Salmonella, and Escherichia.

The original version of this article unfortunately contained a mistake.

Full genome sequence of a polyvalent bacteriophage infecting strains of Shigella, Salmonella, and Escherichia.

A novel lytic bacteriophage, Escherichia phage EcS1, was isolated from sewage samples collected in Higashi-Hiroshima, Japan. The complete genome sequence of EcS1 was determined using the Illumina Miseq System. The whole genome of EcS1 was found to be 175,437 bp in length with a mean G+C content of 37.8%. A total of 295 genes were identified as structural, functional, and hypothetical genes. BLAST analysis of the EcS1 genomic sequence revealed the highest identity (79%; query cover of 73-74%) to three T4-related phages that infect Serratia sp. ATCC 39006. Host range experiments revealed that EcS1 has lytic effects on three pathogenic strains of Shigella spp. and a pathogenic strain of Salmonella enterica as well as on E. coli strains. However, two strains of Serratia marcescens showed resistance to this phage. Phylogenetic trees for phage tail fiber protein sequences revealed that EcS1 is closely related to Enterobacteriaceae-infecting phages. Thus, EcS1 is a novel phage that infects several pathogenic strains of the family Enterobacteriaceae.

Antimicrobial Effects of Blueberry, Raspberry, and Strawberry Aqueous Extracts and their Effects on Virulence Gene Expression in Vibrio cholerae.

The antimicrobial effects of aqueous extracts of blueberry, raspberry, and strawberry on 13 pathogenic bacteria were evaluated. The minimum inhibitory concentrations and minimum bactericidal concentrations of the extracts were determined before and after neutralization to pH 7.03 ± 0.15. Both Gram-positive and Gram-negative pathogenic bacteria were selectively inhibited by the non-neutralized berries. Blueberry was the best inhibitor, and Vibrio and Listeria were the most sensitive bacteria. After neutralization, blueberry affected only Vibrio and Listeria, whereas the antimicrobial activities of raspberry and strawberry were abolished. The total contents of phenolics, flavonoids, and proanthocyanidins in the extracts were measured with colorimetric methods and were highest in strawberry, followed by raspberry, and then blueberry. We also studied the effects of sub-bactericidal concentrations of the three berry extracts on virulence gene expression in Vibrio cholerae. Real-time quantitative reverse transcription-polymerase chain reaction revealed that the three berry extracts effectively repressed the transcription of the tcpA gene. Raspberry also repressed the transcription of the ctxA gene, whereas blueberry and strawberry did not. However, the three berry extracts did not affect the transcription of toxT. These results suggest that the three berry extracts exert potent antimicrobial effects and inhibit the expression of the virulence factors of V. cholerae.

Genetic characterization of antibiotic resistance and virulence factors in Enterococcus spp. from Japanese retail ready-to-eat raw fish.

Little information is available on the diversity and distribution of resistance and virulence factors in enterococci isolated from retail fish. In this study, 200 samples of retail ready-to-eat raw fish (sashimi) collected from the Japanese prefecture of Hiroshima were analyzed for incidence of Enterococcus spp. We recovered 96 enterococcal isolates from 90 (45%, 90/200) samples. Fifty-six strains were identified at the species level: E. faecalis (n = 31), E. faecium (n = 7), E. casseliflavus (n = 7), E. gallinarum (n = 3), E. phoeniculicola (n = 4), E. raffinosus (n = 2), E. saccharolyticus (n = 1), and E. gilvus (n = 1). Twenty-five (26%, 25/96) strains carried antibiotic resistance genes. These included the tet(M), tet(L), tet(K), erm(B), msr(A/B), aph(3'), and blaZ genes, which were detected in 12.5%, 9.3%, 2%, 14.5%, 1%, 1%, and 2% of isolates, respectively. The virulence genes gelE and asa1 were detected in 31 and 24 E. faecalis strains, respectively. Both genes were detected in one E. faecium strain. In conclusion, this is the first study to underscore the importance of sashimi as not only a reservoir of Enterococcus spp. carrying resistance and virulence genes, but also a reservoir for unusual Enterococcus spp.

[Development and Usefulness of Support Package for Learning of Image Interpretation Assistance Using CT and MRI Image in Emergency Medicine].

The Japanese Ministry of Health, Labour and Welfare announced about the expansion of duties by the radiological technologists in team medical care in April, 2010, and the importance of image interpretation assistance by the radiological technologists became higher. In that respect, for improvement in ability of image interpretation assistance by the radiological technologists in emergency medicine, we developed a support package for learning of image interpretation assistance (support package) and evaluated the usefulness for learning of image interpretation assistance by questionnaires. The support package included digital imaging and communications in medicine (DICOM) data of case, explainer video of urgent imaging findings, and DICOM viewer. In 100% of evaluators, the support package was useful for urgent imaging findings in emergency medicine. Moreover, 68.9% of the evaluators had an experience helped by learning to use the support package in the clinical site. In conclusion, we confirmed that the support package was useful for learning of image interpretation assistance by the radiological technologists.

Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers.

Avian pathogenic Escherichia coli (APEC) causes extensive mortality in poultry flocks, leading to extensive economic losses. To date, little information is available on the molecular basis of antimicrobial resistance in APEC in Africa. Therefore, the objective of this study was to characterize the virulence and antimicrobial resistance of multidrug-resistant APEC isolated from septicemic broilers in Egypt at the molecular level. Among 91 non-repetitive E. coli isolates, 73 (80.2%) carried three or more of the APEC virulence genes iroN, ompT, iss, iutA, and hlyF. All 73 APEC isolates showed multidrug resistance phenotypes, particularly against ampicillin, tetracycline, spectinomycin, streptomycin, kanamycin, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 and class 2 integrons in 34 (46.6%) and seven (9.6%) isolates, respectively. The ß-lactamase-encoding genes, bla(TEM-1), bla(TEM-104), bla(CMY-2), bla(OXA-30), bla(CTX-M-15), and bla(SHV-2); tetracycline resistance genes, tet(A), tet(B), tet(C), tet(D), and tet(E); the plasmid-mediated quinolone resistance genes, qnrA1, qnrB2, qnrS1, and aac(6')-Ib-cr, and florfenicol resistance gene, floR, were also identified in 69 (94.5%), 67 (91.8%), 47 (64.4%), and 13 (17.8%) isolates, respectively. To the best of our knowledge, this is the first report of molecular characterization of antimicrobial resistance in APEC strains from Africa.

Predominance of Multidrug-Resistant Gram-Negative Bacteria Isolated from Supermarket Retail Seafood in Japan.

Reports have documented antimicrobial usage in aquaculture, and the aquatic ecosystem can be considered a genetic storage site for antibiotic-resistant bacteria. This study assessed the prevalence of antimicrobial resistance (AMR) among Gram-negative bacteria recovered from retail seafood in Hiroshima, Japan. A total of 412 bacteria were isolated and screened for the presence of ß-lactamases, acquired carbapenemases, and mobile colistin-resistance (mcr) genes. Forty-five (10.9%) isolates were dominated by Morganella (28%), Proteus (22%), Aeromonas (14%), Citrobacter (8%), and Escherichia (8%) and carried AMR genes. The identified AMR genes included those encoded in integrons (19), aac(6՛)-Ib (11), blaTEM-1 (7), blaCTX-M-like (12), blaCTX-M-65 (2), blaSHV-12 (1), blaSHV-27 (1), blaOXA-10 (1), blaOXA-2 (1), and mcr (2). The most common clinical resistances were against ampicillin, colistin, sulfamethoxazole/trimethoprim, tetracycline, and ciprofloxacin. Multidrug resistance (MDR) occurred in 27 (60%) AMR isolates, and multiple antibiotic resistance indices ranged from 0.2 to 0.8. A conjugation experiment showed that 10 of the 11 selected MDR strains harbored conjugable plasmids, although PCR-based replicon typing described seven strains as untypable. IncF replicon was identified in MDR extended-spectrum ß-lactamase-producing Escherichia coli of the pathogenic B2 phylogroup. Our findings suggest that retail seafood harbors MDR bacteria of human interest that require strict resistance surveillance in the seafood production continuum.

Comparative molecular profiling of antimicrobial resistance and phylogenetic characterization of multidrug-resistant Escherichia coli isolated from meat sources in 2009 and 2021 in Japan.

The global spread of antimicrobial resistance (AMR) is alarming. Escherichia coli is a Gram-negative bacterium that causes healthcare-associated infections and is a major threat to public health. Currently, no comprehensive antimicrobial surveillance of multidrug-resistant E. coli of diverse phylogroups along the meat value chain has been implemented in Higashihiroshima, Japan. Therefore, by employing the One Health approach, 1183 bacterial isolates, including 303 recovered from meat samples in 2009, were screened for the presence of antimicrobial resistance determinants using multiplex PCR and DNA sequencing techniques. Seventy-seven non-duplicate E. coli isolates that harbored AMR genes were subjected to antimicrobial susceptibility testing and the detection of integrons. Phylogenetic characterization, which has not been previously investigated, was used to assign E. coli to one of the eight phylogroups. Twenty-six out of 33 (78.8%) and 34 out of 44 (77.3%) E. coli isolates from 2009 and 2021 exhibited multidrug resistance (MDR) phenotypes, respectively. The most common clinical resistance was observed against ampicillin, tetracycline, kanamycin, sulfamethoxazole/trimethoprim, cefotaxime, and chloramphenicol. Overall, 22.1% (17/77) of the E. coli isolates carried extended-spectrum ß-lactamase (ESBL)-encoding genes and showed the ESBL-resistant phenotypes. For the two isolation years, AmpC/ESBL prevalence decreased from 42.4% in 2009 to 20.5% in 2021. The identified AMR genes included blaCTX-M-1, blaCTX-M-2, blaCTX-M-14, blaCTX-M-15, and blaSHV-12 (ESBL-types); blaSHV-1, blaTEM-1, blaTEM-135, and blaTEM-176 (narrow-spectrum types); blaCMY-4, blaADC-32, blaADC-216, blaACT-48, and blaACT-51 (AmpC types); and integrons. All E. coli isolates were negative for carbapenemase-encoding genes, whereas one isolate from 2009 carried mcr-5.1 allele. Approximately 52% of E. coli isolates identified in 2009 were assigned to phylogroup A compared to the 20.5% in 2021. Notably, the highest proportions of E. coli phylogroups exhibiting MDR were groups A, B1, and F, suggesting that members of these groups are mostly associated with drug resistance. This study highlights the role of meat as a significant reservoir of MDR E. coli and potential source for transmission of AMR genes. Our findings emphasize the importance of continuous monitoring to track the changes in the spread of antimicrobial resistance in the food chain.

A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum ß-Lactamases, Plasmid-Mediated AmpC ß-Lactamases, and Integrons in Gram-Negative Bacteria.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum ß-lactamases (ESBLs), plasmid-mediated AmpC ß-lactamases, aac(6')-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant ß-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC ß-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

Emergence of colistin-resistant Enterobacter cloacae and Raoultella ornithinolytica carrying the phosphoethanolamine transferase gene, mcr-9, derived from vegetables in Japan.

IMPORTANCE: Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt.

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The ß-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.

Loads of Coliforms and Fecal Coliforms and Characterization of Thermotolerant Escherichia coli in Fresh Raw Milk Cheese.

The aim of this study was to assess the hygienic status of raw milk cheese and determine the trends of virulence and antimicrobial resistance in thermotolerant Escherichia coli. Two hundred samples of karish, a popular Egyptian fresh raw milk cheese, were analyzed for coliforms and fecal coliforms using a standard most probable number (MPN) technique. Overall, 85% of samples were unsuitable for consumption, as they exceeded Egyptian standards for coliforms (10 MPN/g), and 65% of samples exhibited coliforms at 44.5 °C. Of 150 recovered thermotolerant strains, 140 (93.3%) were identified as E. coli. Importantly, one Shiga toxin-producing E. coli (STEC) strain carrying a striking virulence pattern, stx1-, stx2+, eae-, was detected. Eleven strains (7.8%, 11/140) showed resistance to third-generation cephalosporins. Antibiotic resistance genes included blaSHV, blaCTX-M, qnrS, tet(A), and tet(B), which were present in 4.3%, 2.8%, 0.71%, 2.1%, and 0.71% of isolates, respectively. In conclusion, this study indicated that hygienic-sanitary failures occurred throughout the production process of most retail karish cheese. Furthermore, our findings emphasize the need for adopting third-generation cephalosporin-resistant E. coli as an indicator for monitoring antimicrobial resistance in raw milk cheese to identify the potential public health burden associated with its consumption.