Background and Aim: Raw milk is considered an essential source of nutrition during all stages of human life because it offers a valuable supply of protein and minerals. Importantly, milk is considered a good media for the growth and contamination of many pathogenic bacteria, especially food-borne pathogens such as Escherichia coli. Thus, the objective of this study was to characterize E. coli and detect its virulence factors and antibiotic resistance from raw milk samples. Materials and Methods: Raw milk samples (n=100) were collected from different localities in Qena, Egypt, and investigated for the presence of E. coli using different biochemical tests, IMViC tests, serotyping to detect somatic antigen type, and molecularly by polymerase chain reaction (PCR) tests. The presence of different virulence and antimicrobial genes (hly, eae, stx1, stx2, blaTEM, tetA(A), and tetB genes) in E. coli isolates was evaluated using PCR. Results: The results demonstrated that 10 out of 100 milk samples were contaminated with E. coli. Depending on serology, the isolates were classified as O114 (one isolate), O27 (two isolates), O111 (one isolate), O125 (two isolates), and untypeable (five isolates) E. coli. The sequencing of partially amplified 16S rRNA of the untypeable isolates resulted in one isolate, which was initially misidentified as untypeable E. coli but later proved as Enterobacter hormaechei. Moreover, antibacterial susceptibility analysis revealed that nearly all isolates were resistant to more than 3 families of antibiotics, particularly to β-lactams, clindamycin, and rifampin. PCR results demonstrated that all E. coli isolates showed an accurate amplicon for the blaTEM and tetA(A) genes, four isolates harbored eae gene, other four harbored tetB gene, and only one isolate exhibited a positive stx2 gene. Conclusion: Our study explored vital methods for identifying E. coli as a harmful pathogen of raw milk using 16S rRNA sequencing, phylogenetic analysis, and detection of virulence factors and antibiotic-resistant genes.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen associated with nosocomial infections. In this study, 100 raw milk samples were collected from Qena, Egypt, and subjected to conventional and molecular assays to determine the presence of A. baumannii and investigate their antimicrobial resistance and biofilm formation. Our findings revealed that, among the 100 samples, Acinetobacter spp. were found in 13 samples based on CHROM agar results. We further characterized them using rpoB and 16S-23SrRNA sequencing and gyrB multiplex PCR analysis and confirmed that 9 out of the 13 Acinetobacter spp. isolates were A. baumannii and 4 were other species. The A. baumannii isolates were resistant to β-lactam drugs, including cefotaxime (44%), ampicillin-sulbactam and levofloxacin (33.3% for each), imipenem, meropenem and aztreonam (22.2% for each). We observed different antimicrobial resistance patterns, with a multi-antibiotic resistant (MAR) index ranging from 0.2 to 0.3. According to the PCR results, bla_OXA-51 and bla_OXA-23 genes were amplified in 100% and 55.5% of the A. baumannii isolates, respectively, while the bla_OXA-58 gene was not amplified. Furthermore, the metallo-β-lactamases (MBL) genes bla_IMP and bla_NDM were found in 11.1% and 22.2% of isolates, respectively, while bla_VIM was not amplified. Additionally, eight A. baumannii isolates (88.8%) produced black-colored colonies on Congo red agar, demonstrating their biofilm production capacity. These results showed that, besides other foodborne pathogens, raw milk should also be examined for A. baumannii, which could be a public health concern.

Pseudomonas is a communal motif of environmental associated disease and causes a serious problem in poultry farms, so this study was deliberated to investigate the quandary of Pseudomonas species especially Pseudomonas aeruginosa (P. aeruginosa) which has multifarious virulence genes and plays a major role in poultry outbreaks. Also, it focuses the light on the problem of antimicrobial and disinfectant resistance. A total of 200 samples (100 from dead in shell chicken embryos and 100 from broilers at different ages) were collected from different hatcheries and farms in Luxor governorate, 40 isolates (20%) of Pseudomonas species were isolated and identified serologically as P.cepacia, P.fluorescens, P.putida, P.fragi and P.aeruginosa. PCR inveterate the existence of P.aeruginosa DNA in seven isolates by using 16SrDNA primers at 956bp. P.aeruginosa isolates have different virulence genes such as toxA, exoS, lasB, lasI and oprL gene with incidence rate 71.42% for each of them, except oprL was 100%. Also, Quaternary Ammonium Compounds resistant genes (QACs) were detected in P. aeruginosa isolates with incidence rate (14.28%) for each of qacAB and qacCD genes, while the qacED1 gene incidence was (100%). P. aeruginosa isolates showed an obstacle of antimicrobial resistance for different antimicrobials while most of these isolates cleared susceptibility for ciprofloxacin and norfloxacin. In conclusion, this work described the problem of P. aeruginosa as it proved a high virulence repertoire owned by the P. aeruginosa that confirming its pathogenicity for chicken embryos and broilers. Also, our study is fuelling the concern on disinfectant resistance problem and displaying the relation between QACs and antibiotic resistance. So, the deterrence of the Pseudomonas infection in the poultry housing becomes necessary by applying strict bio-security measures.

Vibrio species can cause foodborne infections and lead to serious gastrointestinal illnesses. The purpose of this research was to detect the Vibrio cholerae and Vibrio parahaemolyticus in raw milk, dairy products, and water samples. Also, it investigated the virulence factors, antibiotic resistance and biofilm formation in isolated bacteria. Conventional and molecular approaches were used to identify the isolates in this study. Vibrio species were detected in 5% of the samples. Vibrio cholerae and Vibrio parahaemolyticus were isolated from 1.25 and 1.5%, respectively, of the total samples. Penicillin resistance was detected in all strains of Vibrio cholerae and Vibrio parahaemolyticus, with a MAR index ranging from 0.16 to 0.5. Four isolates were moderate biofilm producer and three of them were MDR. When Vibrio cholerae was screened for virulence genes, ctxAB, hlyA, and tcpA were found in 80, 60, and 80% of isolates, respectively. However, tdh + /trh + associated-virulence genes were found in 33.3% of Vibrio parahaemolyticus isolates.

Bacteria of the genus Pseudomonas are pathogens in both humans and animals. The most prevalent nosocomial pathogen is P. aeruginosa, particularly strains with elevated antibiotic resistance. In this study, a total of eighteen previously identified Pseudomonas species strains, were isolated from chicken. These strains were screened for biofilm formation and antibiotic resistance. In addition, we evaluated clove oil’s effectiveness against Pseudomonas isolates as an antibiofilm agent. The results showed that Pseudomonas species isolates were resistant to most antibiotics tested, particularly those from the β-lactamase family. A significant correlation (p < 0.05) between the development of multidrug-resistant isolates and biofilms is too informal. After amplifying the AmpC-plasmid-mediated genes (bla_CMY, bla_MIR, DHA, and FOX) and biofilm-related genes (psld, rhlA, and pelA) in most of our isolates, PCR confirmed this relationship. Clove oil has a potent antibiofilm effect against Pseudomonas isolates, and may provide a treatment for bacteria that form biofilms and are resistant to antimicrobials.