Epidemiological study on Listeria monocytogenes in Egyptian dairy cattle farms' insights into genetic diversity of multi-antibiotic-resistant strains by ERIC-PCR.

Listeria monocytogenes (L. monocytogenes) is frequently detected in ruminants, especially dairy cattle, and associated with the sporadic and epidemic outbreak of listeriosis in farms. In this epidemiological study, the prevalence, virulence, antibiotic resistance profiles, and genetic diversity of L. monocytogenes in three Egyptian dairy cattle farms were investigated. The risk factors associated with the fecal shedding of L. monocytogenes were analyzed. The L. monocytogenes strains from the three farms were categorized into distinct genotypes based on sampling site and sample type through enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). A total of 1896 samples were collected from animals, environments, and milking equipment in the three farms. Results revealed that 137 (7.23%) of these samples were L. monocytogenes positive. The prevalence of L. monocytogenes in the animal samples was high (32.1%), and the main environmental source of prevalent genotypes in the three farms was silage. For all sample types, L. monocytogenes was more prevalent in farm I than in farms II and III. Risk factor analysis showed seasonal variation in production hygiene. For all sample types, L. monocytogenes was significantly more prevalent in winter than in spring and summer. The level of L. monocytogenes fecal shedding was high likely because of increasing age, number of parities, and milk yield in dairy cattle. Two virulence genes, namely, hlyA & prfA, were also detected in 93 strains, whereas only one of these genes was found in 44 residual strains. Conversely, iap was completely absent in all strains. The strains exhibited phenotypic resistance to most of the tested antibiotics, but none of them was resistant to netilmicin or vancomycin. According to sample type, the strains from the animal samples were extremely resistant to amoxicillin (95.2%, 80/84) and cloxacillin (92.9%, 78/84). By comparison, the strains from the environmental samples were highly resistant to cefotaxime (86.95%, 20/23). Furthermore, 25 multi-antibiotic resistance (MAR) patterns were observed in L. monocytogenes strains. All strains had a MAR index of 0.22-0.78 and harbored antibiotic resistance genes, including extended-spectrum ß-lactamase (blaCTX-M [92.7%] and blaDHA-1 [66.4%]), quinolones (qnrS [91.2%], qnrA [58.4%], parC [58.4%], and qnrB [51%]), macrolides (erm[B] [76.6%], erm(C) [1.5%], and msr(A) [27%]), trimethoprim (dfrD [65.7%]), and tetracyclines (tet(M) [41.6%], tet(S) [8%], and int-Tn [26.3%]). ERIC-PCR confirmed that the strains were genetically diverse and heterogeneous. A total of 137 isolated L. monocytogenes strains were classified into 22 distinct ERIC-PCR groups (A-V). Among them, ERIC E (10.2%) was the most prevalent group. These results indicated that environment and milking equipment served as reservoirs and potential transmission ways of virulent and multidrug-resistant L. monocytogenes to dairy animals, consequently posing threats to public health. Silage is the main environmental source of prevalent genotypes on all three farms. Therefore, hygienic measures at the farm level should be developed and implemented to reduce L. monocytogenes transmission inside dairy cattle farms.

Enhanced Antibacterial Activity of Silver Nanoparticles Combined with Hydrogen Peroxide Against Multidrug-Resistant Pathogens Isolated from Dairy Farms and Beef Slaughterhouses in Egypt.

PURPOSE: The last few decades have witnessed a rapid and global increase in multidrug-resistant bacteria (MDR) emergence. METHODS: The aim of the current study is to isolate the most common MDR bacteria from dairy farms and beef slaughterhouses followed by evaluation of their antimicrobial resistance pattern and assessment of the antibacterial activity of AgNPs-H2O2 as an alternative to conventional antibiotics. In this regard, 200 samples were collected from two dairy farms and one beef slaughterhouse located in Dakhliya Governorate, Egypt. RESULTS: Interestingly, out of 120 collected samples from dairy farms, the prevalence of the isolated strains was 26.7, 23.3, 21.7, 16.7, and 11.7% for S. typhimurium, E. coli O157:H7, L. monocytogenes, K. pneumoniae and P. aeruginosa, respectively. Meanwhile, the overall prevalence was 30, 25, 22.5, 17.5, and 5% for E. coli O157:H7, L. monocytogenes, S. typhimurium, P. aeruginosa, and K. pneumoniae, respectively, for the 80 samples collected from a beef slaughterhouse. The antimicrobial susceptibility pattern elucidated that all isolated strains exhibited resistance to at least four of the tested antimicrobials, with multiple-antibiotic resistance index values (MAR) ranging between 0.44 and 0.88. Furthermore, the commercial AgNPs-H2O2 product was characterized by transmission electron microscopy (TEM) and zeta potential that showed spherical particles with a surface charge of -0.192 mV. The antimicrobial activity of synergized nano-silver (AgNP) with H2O2 product toward MDR strains was assessed via measuring minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill curve. CONCLUSION: The present data report high prevalence rates of MDR pathogens in dairy farms and abattoirs. More importantly, AgNPs-H2O2 exerted broad-spectrum bactericidal activity toward MDR bacterial strains, suggesting their promising usage as safe, ecofriendly, cost-effective antibacterial agents. To our knowledge, this study is a pioneer in investigating the potential alternative antimicrobial role of silver nanoparticles for control of multiple drug-resistant pathogens in Egypt.

Novel eradication methods for Staphylococcus aureus biofilm in poultry farms and abattoirs using disinfectants loaded onto silver and copper nanoparticles.

Recent developments in the nanotechnology field have created opportunities to design new biomaterials for Staphylococcus aureus biofilm eradication. These biomaterials including disinfectant-loaded nanoparticles could overcome the limitations of conventional disinfectants. The objective of this study was to assess the biocidal activity of five commercial disinfectants (DC&R®, VirkonS®, TH4++, Tek-Trol, and peracetic acid) alone and as with silver and copper nanocomposites on S. aureus biofilm at different concentrations and exposure times. Consequently, 227 samples were collected from two broiler farms, two-layer farms, and three abattoirs at El-Dakahlia Province, Egypt, during summer 2018. The samples were collected from birds as well as the surrounding environment. S. aureus strains were isolated and biofilm producers were phenotypically evaluated by Congo red agar (CRA) test. Besides, 4 biofilm-associated genes including bap, fnbA, cna, and ebps were genotypically detected by PCR technology. Out of 227 collected samples, 141 (62.1%) strains were identified as S. aureus, while 127 strains (90.1%) were S. aureus biofilm producers for all examined samples except for hand swabs of abattoir workers. The prevalence of fnbA and bap genes was 79.5% (101/127) and 20.5% (26/127), respectively but, no strains harbored cna or ebps genes. Tested nanocomposites were prepared using an aqueous solution of metal salts such as copper sulfate and silver nitrate and added to the same amount of disinfectant solution. The obtained nanocomposites were characterized by transmission electron microscopy (TEM) and zeta potential which showed spherical and elongated particles and with a surface charge of disinfectants-silver and copper nanocomposites-of 2.92 and 3.43 mV, respectively. Complete eradication of S. aureus biofilm was observed after treatment with disinfectants loaded onto silver (AgNPs) and copper (CuNPs) nanoparticles in varying concentrations as well as at different exposure times in comparing to disinfectants alone. Our results exhibited the potential applications of disinfectant nanocomposites in complete eradication of S. aureus biofilm in farms and abattoirs without developing of disinfectant resistant bacteria.

Antimicrobial resistance profiles and virulence genotyping of Salmonella enterica serovars recovered from broiler chickens and chicken carcasses in Egypt.

BACKGROUND: This study aimed to survey the prevalence, antimicrobial resistance, and virulence-associated genes of Salmonella enterica recovered from broiler chickens and retail shops at El-Sharkia Province in Egypt. Salmonella virulence factors were determined using the polymerase chain reaction assays targeting the invA, csgD, hilC, bcfC, stn, avrA, mgtC, ompF, sopE1 and pefA genes. RESULTS: One hundred tweenty out of 420- samples from broiler chickens' cloacal swabs, farm environmental samples, and freshly dressed whole chicken carcasses were positive Salmonella species. The isolates were serotyped as S. Enteritidis as the most dominant serotypes. Interestingly, none of the isolates were resistant to imipenem. The multidrug resistance was determined in 76.7% of the isolates with multidrug antibiotic resistance index of 0.2-0.6. Eight virulence genes (invA, csgD, hilC, stn, bcfC, mgtC, avrA, and ompf) were characterized among 120 S. enterica isolates with variable frequencies, while sopE1and pefA genes that were completely absent in all isolates. Based on the combination of presence and absence of virulence genes, the most common genetic profile (P7, 30%) was invA and csgD genes. CONCLUSION: S. Enteritidis and S. Typhimurium were the most common identified serotypes in the examined sources. Circulation of such strains in broiler farms required introducing special biosecurity and biocontrol measures for control of Salmonella. Such measures might limit the adverse effects of antibiotics and ensure the safety of the environment and animal-derived food.