Antimicrobial resistance and virulence characterization of Staphylococcus aureus and coagulase-negative staphylococci from imported beef meat.

BACKGROUND: The objectives of this study were to characterize the diversity and magnitude of antimicrobial resistance among Staphylococcus species recovered from imported beef meat sold in the Egyptian market and the potential mechanisms underlying the antimicrobial resistance phenotypes including harboring of resistance genes (mecA, cfr, gyrA, gyrB, and grlA) and biofilm formation. RESULTS: The resistance gene mecA was detected in 50% of methicillin-resistant non-Staphylococcus aureus isolates (4/8). Interestingly, our results showed that: (i) resistance genes mecA, gyrA, gyrB, grlA, and cfr were absent in Staphylococcus hominis and Staphylococcus hemolyticus isolates, although S. hominis was phenotypically resistant to methicillin (MR-non-S. aureus) while S. hemolyticus was resistant to vancomycin only; (ii) S. aureus isolates did not carry the mecA gene (100%) and were phenotypically characterized as methicillin- susceptible S. aureus (MSS); and (iii) the resistance gene mecA was present in one isolate (1/3) of Staphylococcus lugdunensis that was phenotypically characterized as methicillin-susceptible non-S. aureus (MSNSA). CONCLUSIONS: Our findings highlight the potential risk for consumers, in the absence of actionable risk management information systems, of imported foods and advice a strict implementation of international standards by different venues such as CODEX to avoid the increase in prevalence of coagulase positive and coagulase negative Staphylococcus isolates and their antibiotic resistance genes in imported beef meat at the Egyptian market.

Characterization and susceptibility of streptococci and enterococci isolated from Nile tilapia (Oreochromis niloticus) showing septicaemia in aquaculture and wild sites in Egypt.

BACKGROUND: The present investigation was an endeavor into the elucidation of the disease-causing pathogen of streptococcosis in Nile tilapia (Oreochromis niloticus) in Egypt affecting adult fish cultured and wild fish in the Nile river. Fish were obtained from commercial fishermen, collected as part of their routine fishing activities. The researchers observed the routine fishing process and selected fish for use in the study, at the point of purchase from the fisherman. RESULTS: Diseased fish showed exophthalmia with accumulation of purulent and haemorrhagic fluid around eyes, and ventral petechial haemorrhages. The Post mortem examination revealed, abdominal fat haemorrhage, pericarditis and enlargement of the liver, spleen and kidney. Gram-stained smears revealed the presence of Gram-positive cocci, ß-hemolytic, oxidase and catalase negative. Analysis of the 16S rRNA gene confirmed that the 17 tilapia isolates studied were 6/17 Enterococcus faecalis, 2/17 Enterococcus gallinarum, 3/17 Streptococcus pluranimalium, 2/17 Aerococcus viridans, 1/17 isolate of each Streptococcus dysgalactiae, Streptococcus anginosus, Lactococcus garvieae and Granulicetella elegans/Leuconostoc mesenteroides cremoris. It should be noted that there was no mixed infection. Multiple resistance was observed and the most frequent antibiotic combination was penicillin, ampicillin, vancomycin, chloramphenicol, rifampicin, ofloxacin, clindamycin, erythromycin and tetracycline representing eight classes. CONCLUSIONS: Consequently, we concluded that Streptococcus species are an emerging pathogen for Nile tilapia aquaculture in Egypt and to be considered as a new candidate in the warm water fish diseases in Egypt with special reference to L. garvieae, S. dysgalactiae in addition to L. mesenteroides cremoris which was not reported before from tilapia and taking into consideration their zoonotic implications for public health.

Risk factors associated with E. coli causing neonatal calf diarrhea.

Calf diarrhea is one of the major health challenges in cattle herds. The bacteriological examination of fecal samples collected from apparently healthy and diarrheic calves' revealed isolation of 26 E. coli isolates out of 56 calves with an incidence of 46.4%. Serogroups O1, O26, O44, O55, O115, O119, O125, O146, and O151 were identified from the collected fecal samples. Using PCR all isolates was positive for ompA gene species specific for E. coli. While stx1 and eaeA genes detected with incidence of 3.8 and 19.2% respectively from the isolates. The presence of stx2 gene was negative in the fecal isolates. Among colostrum samples 4 E. coli isolates were detected and serogrouped to O26, O55 and O119. They were negative for eaeA, stx1 and stx2 except strain number 4 (O55) was positive for stx1. E. coli strains were sensitive to norfloxacin (80.7%) and resistant to ampicillin and cefotaxime (100% each). Based on our findings, there was no association between occurrence of E. coli and age of calf (2-14 days), while bottle feeding calf colostrum may be a source of E. coli contamination.

Urinary tract infection attributed to Escherichia coli isolated from participants attending an unorganized gathering.

AIM: Participants in an unorganized gathering are potential hosts of diseases, bringing diseases from around the world to be introduced to a large at-risk population. Therefore, we investigated the gene repertoire in 29 Escherichia coli strains linked to urinary tract infection isolated from patients transferred to the hospital after attending an unorganized gathering in Cairo. MATERIALS & METHODS: Virulence and resistance determinants, phenotypic antibiotic resistance, biofilm formation, their serotypes and phylogenetic relationships were analyzed. RESULTS: The 29 tested serovars were phenotypically virulent, with the prevalence of group B2, and resistant to tetracycline, naldixic acid, ampicillin, trimethoprim, neomycin, oxytetracycline and erythromycin encoding the iss virulent gene. CONCLUSION: A One Health approach is a must to monitor and control E. coli urinary tract infections.

An alternative approach for evaluating the phenotypic virulence factors of pathogenic Escherichia coli.

Escherichia coli is a recognized zoonotic food-borne pathogen; however, the use of polymerase chain reaction (PCR) in the underdeveloped countries to differentiate pathogenic from non-pathogenic E. coli is a problematic issue. Our grail was to assess the phenotypic virulence markers motility, hemolysin, congo red agar, embryo lethality assay and serum resistance for pathogenic E. coli (PEC) correlated to PCR tests which is currently used world-wide to evaluate the PEC. The 448 strains of Escherichia coli that were isolated from different sources, were characterized for phenotypic virulence factors such as motility, hemolysin, Congo red binding, Embryo Lethality assay (ELA) and serum resistance, as well as antibiotic susceptibility using disc diffusion method to 23 antibiotics. Results exhibited 100% motility and Congo red binding, 97.1% for hemolysin production and 90.2% in the ELA. As a result, we were able to hypothetically conclude that the aforementioned virulence markers are plain, straightforward, economical, rapid, more dynamic, uncomplicated methodology, duplicatable and cost next to nothing when compared to the molecular PCR. Their implementation in a diagnostic microbiology laboratory for vetting is a rewarding task in the underdeveloped countries. It augments endeavors to minimize the use of PCR in our investigations especially during epidemiological and outbreak investigations of PEC.

Poultry and beef meat as potential seedbeds for antimicrobial resistant enterotoxigenic Bacillus species: a materializing epidemiological and potential severe health hazard.

Although Bacillus cereus is of particular concern in food safety and public health, the role of other Bacillus species was overlooked. Therefore, we investigated the presence of eight enterotoxigenic genes, a hemolytic gene and phenotypic antibiotic resistance profiles of Bacillus species in retail meat samples. From 255 samples, 124 Bacillus isolates were recovered, 27 belonged to B. cereus and 97 were non-B. cereus species. Interestingly, the non-B. cereus isolates carried the virulence genes and exhibited phenotypic virulence characteristics as the B. cereus. However, correlation matrix analysis revealed the B. cereus group positively correlates with the presence of the genes hblA, hblC, and plc, and the detection of hemolysis (p < 0.05), while the other Bacillus sp. groups are negatively correlated. Tests for antimicrobial resistance against ten antibiotics revealed extensive drug and multi-drug resistant isolates. Statistical analyses didn't support a correlation of antibiotic resistance to tested virulence factors suggesting independence of these phenotypic markers and virulence genes. Of special interest was the isolation of Paenibacillus alvei and Geobacillus stearothermophilus from the imported meat samples being the first recorded. The isolation of non-B. cereus species carrying enterotoxigenic genes in meat within Egypt, suggests their impact on food safety and public health and should therefore not be minimised, posing an area that requires further research.

Poultry hatcheries as potential reservoirs for antimicrobial-resistant Escherichia coli: A risk to public health and food safety.

Hatcheries have the power to spread antimicrobial resistant (AMR) pathogens through the poultry value chain because of their central position in the poultry production chain. Currently, no information is available about the presence of AMR Escherichia coli strains and the antibiotic resistance genes (ARGs) they harbor within hatchezries. Therefore, this study aimed to investigate the possible involvement of hatcheries in harboring hemolytic AMR E. coli. Serotyping of the 65 isolated hemolytic E. coli revealed 15 serotypes with the ability to produce moderate biofilms, and shared susceptibility to cephradine and fosfomycin and resistance to spectinomycin. The most common ß-lactam resistance gene was blaTEM, followed by blaOXA-1, blaMOX-like, blaCIT-like, blaSHV and blaFOX. Hierarchical clustering of E. coli isolates based on their phenotypic and genotypic profiles revealed separation of the majority of isolates from hatchlings and the hatchery environments, suggesting that hatchling and environmental isolates may have different origins. The high frequency of ß-lactam resistance genes in AMR E. coli from chick hatchlings indicates that hatcheries may be a reservoir of AMR E. coli and can be a major contributor to the increased environmental burden of ARGs posing an eminent threat to poultry and human health.

Molecular characterization of the capsular antigens of Pasteurella multocida isolates using multiplex PCR.

The use of molecular techniques for detection and characterization of the Pasteurella multocida is very important for rapid and specific detection and characterization of the organism. During the period from 15th February, 2014 to 15th April, 2015, 425 nasopharyngeal swabs and 175 lung and spleen samples were collected and examined by conventional methods, 80 strains (18.82%) of P. multocida were isolated from the calves, sheep and goat with respiratory manifestation. Meanwhile, 77 strains (44%) were isolated from emergency slaughtered animals. All the recovered strains were positive for specific PCR for detection of P. multocida strains previously identified as P. multocida by standard microbiological techniques. Multiplex PCR for molecular typing of the capsular antigens of the recovered P. multocida revealed positive amplification of 1044 bp fragments specific to the capsular antigen type A with 105 strains (66.88%), and amplification 511 bp fragments of the capsular antigen type E with 52 strain (33.12%) and absence of B, D and F antigens. Multiplex PCR for molecular typing of the capsular antigens of P. multocida can be used as a simple, sensitive, rapid, reliable technique instead of the serological techniques for identification of the capsular antigens of P. multocida.

Comparative studies for serodiagnosis of haemorrhagic septicaemia in cattle sera.

Haemorrhagic septicaemia caused by Pasteurella multocida is a major epizootic disease in cattle and buffaloes in developing countries with high morbidity and mortality rate. In the present study, a total of 88 P. multocida isolates were isolated from 256 nasopharyngeal swabs and lung tissues samples (34.4%) during the period from January, 2013 to March, 2014 from different governorates located in Egypt. Dead calves showed the highest percentage of P. multocida isolation followed by the emergency slaughtered calves, diseased calves then apparently healthy ones. These isolates were confirmed as P. multocida microscopically, biochemically by traditional tests and by API 20E commercial kit then by PCR. The percentages of positive serum samples using somatic antigen and micro-agglutination test at 1/1280 diluted serum were 10%, 54.49% and 0% in apparently healthy, diseased and emergency slaughtered samples, respectively whereas, the percentages using capsular antigen and indirect haemagglutination test were 40%, 60.89% and 60% in apparently healthy, diseased and emergency slaughtered samples, respectively. The ELISA showed the highest sensitivity for diagnosing P. multocida in apparently healthy, diseased and emergency slaughtered animals with percentages of 42%; 92.9% and 80%, respectively. The obtained results revealed that the ELISA using capsular antigen of P. multocida is a more sensitive and specific serological test for diagnosis of haemorrhagic septicaemia.

Phenotypic and genotypic analysis of pathogenic Escherichia coli virulence genes recovered from Riyadh, Saudi Arabia.

The current study was carried out to evaluate the phenotypic and genotypic characterization of avian pathogenic Escherichia coli recovered from Riyadh, Saudi Arabia. During the period of 10th February-30th May 2015, 70 E. coli strains were isolated from chicken farms located in Riyadh, Saudi Arabia. All strains were tested phenotypically by standard microbiological techniques, serotyped and the virulence genes of such strains were detected by polymerase chain reaction (PCR). Most of the recovered strains from chickens belonged to serotype O111:K58 25 strains (35.7%), followed by serotype O157:H7 13 strains (18.57%), followed by serotype O114:K90 10 strains (14.29%), then serotype O126:K71 9 strains (12.9%), serotype O78:K80 8 strains (11.43%) and in lower percentage serotype O114:K90 and O119:K69 5 strains (7.14%). The virulence genotyping of E. coli isolates recovered from broilers revealed the presence of the uidA gene in all the field isolates (6 serovars) examined in an incidence of 100%, as well as the cvaC gene was also present in all field isolates (6 serovars), while the iutA gene and the iss gene were detected in 5 out of 6 field serovars in an incidence of 81.43% and 64.29%, respectively. Phenotypical examination of the other virulence factors revealed that 65 isolates were hemolytic (92.9%), as well as 15 isolates (21.42%) were positive for enterotoxin production. Meanwhile, 21 isolates (30%) were positive for verotoxin production, 58 isolates (82.86%) for the invasiveness and 31 isolates (44.29%) for Congo red binding activities of the examined serotypes.

Vaccination against Corynebacterium pseudotuberculosis infections controlling caseous lymphadenitis (CLA) and oedematousskin disease.

Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a causative organism of caseous lymphadenitis (CLA) in sheep and acute disease in buffaloes known as oedematous skin disease (OSD). Human affected with the disease show liver abscess and abscess in the internal lymph nodes. The vaccination against CLA up till now occurs by using formalin inactivated whole cells of biovar 1 (sheep strain). Combined vaccine composed of formalin inactivated whole cells of sheep strain and recombinant phospholipase D (rPLD) and another vaccine composed of formalin inactivated whole cells (buffalo origin) and rPLD were prepared in Biotechnology center for services and Researches laboratory at Cairo university and applied for protection against CLA. Both vaccines induced complete protection (100%) against challenge with virulent biovar 1 or biovar 2. Also vaccination against OSD was performed by two types of vaccines. Vaccine-1 was composed of formalin inactivated whole cell biovar 1 combined with rPLD and the second vaccine was composed of formalin inactivated whole cells of biovar 2 combined with rPLD. No lesions developed in vaccinated and non vaccinated buffaloes challenged with C. pseudotuberculosis biovar revealing that biovar 1 C. pseudotuberculosis is not infective for buffaloes. Buffaloes vaccinated with the second vaccine and control non vaccinated animals challenged with biovar 2 (buffalo origin) resulted in development of OSD in all animals. This indicates that OSD results due to production of toxin (s) other than PLD. Discovering this toxin (s) is of value in formulation of a future vaccine against OSD.

Prevalence of the Antibiotic Resistance Genes in Coagulase-Positive-and Negative-Staphylococcus in Chicken Meat Retailed to Consumers.

The use of antibiotics in farm management (growing crops and raising animals) has become a major area of concern. Its implications is the consequent emergence of antibiotic resistant bacteria (ARB) and accordingly their access into the human food chain with passage of antibiotic resistance genes (ARG) to the normal human intestinal microbiota and hence to other pathogenic bacteria causative human disease. Therefore, we pursued in this study to unravel the frequency and the quinolone resistance determining region, mecA and cfr genes of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), methicillin-resistant coagulase-negative staphylococci (MRCNS) and methicillin-susceptible coagulase-negative staphylococci (MSCNS) isolated from the retail trade of ready-to-eat raw chicken meat samples collected during 1 year and sold across the Great Cairo area. The 50 Staphylococcus isolated from retail raw chicken meat were analyzed for their antibiotic resistance phenotypic profile on 12 antibiotics (penicillin, oxacillin, methicillin, ampicillin-sulbactam, erythromycin, tetracycline, clindamycin, gentamicin, ciprofloxacin, chloramphenicol, sulfamethoxazole-trimethoprim, and vancomycin) and their endorsement of the quinolone resistance determining region, mecA and cfr genes. The isolation results revealed 50 isolates, CPS (14) and CNS (36), representing ten species (S. aureus, S. hyicus, S. epidermedius, S. lugdunensis, S. haemolyticus, S. hominus, S. schleiferi, S. cohnii, S. intermedius, and S. lentus). Twenty seven isolates were methicillin-resistant. Out of the characterized 50 staphylococcal isolates, three were MRSA but only 2/3 carried the mecA gene. The ARG that bestows resistance to quinolones, ß-lactams, macrolides, lincosamides, and streptogramin B [MLS(B)] in MRSA and MR-CNS were perceived. According to the available literature, the present investigation was a unique endeavor into the identification of the quinolone-resistance-determining-regions, the identification of MRSA and MR-CNS from retail chicken meat in Egypt. In addition, these isolates might indicate the promulgation of methicillin, oxacillin and vancomycin resistance in the community and imply food safety hazards.

Molecular characterization of Escherichia coli O157:H7 recovered from meat and meat products relevant to human health in Riyadh, Saudi Arabia.

Raw meat can harbor pathogenic bacteria, potentially harmful to humans such as Escherichia coli O157:H7 causing diarrhea and hemolytic-uremic syndrome (HS). Therefore, the current study was carried out to evaluate the prevalence and the molecular detection characterization of E. coli serotype O157:H7 recovered from raw meat and meat products collected from Saudi Arabia. During the period of 25th January 2013 to 25th March 2014, 370 meat samples were collected from abattoirs and markets located in Riyadh, Saudi Arabia "200 raw meat samples and 170 meat products". Bacteriological analysis of the meat samples and serotyping of the isolated E. coli revealed the isolation of 11 (2.97%) strains of E. coli O157:H7. Isolation of E. coli O157:H7 in raw beef, chicken and mutton were 2%, 2.5%, and 2.5%, respectively, however, there was no occurrence in raw turkey. The incidences of E. coli O157:H7 in ground beef, beef burgers, beef sausage, ground chicken and chicken burgers were 5%, 10%, 0.0%, 5% and 0.0%, respectively. The multiplex PCR assay revealed that 3 (27.27%) out of 11 E. coli O157:H7 isolates from raw beef, chicken and mutton had stx1, stx2, and eae while 5 (45.45%) E. coli O157:H7 isolates from ground beef, ground chicken, and raw beef had both stx1 and stx2. However, from beef burgers, only one E. coli O157:H7 isolate had stx1 while two were positive for hlyA gene. These results call for urgent attention toward appropriate controls and good hygienic practices in dealing with raw meat.

Molecular and serotyping characterization of shiga toxogenic Escherichia coli associated with food collected from Saudi Arabia.

Shiga toxin-producing Escherichia coli (STEC) strains are considered as one of the major food-borne disease agents in humans worldwide. STEC strains, also called verotoxin-producing E. coli strains. The objectives of the present study were serotyping and molecular characterization of shiga toxigenic E. coli associated with raw meat and milk samples collected from Riyadh, Saudi Arabia. A total of 540 milk samples were collected from 5 dairy farms and 150 raw meat samples were collected from different abattoirs located in Riyadh, Saudi Arabia. E. coli were recovered from 86 milk samples (15.93%), serotyping of E. coli isolates revealed, 26 (4.81%) strains O157: H7, 23 (4.26%) strains O111, 20 (3.70%) strains O113: H21, 10 (1.85%) strains O22: H8 and 7 (1.3%) strains O172: H21. Meanwhile, 17 (11.33%) strains of E. coli were recovered from raw meat samples, serotyping of E. coli isolates revealed, 6 (4%) strains O157: H7, 5 (3.33%) strains O111 and 4 (2.67%) strains O174: H2 and only two (1.33%) strains were identified as O22: H8. Shiga toxin2 was detected in 58 (67.44%) serotypes of E. coli recovered from milk samples and 16 (94.12%) serotypes of E. coli recovered from meat samples, while intimin gene was detected in 38 (44.186%) serotypes of E. coli recovered from milk samples and in 10 (58.82%) serotypes of E. coli recovered from meat samples. The results of this study revealed the efficiency of combination between serotyping and molecular typing of E. coli isolates recovered from food of animal origin for rapid detection and characterization of STEC.