Molecular characterization of enterohemorrhagic Escherichia coli isolated from diarrhea samples from human, livestock, and ground beef in North Jordan.

BACKGROUND AND AIM: Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen with worldwide distribution. Data regarding its presence, distribution, virulence, and antimicrobial susceptibility among various animal species and humans in Jordan are lacking. Therefore, the objectives of this study were to isolate and characterize EHEC from human and animal diarrhea fecal samples and ground beef samples. MATERIALS AND METHODS: A total of 100 and 270 diarrhea fecal samples from humans and animals, respectively, were collected. In addition, 40 ground beef meat samples were collected from retail markets. EHEC was positively identified by detecting Shiga toxins (stx1 and stx2) genes using multiplex polymerase chain reaction (PCR). Antimicrobial susceptibility patterns were determined using the disk diffusion test. Beta-lactamase production was detected using the double disk diffusion test and the extended-spectrum beta-lactamases (ESBLs) were identified by detection of blaTEM , blaSHV , and OXA-1 genes using multiplex PCR. Pulsed-field gel electrophoresis (PFGE) was used to investigate the relatedness of EHEC isolates from different sources. RESULTS: Out of 410 samples, 194 E. coli isolates were positively identified, of which 57 isolates (29%) were classified as EHEC. Thirty-five (61%) of EHEC isolates were serotyped as O157 (19: O157:H7 and 16: O157:NM). The stx1 gene was detected only among the sheep and goats isolates at a rate of 7.6% and 5.2%, respectively, while the stx2 gene was detected in only one ground beef meat sample. EHEC isolates showed high resistance patterns against amoxicillin, gentamycin, cephalexin, and doxycycline. Twenty-four out of 32 EHEC isolates were determined as ESBL producers, among which 14 isolates expressed the blaSHV gene and 19 isolates expressed the blaTEM while four expressed both genes. PFGE analysis revealed two clusters with high similarity (92%) originated from ground beef meat and cattle fecal samples. No similarities were found between human and animal E. coli isolates. CONCLUSION: Results of this study indicate widespread ESBL EHEC among humans, animals, and ground beef meat samples. These results represent an important alarm that requires the implementation of appropriate preventative measures by both human and animal health sectors to prevent the transmission of this important foodborne pathogen.

Molecular characterization of Salmonella spp. isolates from river and dam water, irrigated vegetables, livestock, and poultry manures in Jordan.

BACKGROUND AND AIM: Salmonellosis is an important food-borne and zoonotic disease with high morbidity and mortality rates. The objectives of this study were to isolate, serotype, and genetically characterize Salmonella spp. from Zarqa river and King Talal dam waters, vegetables irrigated by such waters, and manure of poultry and livestock farms located in the Zarqa river basin in Jordan. In addition, certain virulence factors and antimicrobial resistance patterns of isolated Salmonella strains were determined. MATERIALS AND METHODS: A total of 250 samples were cultured using routine microbiological methods. Suspected Salmonella spp. were identified based on colony morphology and confirmed using biochemical and molecular methods. Virulence genes including invA, stn, and pCT plasmid were detected using multiplex PCR. Phylogenetic analysis was performed using pulsed-field gel electrophoresis (PFGE). RESULTS: In total, 32/250 (12.8%) Salmonella spp. isolates were recovered from different sources. Of these, the most common serotype was Salmonella subspecies 1 (23 isolates), followed by Salmonella enterica serovar Typhimurium (4 isolates), Salmonella enterica serovar Typhi (3 isolates), and finally Salmonella enterica serovar Enteritidis (2 isolates). The PFGE indicated that Salmonella enterica serovar Typhimurium isolated from poultry manure and from parsley were closely related (84.6%). Salmonella enterica serovar Enteritidis isolated from the dam water was closely related to Salmonella enterica serovar Enteritidis isolated from spearmint (73.8%). Salmonella enterica serovar Typhi isolated from the river and dam water were 100% related to Salmonella enterica serovar Typhi isolated from lettuce. In the antimicrobial sensitivity test, 14 out of 32 (43.8%) isolated Salmonella strains were resistant to two or more of the major antimicrobial agent groups. However, the majority of isolates were sensitive to ceftriaxone, ciprofloxacin, cefuroxime, and gentamicin (97%, 93.8%, and 87.5%, 84.4%, respectively). All isolates were resistant to erythromycin and amoxicillin. CONCLUSION: Results of this study indicate a serious potential threat to public health associated with consuming leafy green vegetables grown on the banks of Zarqa river and its dam because of widespread Salmonella spp. contamination. Appropriate monitoring of irrigation water must be applied to reduce the possibility of cross-contamination.

Antimicrobial susceptibility of multidrug-resistant Pseudomonas aeruginosa isolated from drinking water and hospitalized patients in Jordan.

Pseudomonas aeruginosa (P. aeruginosa) is an important environmental, opportunistic and nosocomial pathogen with a significant threat to public health. The objectives of this study were to determine the in vitro antimicrobial susceptibility patterns of, and antibiotic drug combinations with synergistic effects against P. aeruginosa isolated from drinking water and hospitalized patients in Jordan. A total of 16 P. aeruginosa isolates were obtained from hospitalized patients and 15 were isolated from bottled drinking water were used in the study. Bacterial isolation and identification was performed using routine microbiological methods and confirmed using PCR technique targeting the 16S rDNA gene. The antimicrobial susceptibility patterns were determined by measuring the minimum inhibitory concentration (MIC) using the 2-fold microdilution method. Synergy interaction between various antimicrobials was determined using the checkerboard method and fractional inhibitory concentration index (FICI). The majority of water isolates were sensitive to gentamicin (93.3%), ticarcillin (86.7%) and ciprofloxacin, levofloxacin, amikacin, colistin, piperacillin, azlocillin, aztreonam, ceftazidime and imipenem (100% each). All water isolates (100%) were resistant to amoxicillin, oxytetracycline and doxycycline (93.3% and 86.7, respectively). For the clinical isolates, all (100%) were sensitive to ceftazidime, 81.3% were sensitive to aztreonam, while 62.5% were sensitive to ciprofloxacin, levofloxacin, gentamicin, amikacin, colistin, piperacillin, ticracillin, azlocillin, and imipenem. All clinical isolates (100%) were resistant to oxytetracycline, doxycycline and amoxicillin. Analysis of the checkerboard synergy assay of multi-drug resistant isolates (n=26) showed significant synergism (P ≤ 0.05) when ciprofloxacin or gentamicin were included in the combination. There were no significant differences in synergistic activity between ciprofloxacin and levofloxacin when combined with other antimicrobial agents of the beta-lactams or aminoglycosides classes. There were no significant differences in the synergistic activities between beta lactams - aminoglycoside and beta lactams - fluoroquinolone combinations. Results of this study indicate an alarming widespread presence of multidrug-resistant P. aeruginosa associated with chronic suppurative infections in hospitalized patients and apparently clean drinking water in Jordan. Treatment of clinical suppurative lesions must be based on culture and in vitro susceptibility testing using potent antimicrobial combinations to avoid emergence of resistant strains and to improve the clinical outcome of treated patients.

Identification of Escherichia coli from broiler chickens in Jordan, their antimicrobial resistance, gene characterization and the associated risk factors.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is the principle cause of colibacillosis affecting poultry. The main challenge to the poultry industry is antimicrobial resistance and the emergence of multidrug resistant bacteria that threaten the safety of the food chain. Risk factors associated with emergence of antimicrobial resistance among avian pathogenic E. coli were correlated with the inappropriate use of antimicrobials along with inadequate hygienic practices, which encourages the selection pressure of antimicrobial resistant APEC. The aim of this study was to isolate, identify, serogroup and genotype APEC from broilers, assess their antibiotic resistance profile, expressed genes and the associated risk factors. RESULTS: APEC was isolated from the visceral organs of sick chickens with a prevalence of 53.4%. The most prevalent serotypes were O1, O2, O25 and O78, in percentage of 14.8, 12.6, 4.4 and 23.7%, respectively. Virulence Associated Genes; SitA, iss, iucD, iucC, astA, tsh cvi and irp2 were detected in rate of 97.4, 93.3, 75, 74, 71, 46.5, 39 and 34%, respectively and 186 (69.2%) isolates possess > 5-10 genes. The highest resistance was found against sulphamethoxazole-trimethoprim, florfenicol, amoxicillin, doxycycline and spectinomycin in percentage; 95.5, 93.7, 93.3, 92.2 and 92.2%, respectively. Sixty-eight percent of APEC isolates were found to have at least 5 out of 8 antimicrobial resistant genes. The most predominant genes were Int1 97%, tetA 78.4%, bla TEM 72.9%, Sul1 72.4%, Sul2 70.2%. Two risk factors were found to be associated with the presence of multi-drug resistant APEC in broiler chickens, with a P value ≤0.05; the use of ground water as source of drinking water and farms located in proximity to other farms. CONCLUSIONS: This study characterized the VAGs of avian pathogenic E. coli and establish their antimicrobial resistance patterns. The widespread of antimicrobial resistance of APEC isolates and detection of ARGs highlighted the need to monitor the spread of ARGs in poultry farms and the environment in Jordan. Use of ground water and closely located farms were significant risk factors associated with the presence of MDR APEC in broiler chickens in Jordan.

Herd- and individual-level prevalences of and risk factors for Salmonella spp. fecal shedding in dairy farms in Al-Dhulail Valley, Jordan.

Salmonellosis is an important disease frequently associated with diarrhea in calves. From January to September 2009, a cross-sectional study involving 91 dairy farms was conducted to determine the prevalence of Salmonella spp. infection in cattle in Al-Dhulail Valley, Jordan. A total of 910 calve and cow fecal samples were collected. Information on farm management practices was obtained through personal interviews using a standardized questionnaire and was tested as risk factors for Salmonella spp. positivity in farms by using logistic regression analysis. Standard conventional methods for Salmonella isolation and serotyping were used, and the disk agar diffusion test was used for antimicrobial testing. The herd-level prevalence of Salmonella spp. in calves, cows, and dairy farms was 12, 12, and 23 %, respectively, and the individual-level prevalence was 4 % for calves, cows, and dairy farms. Forty-six percent of the dairy farms had calf diarrhea, and 4 % had cow diarrhea. Seven (17 %) of the 42 farms with calf diarrhea had Salmonella. However, only 7 % (95 % CI: 4, 10) of the 221 diarrheic and 1 % (95 % CI: 0.2, 4) of the 234 of non-diarrheic calves had Salmonella. A total of 33 Salmonella isolates were obtained from the fecal samples: 12 isolates were Salmonella typhimurium, 6 were Salmonella montevideo, 6 were Salmonella anatum, 2 were Salmonella enteritidis, and 7 isolates were not serotyped. All isolates were susceptible to ciprofloxacin, trimethoprim-sulfamethoxazole, gentamycin, neomycin, colisitin, and amoxicillin at 100, 91, 85, 79, 79, and 70 %, respectively. Out of the 11 variables/categories, the frequency of cleaning every 2 months or more was associated with high odds of infection among calves (OR = 5.6) and farms (OR = 7.0).

Neutrophil gelatinase-associated lipocalin (NGAL) and insulin-like growth factor (IGF)-1 association with a Mannheimia haemolytica infection in sheep.

This study was aimed at mapping the tissue distribution of some inflammatory parameters associated with a Mannheimia haemolytica (M. haemolytica) infection in sheep. The M. haemolytica was isolated and characterized from the affected lungs of slaughtered animals. Cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, insulin-like growth factor (IGF)-1, as well as the acute-phase protein, neutrophil gelatinase-associated lipocalin (NGAL), were identified in the lung tissues, the serum, and the lymph nodes of M. haemolytica infected sheep, by enzyme-linked immunosorbent assay (ELISA). NGAL and IGF-1 pointed to an innate immune response, and epithelial cell repairing, respectively. The adaptive immune response was identified through the type of cytokines present in the affected sheep, as TNF-α represents the pro-inflammatory cytokines, and IL-10 represents the anti-inflammatory cytokines. M. haemolytica isolates were confirmed by polymerase chain reaction (PCR) and DNA sequences. There was a significant difference in the concentrations of NGAL, IGF-1, TNF-α, and IL-10, as observed in the affected sheep when compared to the healthy sheep. This study, for the first time, closely describes the distribution of some key and new inflammatory parameters in the tissue homogenate of affected lungs.

Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii.

Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30 min or 11.25 for 5 min), cold (4°C for 24h), heat (55°C for 5 min), and desiccation (cells were dried at 40°C for 2h and held at 21°C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB.