Dissemination of mcr-1 and ß-lactamase genes among Pseudomonas aeruginosa: molecular characterization of MDR strains in broiler chicks and dead-in-shell chicks infections.

OBJECTIVES: Pseudomonas aeruginosa (P. aeruginosa) is one of the most serious pathogens implicated in antimicrobial resistance, and it has been identified as an ESKAPE along with other extremely significant multidrug resistance pathogens. The present study was carried out to explore prevalence, antibiotic susceptibility phenotypes, virulence-associated genes, integron (int1), colistin (mcr-1), and ß-lactamase resistance' genes (ESBls), as well as biofilm profiling of P. aeruginosa isolated from broiler chicks and dead in-shell chicks. DESIGN: A total of 300 samples from broiler chicks (n = 200) and dead in-shell chicks (n = 100) collected from different farms and hatcheries located at Mansoura, Dakahlia Governorate, Egypt were included in this study. Bacteriological examination was performed by cultivation of the samples on the surface of both Cetrimide and MacConkey's agar. Presumptive colonies were then subjected to biochemical tests and Polymerase Chain Reaction (PCR) targeting 16S rRNA. The recovered isolates were tested for the presence of three selected virulence-associated genes (lasB, toxA, and exoS). Furthermore, the retrieved isolates were subjected to phenotypic antimicrobial susceptibility testing by Kirby-Bauer disc diffusion method as well as phenotypic detection of ESBLs by both Double Disc Synergy Test (DDST) and the Phenotypic Confirmatory Disc Diffusion Test (PCDDT). P. aeruginosa isolates were then tested for the presence of antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, OXA-2, VEB-1, SHV, TEM, and CTX-M). Additionally, biofilm production was examined by the Tube Adherent method (TA) and Microtiter Plate assay (MTP). RESULTS: Fifty -five isolates were confirmed to be P. aeruginosa, including 35 isolates from broiler chicks and 20 isolates from dead in-shell chicks. The three tested virulence genes (lasB, toxA, and exoS) were detected in all isolates. Antibiogram results showed complete resistance against penicillin, amoxicillin, ceftriaxone, ceftazidime, streptomycin, erythromycin, spectinomycin, and doxycycline, while a higher sensitivity was observed against meropenem, imipenem, colistin sulfate, ciprofloxacin, and gentamicin. ESBL production was confirmed in 12 (21.8%) and 15 (27.3%) isolates by DDST and PCDDT, respectively. Antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, SHV, TEM, and CTX-M), were detected in 87.3%, 18.2%, 16.4%, 69.1%, 72.7%, and 54.5% of the examined isolates respectively, whereas no isolate harbored the OXA-2 or VEB-1 genes. Based on the results of both methods used for detection of biofilm formation, Kappa statistics [kappa 0.324] revealed a poor agreement between both methods. CONCLUSIONS: the emergence of mcr-1 and its coexistence with other resistance genes such as ß-lactamase genes, particularly blaOXA-10, for the first time in P. aeruginosa from young broiler chicks and dead in-shell chicks in Egypt pose a risk not only to the poultry industry but also to public health.

Prevalence and characterization of quinolone-resistant Escherichia coli isolated from retail raw beef and poultry meat in Egypt.

Objective: The goal of this study was to look at quinolone-resistant (QR) Escherichia coli (E. coli) from retail beef and poultry meat in Egypt by looking at the QR mechanisms in the resistant strains. Materials and Methods: In total, 120 samples of raw poultry meat (n = 60) and beef meat (n = 60) were purchased from Mansoura retail stores between January and March 2021, and evaluated microbiologically for E. coli. Then, an antimicrobial sensitivity test was applied to all isolates. The prevalence of QR E. coli with concern for the QR determinants, including quinolone resistance-determining regions (QRDRs) mutations, the plasmid-mediated quinolone resistance gene (PMQR), and the efflux pump activity were determined. Results: The total prevalence of E. coli was 34.2% (41/120). Noticeably, the prevalence of E. coli in poultry meat (40%, 24/60) was higher than that of beef (28%, 17/60). All strains were assessed for their antimicrobial susceptibility using the disc diffusion technique; the highest rate of resistance (100%) was displayed to clindamycin and cefuroxime, followed by ampicillin (97.6%), doxycycline (92.7%), amoxicillin-clavulanate (92.7%), nalidixic acid (NA) (80.5%), sulfamethoxazole/trimethoprim (70.7%), chloramphenicol (63.4%), gentamicin, and azithromycin (58.5% each). Multiple antimicrobial resistance (strains resistant to three or more antimicrobial classes) was displayed by 97.6% of E. coli isolates. Regarding QR, 37 isolates could resist at least one of the examined quinolones. Regarding PMQR genes, qnrS was determined in 70% (7/10) of QR E. coli, while qnrA, qnrB, and qnrD were not identified. While the mutations determined regions of QR in the resistant E. coli isolates, S83L was the most prevalent in gyrase subunit A either alone or combined with D87N and D87Y, and three isolates of QR E. coli isolates revealed a topoisomerase IV subunit mutation harboring S80I. 20% of the isolates displayed efflux activity, as NA showed a considerable difference between its zones of inhibition. Conclusion: The high prevalence of antimicrobial-resistant E. coli, with concern for QR strains harboring different resistance mechanisms in poultry meat and beef, threatens the public's health. Thus, standard manufacturing procedures and adequate hygiene conditions must be followed in all phases of meat preparation, production, and consumption, and public knowledge should be improved.

Genotypic characterization, antimicrobial susceptibility and virulence determinants of Campylobacter jejuni and Campylobacter coli isolated from pastured poultry farms.

Aim: Campylobacter is the leading bacterial pathogen that causes foodborne illnesses worldwide. Pasture farming is regarded as an important source of agricultural production for small farming communities. Consumer preference for pasture-raised animal products has increased; however, there is a paucity of information on the microbiological quality of pasture-raised poultry products. The purpose of this study was to explore genetic relatedness of thermophilic Campylobacter isolates, to assess antibiotic resistance phenotypically and genotypically, and to screen the presence of virulence determinants of Campylobacter isolates from pasture-raised poultry farms from southeastern United States. Methods: Ninety-seven Campylobacter isolates previously identified by Q7 BAX® System Real-Time PCR were genotyped by multilocus sequence typing (MLST). Campylobacter isolates were then evaluated for their phenotypic antimicrobial susceptibility against nine antimicrobial agents using Sensititre plates. Additionally, Campylobacter isolates were tested for the presence of antimicrobial resistance-associated elements. Furthermore, Campylobacter isolates were screened for the presence of 13 genes encoding putative virulence factors by PCR. These included genes involved in motility (flaA and flhA), adhesion and colonization (cadF, docC, racR, and virB11), toxin production (cdtA, cdtB, cdtC, wlaN, and ceuE) and invasion (ciaB and iamA). Results: Among 97 Campylobacter isolates, Campylobacter jejuni (n = 79) and Campylobacter coli (n = 18) were identified. By MLST, C. jejuni isolates were assigned to seven clonal complexes. Among them, ST-353, ST-607 and ST-21 were the most common STs recognized. All C. coli (n = 18) isolates were included in CC-828. Interestingly, eight STs identified were not belonging any previous identified clonal complex. Campylobacter isolates displayed a high resistance rate against tetracycline (81.4%), while a low rate of resistance was observed against macrolides (azithromycin and erythromycin), quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin), aminoglycosides (gentamicin), ketolide (telithromycin), amphenicol (florfenicol) and lincomycin (clindamycin). Thirteen isolates (13.54%) were pan-susceptible to all tested antibiotics, while nine isolates were multi-antimicrobial resistant (MAR; resist to three or more antimicrobial classes). Interestingly, there were no isolates resistant to all antimicrobial classes. Thr86Ile mutation was identified in all quinolones resistant strains. Erythromycin encoding gene (ermB) was identified in 75% of erythromycin resistant isolates. The A2075 mutation was detected in one erythromycin resistant strain, while A2074 could not be identified. The tetO gene was identified in 93.7% of tetracycline resistant isolates and six tetracycline susceptible isolates. In conclusion, the results of this study revealed that Campylobacter isolates from pasture-raised poultry farms showed the ST relatedness to Campylobacter isolates commonly associated with humans, indicating pasture-raised broiler flocks, similar to conventionally-reared broiler flocks, as a potential vector for antibiotic-resistant and pathogenic strains of thermophilic Campylobacter to humans.

Effects of dietary nano-selenium supplementation on Riemerella anatipestifer vaccinated and challenged Pekin ducklings (Anas platyrhynchos).

Riemerella anatipestifer (RA) is a common disease causing economic losses to duck farms worldwide. Novel supplements are crucially needed to control this bacterium, enhance poultry performance, and produce synergistic effects with vaccines in stimulating the immune system. This study investigated the effect of nano-selenium (Nano-Se) on the vaccinated (VAC) and challenged (Ch) Pekin ducklings (Anas platyrhynchos) with RA. Five experimental groups (G1-G5) were included in this study: G1 was the control group, G2 was the RA-challenged group, G3 was the Nano-Se+Ch group, G4 was the VAC+Ch group, and G5 was the Nano-Se+VAC+Ch group. The Nano-Se (0.3 mg/kg diet) was supplemented for 5 weeks post-vaccination (PV). The ducklings were vaccinated subcutaneously with the RA vaccine at 7 days of age and challenged with RA at the 3rd week PV. Blood, pharyngeal swabs and tissue samples were collected at the 3rd week PV and at different times post-challenge (PC). The growth performance (weight gain and feed conversion ratio), clinical signs, gross lesions, mortality, bacterial shedding, haematological, immunological, and biochemical parameters, cytokines production, and histopathological lesion scores showed significant differences (P < 0.05) between the challenged (G2) group and the supplemented (G3 & G5) groups. G5 showed the highest (P < 0.05) growth performance, phagocytic activity, IgM and IgG, splenic interleukin-2 (IL-2), IL-10, and interferon-gamma (IFN-γ) gene expressions, and the lowest mortality, bacterial shedding, hepatic and renal damage, heterophil/lymphocyte ratio and lesion scores compared to the other groups. In conclusion, the supplementation of nano-selenium for five weeks in the diet can improve the growth performance, immune status, and cytokines production in ducklings vaccinated and challenged with RA.

Immunological and Oxidative Biomarkers in Bovine Serum from Healthy, Clinical, and Sub-Clinical Mastitis Caused by Escherichia coli and Staphylococcus aureus Infection.

The study aimed to investigate the mastitis' emerging causative agents and their antimicrobial sensitivity, in addition to the hematological, biochemical indicators, oxidative biomarkers, acute phase protein (APP), and inflammatory cytokine changes in dairy farms in Gamasa, Dakahlia Governorate, Egypt. One hundred Holstein Friesian dairy cattle with clinical and subclinical mastitis were investigated and were allocated into three groups based on a thorough clinical examination. Escherichia coli and Staphylococcus aureus were found responsible for the clinical and subclinical mastitis in dairy farms, respectively. Multiple drug resistance (MDR) was detected in 100%, and 94.74% of E. coli and S. aureus isolates, respectively. Significantly low RBCs count, Hb, and PCV values were detected in mastitic cows compared with both subclinical mastitic and control groups; moreover, WBCs, lymphocytes, and neutrophil counts were significantly diminished in mastitic cows compared to the controls. Significantly higher levels of AST, LDH, total protein, and globulin were noticed in both mastitic and subclinical mastitic cows. The haptoglobin, fibrinogen, amyloid A, ceruloplasmin, TNF-α, IL-1ß, and IL-6 levels were statistically increased in mastitic cows compared to the controls. Higher MDA levels and reduction of TAC and catalase were identified in all the mastitic cases compared to the controls. Overall, the findings suggested potential public health hazards due to antimicrobial resistance emergence. Meanwhile, the APP and cytokines, along with antioxidant markers can be used as early indicators of mastitis.

Workplace violence at emergency departments, Ain Shams University Hospitals, Cairo, Egypt.

BACKGROUND: The present study aimed to determine the prevalence and forms of workplace violence (WPV) at the emergency departments (EDs) of Ain Shams University Hospitals (ASUH), Cairo and identify risk factors for WPV. METHODS: A cross-sectional study was conducted at the EDs of ASUH comprising attending physicians and nurses using a self-administered structured questionnaire. Interviews were conducted with patients and relatives attending these departments to explore attitudes toward WPV against healthcare workers. RESULTS: The present study comprised 108 healthcare professionals working in EDs. Verbal violence was the most common type of WPV (86.1%), followed by sexual (48.1%) and physical violence (34.3%). Patient relatives were the most common perpetrator of all types of violence. A lack of facilities was the most common risk factor for violence (82.4%), followed by overcrowding (50.9%) and patient culture (47.2%). On the other hand, approximately 78% of interviewed patients and relatives agreed that the occurrence of violence at EDs was due to several triggering factors, including improper manner of communication by healthcare workers (63.2%), lack of facilities (32.4%), waiting time (22.1%), and unmet expectations (22.1%). CONCLUSION: WPV represents a significant issue in EDs with violent behavior against healthcare workers widely accepted by attending patients.

Phylotypic Profiling, Distribution of Pathogenicity Island Markers, and Antimicrobial Susceptibility of Escherichia coli Isolated from Retail Chicken Meat and Humans.

Escherichia coli (E.coli) found in retail chicken meat could be causing a wide range of infections in humans and constitute a potential risk. This study aimed to evaluate 60 E. coli isolates from retail chicken meat (n = 34) and human urinary tract infections (UTIs, n = 26) for phylogenetic diversity, presence of pathogenicity island (PAI) markers, antimicrobial susceptibility phenotypes, and antimicrobial resistance genes, and to evaluate their biofilm formation capacity. In that context, confirmed E.coli isolates were subjected to phylogrouping analysis using triplex PCR, antimicrobial susceptibility testing using the Kirby-Bauer disc diffusion method; PAI distribution was investigated by using two multiplex PCRs. Most of the chicken isolates (22/34, 64.7%) were identified as commensal E. coli (A and B1), while 12 isolates (35.3%) were classified as pathogenic virulent E. coli (B2 and D). Similarly, the commensal group dominated in human isolates. Overall, 23 PAIs were detected in the chicken isolates; among them, 39.1% (9/23) were assigned to group B1, 34.8% (8/23) to group A, 4.34% (1/23) to group B2, and 21.7% (5/23) to group D. However, 25 PAIs were identified from the human isolates. PAI IV536 was the most prevalent (55.9%, 69.2%) PAI detected in both sources. In total, 37 (61.7%) isolates of the chicken and human isolates were biofilm producers. Noticeably, 100% of E. coli isolates were resistant to penicillin and rifamycin. Markedly, all E. coli isolates displayed multiple antibiotic resistance (MAR) phenotypes, and the multiple antibiotic resistance index (MARI) among E. coli isolates ranged between 0.5 and 1. Several antibiotic resistance genes (ARGs) were identified by a PCR assay; the sul2 gene was the most prevalent (38/60, 63.3%) from both sources. Interestingly, a significant positive association (r = 0.31) between biofilm production and resistance to quinolones by the qnr gene was found by the correlation analysis. These findings were suggestive of the transmission of PAI markers and antibiotic resistance genes from poultry to humans or humans to humans through the food chain. To avoid the spread of virulent and multidrug-resistant E. coli, intensive surveillance of retail chicken meat markets is required.

Genetic Characterization and Antimicrobial-Resistant Profiles of Staphylococcus aureus Isolated from Different Food Sources.

This study determined the prevalence of Staphylococcus aureus in food of animal origin, investigated its antimicrobial susceptibility profiles and antimicrobial-resistant genes encoding resistance to methicillin (mecA), penicillin (blaZ), and vancomycin (vanA). Two hundred and sixty food samples, including raw retail milk, meat, and meat products, were obtained from local retail shops in Mansoura city, Egypt. The overall prevalence of S. aureus in the total examined food samples was 32.69% (85/260). Methicillin-resistant S. aureus (MRSA) was identified in 11.15% (29/260) of the tested food samples. S. aureus indicated a high resistance to nalidixic acid, penicillin, ampicillin, cefuroxime, trimethoprim/sulfamethoxazole, and azithromycin. The multiple antibiotic resistance (MAR) rate was 89.4% of the total S. aureus isolates, and MARindex ranges from 0.05-0.64. Genotypically, mecA and blaZ genes were identified in a percentage of 34.11% and 82.35%, respectively, while no isolates harbored the vanA gene. The presence of MAR S. aureus particularly, MRSA in food samples, is of great concern and represents a possible threat to the community. Therefore, the study's findings highlight the importance of establishing vigilant food safety practices for food handlers to inhibit the transmission of S. aureus through the food chain to reduce public health risks.

Prevalence and Characterization of PVL-Positive Staphylococcus aureus Isolated from Raw Cow's Milk.

The present study aimed to investigate the prevalence, antibiotic susceptibility profiles, and some toxin genes of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus (S. aureus) in unpasteurized raw cow's milk collected from retail outlets located at Mansoura, Dakahliya governorate, Egypt. In that context, a total of 700 raw cow's milk samples were investigated for the presence of S. aureus, which was identified in 41.1% (288/700) of the samples. Among the S. aureus isolates, 113 PVL-positive S. aureus were identified and subjected for further analysis. The PVL-positive S. aureus were investigated for the existence of toxin-related genes, including hemolysin (hla), toxic shock syndrome toxin-1 (tst), and enterotoxins (sea, seb, sec, see, seg, sei, and selj). Genotypic resistance of PVL-positive strains was performed for the detection of blaZ and mecA genes. Among the PVL-positive S. aureus, sea, seb, and sec were detected in 44.2, 6.2%, and 0.9%, respectively, while the hla and tst genes were identified in 54.9% and 0.9%, respectively. The blaZ and mecA genes were successfully identified in 84.9 (96/113) and 32.7% (37/113) of the total evaluated S. aureus isolates, respectively. PVL-positive S. aureus displayed a high level of resistance to penicillin, ampicillin, and trimethoprim-sulfamethoxazole. Multidrug resistance (resistant to ≥3 antimicrobial classes) was displayed by all methicillin-resistant S. aureus (MRSA) and 38.2% of methicillin-sensitive S. aureus (MSSA) isolates. The obtained findings are raising the alarm of virulent PVL-positive MRSA clones in retail milk in Egypt, suggesting the requirement for limiting the use of ß-lactam drugs in food-producing animals and the importance of implementing strong hygiene procedures in dairy farms and processing plants.

Molecular Characterization, Virulence and Antimicrobial Susceptibility Testing of Riemerella anatipestifer Isolated from Ducklings.

This pilot study aimed to characterize Riemerella anatipestifer from ducklings, testing their susceptibility to antimicrobial agents and to detect their virulence markers. Seven R. anatipestifer isolates with 11.67% infection rate were identified out of sixty freshly dead ducklings and confirmed by PCR assay targeting gyrB gene. The gyrB gene sequences of R. anatipestifer isolates were 100% identical to each other and also showed 100% sequence similarity to the published gyrB genes. Four virulence genes namely ompA, prtC, hagA, and sspA were identified in all isolates except sspA was detected in 5 isolates. The antibiogram revealed higher sensitive to imipenem, amikacin, and rifampin, while, a remarkably high resistance was displayed against ampicillin, penicillin, cefipime, trimethoprim/sulfamethoxazole, gentamicin, ceftazidime, streptomycin and cefoperazone. Proper and rapid identification of R. anatipestifer with detection of their antimicrobial susceptibility and its virulence potential is essential for understanding the epidemiology of R. anatipestifer and to apply the effective control strategies.

Detection of Campylobacter jejuni diversity by clustered regularly interspaced short palindromic repeats (CRISPR) from an animal farm.

BACKGROUND: Campylobacter jejuni is the leading bacterial pathogen that causes foodborne illness worldwide. Because of genetic diversity and sophisticated growth requirements of C. jejuni, several genotyping methods have been investigated to classify this bacterium during the outbreaks. One of such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). OBJECTIVES: The goal of this study was to explore the diversity of C. jejuni isolates with CRISPR from an animal farm. METHODS: Seventy-seven C. jejuni isolates from an animal farm were used in this study. The day-old broilers were reared with other poultry and farm animals, including layer hens, guinea hens, dairy goats and sheep. A small swine herd was also present on an adjacent, but separate plot of land. Isolation and identification of C. jejuni were performed according to the standard procedures. The CRISPR type 1 was PCR amplified from genomic DNA, and the amplicons were sequenced by the Sanger dideoxy method. The direct repeats (DRs) and spacers of the CRISPR sequences were identified using the CRISPRFinder. RESULTS: The CRISPR sequences were detected in all 77 isolates. One type of DRs was identified in these 77 isolates. The lengths of the CRISPR locus ranged from 100 to 560 nucleotides, whereas the number of spacers ranged from one to eight. The distributions of the numbers of CRISPR spacers from different sources seemed to be random. Overall, 17 out of 77 (22%) C. jejuni isolates had two and five spacers, whereas 14 out of 77 (18%) isolates had three spaces in their genomes. By further analysis of spacer sequences, a total of 266 spacer sequences were identified in 77 C. jejuni isolates. By comparison with known published spacer sequences, we observed that 49 sequences were unique in this study. The CRISPR sequence combination of Nos. 16, 19, 48 and 57 was found among a total of 15 C. jejuni isolates containing various multi-locus sequence typing (MLST) types (ST-50, ST-607, ST-2231 and ST-5602). No. 57 spacer sequence was unique from this study, whereas the other three (Nos. 16, 19 and 48) sequences were found in previous reports. Combination of Nos. 5, 9, 15, 30 and 45 was associated with ST-353. To compare the CRISPR genotyping with other methods, the MLST was selected due to its high discriminatory power to differentiate isolates. Based on calculation of the Simpson's index of diversity, a combination of both methods had higher Simpson's index value than those for CRISPR or MLST, respectively. CONCLUSIONS: Our results suggest that the MLST from C. jejuni isolates can be discriminated based on the CRISPR unique spacer sequences and the numbers of spacers. In the future, investigation on the CRISPR resolution for C. jejuni identification in outbreaks is needed. A database that integrates both MLST sequences and CRISPR sequences and is searchable is greatly in demand for tracking outbreaks and evolution of this bacterium.

Effect of tactile/kinesthetic massage therapy on growth and body composition of preterm infants.

Massage therapy (MT) improves growth parameters in preterm infants. The growth of lean mass rather than fat mass has been associated with better long-term outcomes. We aimed to study the effect of tactile/kinesthetic MT on growth and body composition parameters in preterm infants. Preterm (< 32 weeks gestation) infants were randomly assigned at corrected gestational age of 35 weeks to receive 3 consecutive, 15-min, sessions of MT over 5 days or routine care. Primary outcome was mean daily weight gain. Secondary outcomes included anthropometric measurements and body composition parameters assessed by dual X-ray absorptiometry (DXA) scan. Out of 218 infants screened, 86 were eligible and 60 infants (30 in each group) were recruited after parental consent. MT was associated with significant increase in daily weight gain [19.3 (10-34.3) versus 6.2 (2.5-18.4) g/day, p = 0.01] and growth velocity [12.5 (6-21) versus 3.6 (1.6-12.6) g/kg/d, p = 0.01] compared with routine care. Infants on MT showed significant increase in total body mass, fat mass (total/legs), lean mass (total/arms/legs/trunk), and bone mineral density (arms/legs/trunk) values compared with routine care group. In conclusions, MT improves growth quality as evident by increased total and regional lean masses, increased bone mineral density, and peripheral rather than central fat distribution. What is known on this subject? • Massage therapy (MT) for preterm infants leads to achievement of faster independent oral feeding, increased weight gain, less stress, less response to pain, less occurrence of sepsis, and shorter hospital stay. • Growth of lean mass rather than fat mass has been associated with better long-term outcomes. What this study adds? • Tactile/kinesthetic massage therapy in preterm infant is associated with improved growth parameters and anthropometric measures. • Tactile/kinesthetic massage therapy increased total body mass, fat mass (total/legs), lean mass (total/arms/legs/trunk), and bone mineral density (arms/legs/trunk) values.

Phenotypes, antibacterial-resistant profile, and virulence-associated genes of Salmonella serovars isolated from retail chicken meat in Egypt.

AIM: The present study was designed to investigate the occurrence and distribution of Salmonella serotypes in chicken meat samples, and to explore the susceptibility of the strains to antimicrobials, as well as their virulence-associated genes. MATERIALS AND METHODS: Two-hundred retail chicken meat samples from different shops, as well as 25 stool specimens from retail shop workers, were included in the study. The collected samples were examined bacteriologically for the presence of salmonellae. Salmonella isolates were serotyped using a slide agglutination test for O and H antigens and were screened for the presence of five virulence genes (stn, pef, inv A , sop B , and avrA) using a uniplex polymerase chain reaction assay and for their susceptibility to 18 antimicrobial agents using the disk diffusion method. RESULTS: Thirty-one Salmonella isolates belonging to 12 different serovars were identified. Salmonella Enteritidis and Salmonella Kentucky were the dominant serovars (22.6% each). Salmonella isolates displayed a high antibiotic resistance against erythromycin, sulfamethoxazole/trimethoprim, doxycycline, cephalexin, cefaclor, tetracycline, polymyxin B, cefuroxime, vancomycin, and streptomycin. All Salmonella isolates exhibited multidrug resistance (MDR) and demonstrated different virulence genes. The majority of Salmonella serovars (87.1%) harbored sopB gene, 54.8% carried avrA and pef genes, while all isolates carried invA and stn genes. CONCLUSION: The presence of virulent MDR Salmonellae in raw chicken meat could allow the possibility of transmission of these resistant serovars to humans. Therefore, strict hygienic measures should be followed on the whole poultry production chain to decrease the potential transmission of Salmonella infection from poultry meat to humans.

Antimicrobial Resistance, Genetic Diversity and Multilocus Sequence Typing of Escherichia coli from Humans, Retail Chicken and Ground Beef in Egypt.

Contamination of retail foods with foodborne pathogens, particularly the antimicrobial resistant ones, poses a persistent threat to human health. There is a dearth of information about the overlapping Escherichia coli (E. coli) lineages circulating among retail foods and humans in Egypt. This study aimed to determine the clonal diversity of 120 E. coli isolates from diarrheic patients (n = 32), retail chicken carcasses (n = 61) and ground beef (n = 27) from Mansoura, Egypt using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Simpson's index of diversity was calculated to compare the results of both typing methods. Antimicrobial resistance phenotypes, genotypes and phylogrouping of the isolates were also determined. Higher frequencies of antimicrobial resistance were found among chicken isolates compared to beef and human isolates; regardless of isolate source, the predominant antimicrobial resistances were found against ampicillin (87/120, 72.5%), tetracycline and sulfisoxazole (82/120, 68.3%, each), and streptomycin (79/120, 65.8%). None of the isolates displayed resistance to meropenem. The prevalent genes detected were tetA (64.2%), blaTEM (62.5%), sul1 (56.7%), floR (53.3%), sul2 (50%), strB (48.3%) and strA (47.5%) corresponding with resistance phenotypes. Alarmingly, blaCTX was detected in 63.9% (39/61) of chicken isolates. The majority of E. coli isolates from humans (90.6%), beef (81.5%) and chicken (70.5%) belonged to commensal phylogroups (A, B1, C). Using PFGE analysis, 16 out of 24 clusters (66.7%) contained isolates from different sources at a similarity level ≥75%. MLST results assigned E. coli isolates into 25, 19 and 13 sequence types (STs) from chicken, human and beef isolates, respectively. Six shared STs were identified including ST1011, ST156, ST48, ST224 (chicken and beef), ST10 (human and chicken) and ST226 (human and beef). Simpson's index of diversity was higher for MLST (0.98) than PFGE (0.94). In conclusion, the existence of common genetic determinants among isolates from retail foods and humans in Egypt as well as the circulation of shared STs indicates a possible epidemiological link with potential zoonotic hazards.

Genotyping of Campylobacter jejuni Isolates from Poultry by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Campylobacter jejuni is the leading bacterial foodborne pathogen that causes human acute gastrointestinal illness worldwide. Due to its genetic diversity, fastidious growth and sophisticated biochemical requirements, classification of Campylobacter by traditional techniques is problematic. Several molecular typing methods have been explored in this bacterium. One such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). These CRISPRs consist of a direct repeat interspaced with nonrepetitive spacer sequences. In this study, we applied this genotyping method to explore the genetic diversity of C. jejuni isolated from poultry sources. Ninety-nine C. jejuni isolates from poultry environments in four different US states were used. Genomic DNA of the isolates were extracted from cultures using a commercial kit. PCR primers and conditions for CRISPR type 1 amplification were described previously. The amplicons were purified and sequenced by the Sanger dideoxy sequencing method. The direct repeats (DR) and spacers of the CRISPR sequences were identified using the CRISPRFinder. The results show there were 21% isolates no detectable, 30% isolates questionable, and 49% isolates confirmed CRISPR, respectively. The lengths of CRISPR range from 100 to 695 nucleotides. One type of DR was found in CRISPR of these isolates. The number of spacers in CRISPR ranges from 1 to 10 with various sequences. A total of 55 distinctive spacer sequences were identified in 78 isolates. Among them, 33 sequences were found unique in this study. In addition, the CRISPR genotyping had higher the Simpson's index of diversity value than that from flaA nucleotide typing. The results of our study show the CRISPR genotyping on C. jejuni may be complementary to the other genotyping methods.

Dairy Products and Dairy-Processing Environments as a Reservoir of Antibiotic Resistance and Quorum-Quenching Determinants as Revealed through Functional Metagenomics.

Here, the role of the dairy-processing chain as a reservoir of antimicrobial resistance (AR) determinants and a source of novel biocontrol quorum-sensing inhibitors is assessed through a functional metagenomics approach. A metagenomic library comprising ∼22,000 recombinant clones was built from DNA isolated from raw milk, raw milk cheeses, and cheese-processing environment swab samples. The high-throughput sequencing of 9,216 recombinant clones showed that lactic acid bacteria (LAB) dominated the microbial communities of raw milk cheese, while Gram-negative microorganisms of animal or soil origin dominated the microbiota of raw milk and cheese-processing environments. Although functional screening of the metagenomic library did not recover potential quorum-sensing inhibitors, in silico analysis using an in-house database built specifically for this study identified homologues to several genes encoding proteins with predicted quorum-quenching activity, among which, the QsdH hydrolase was the most abundant. In silico screening of the library identified LAB, and especially Lactococcus lactis, as a relevant reservoir of AR determinants in cheese. Functional screening of the library allowed the isolation of 13 recombinant clones showing an increased resistance toward ampicillin, which in all cases was accompanied by a reduced susceptibility to a wide range of ß-lactam antibiotics. This study shows that the dairy-processing environment is a rich reservoir of AR determinants, which vary by sample source, and suggests that combining next-generation sequencing with functional metagenomics can be of use in overcoming the limitations of both approaches.IMPORTANCE The study shows the potential of functional metagenomics analyses to uncover the diversity of functions in microbial communities prevailing in dairy products and their processing environments, evidencing that lactic acid bacteria (LAB) dominate the cheese microbiota, whereas Gram-negative microorganisms of animal or soil origin dominate the microbiota of milk and cheese-processing environments. The functional and in silico screening of the library allowed the identification of LAB, and especially Lactococcus lactis, as a relevant reservoir of antimicrobial resistance (AR) determinants in cheese. Quorum-quenching (QQ) determinants were not recovered through the execution of wet-lab function-based screenings but were detected through in silico sequencing-based analyses.

Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt.

The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants to assess the virulence potential as well as multidrug resistance (MDR) genotypes. We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers (n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types. From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes associated with beta lactam resistance 27/47 (57.5 %). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources.

Yersinia enterocolitica: Prevalence, virulence, and antimicrobial resistance from retail and processed meat in Egypt.

BACKGROUND AND AIM: The objectives of this study were to investigate the prevalence of Yersinia enterocolitica in retail chicken meat, ground and processed beef meat, determine their virulence-associated genes, antimicrobial susceptibility pattern, molecular detection of extended-spectrum ß-lactamases, and their capability of biofilm formation in vitro. MATERIALS AND METHODS: A total of 210 samples (120 retail chicken meat, 30 ground beef, 30 beef burger, and 30 sausage samples) were collected from different retail chicken outlets and markets located at Mansoura city between December 2016 and April 2017. Meat samples were examined bacteriologically for the existence of Y. enterocolitica; bacterial colonies that displayed positive biochemical properties were subjected to polymerase chain reaction targeting 16 rRNA gene. Y. enterocolitica isolates were tested for their susceptibility to six antimicrobial agents using disk diffusion method. Uniplex PCR was used for screening Y. enterocolitica isolates for the presence of two virulence chromosome-associated genes (ail and yst), and ß-lactamases (bla TEM and bla SHV). The capability of Y. enterocolitica to form biofilms was detected by tube method. RESULTS: Thirty Y. enterocolitica isolates (14.29%) were recovered including 19 (15.83%) isolates from chicken meat, 3 (10%) from ground beef, 5 (16.67%) from beef burger, and 3 (10%) from sausage samples. Regarding ail gene, it was detected in 6.67% (2/30), while yst gene detected in 20% (6/30) Y. enterocolitica isolates. About 80%, 70%, 63.33%, and 50% of Y. enterocolitica isolates were sensitive to ciprofloxacin, gentamicin, cefotaxime, and streptomycin, respectively, while 83.33% of Y. enterocolitica isolates were resistant to both ampicillin and cephalothin. Interestingly, 21 (70%) isolates had the capability of biofilms formation in vitro. Among the multidrug-resistant (MDR) strains, a significant difference (p<0.05) was found between MDR and biofilm formation. However, biofilm formation was correlated with the resistance of the isolates to ß-lactam antimicrobials and the presence of ß-lactam-resistant genes. CONCLUSION: The presence of Y. enterocolitica in chicken meat, ground and processed beef meat represents a significant health risk for meat consumers, which reflects the contamination of slaughterhouses and processing operations, therefore, strict hygienic measures should be applied to minimize carcasses contamination.

Multilocus Sequence Typing (MLST) of Campylobacter jejuni Isolated From Broiler Meat in Egypt.

BACKGROUND AND OBJECTIVES: Infection with Campylobacter jejuni is one of the most common causes of bacterial gastroenteritis. Infections are mostly acquired due to consumption of raw or undercooked poultry. The aim of this pilot study is to determine the prevalence and the sequence types (STs) distribution of C. jejuni isolated from broiler meat in Egypt. MATERIALS AND METHODS: A total of 190 broiler meat samples were collected from retail chicken shops located at Mansoura, Egypt and examined bacteriologically for the presence of Campylobacter spp. The biochemically identified Campylobacter isolates were confirmed by Multiplex PCR (m-PCR). In addition, multilocus sequencing typing (MLST) was used for genotyping of C. jejuni isolates. RESULTS: Thirty two Campylobacter isolates divided into C. coli (25 isolates) and C. jejuni (7 isolates) were recovered. Multiplex PCR results found to be 100% in line with biochemical identification. Out of 7 C. jejuni isolates genotyped by MLST, 4 isolates were assigned to ST21, 2 isolates were assigned to ST48 and one isolate was assigned to ST464. CONCLUSION: This study provides valuable information concerning the prevalence of thermophilic Campylobacter spp. and sequence types distribution of C. jejuni recovered from broiler meat for the first time in Egypt. The identified sequence types from this study were frequently reported in human illnesses. Thus, the present results highlight the importance of the retail broiler meat as a significant source for human Campylobacter infection.

Can a midwife-led continuity model improve maternal services in a low-resource setting? A non-randomised cluster intervention study in Palestine.

OBJECTIVES: To improve maternal health services in rural areas, the Palestinian Ministry of Health launched a midwife-led continuity model in the West Bank in 2013. Midwives were deployed weekly from governmental hospitals to provide antenatal and postnatal care in rural clinics. We studied the intervention's impact on use and quality indicators of maternal services after 2 years' experience. DESIGN: A non-randomised intervention design was chosen. The study was based on registry data only available at cluster level, 2 years before (2011and2012) and 2 years after (2014and2015) the intervention. SETTING: All 53 primary healthcare clinics in Nablus and Jericho regions were stratified for inclusion. PRIMARY AND SECONDARY OUTCOMES: Primary outcome was number of antenatal visits. Important secondary outcomes were number of referrals to specialist care and number of postnatal home visits. Differences in changes within the two groups before and after the intervention were compared by using mixed effect models. RESULTS: 14 intervention clinics and 25 control clinics were included. Number of antenatal visits increased by 1.16 per woman in the intervention clinics, while declined by 0.39 in the control clinics, giving a statistically significant difference in change of 1.55 visits (95% CI 0.90 to 2.21). A statistically significant difference in number of referrals was observed between the groups, giving a ratio of rate ratios of 3.65 (2.78-4.78) as number of referrals increased by a rate ratio of 3.87 in the intervention group, while in the control the rate ratio was only 1.06.Home visits increased substantially in the intervention group but decreased in the control group, giving a ratio of RR 97.65 (45.20 - 210.96) CONCLUSION: The Palestinian midwife-led continuity model improved use and some quality indicators of maternal services. More research should be done to investigate if the model influenced individual health outcomes and satisfaction with care. TRIAL REGISTRATION NUMBER: NCT03145571; Results.