Ashraf M. Ahmed

Zoo Animals as Reservoirs of Gram-Negative Bacteria Harboring Integrons and Antimicrobial Resistance Genes

ABSTRACT A total of 232 isolates of gram-negative bacteria were recovered from mammals, reptiles, and birds housed at Asa Zoological Park, Hiroshima prefecture, Japan. Forty-nine isolates (21.1%) showed multidrug resistance phenotypes and harbored at least one antimicrobial resistance gene. PCR and DNA sequencing identified class 1 and class 2 integrons and many β-lactamase-encoding genes, in addition to a novel AmpC β-lactamase gene, blaCMY-26. Furthermore, the plasmid-mediated quinolone resistance genes qnr and aac(6′)-Ib-cr were also identified.

Isolation and Molecular Characterization of Multidrug‐Resistant Strains of Escherichia coli and Salmonella from Retail Chicken Meat in Japan

Sixty-nine Escherichia coli and 10 Salmonella isolates, recovered from retail chicken meat in Hiroshima prefecture, Japan, were assayed for antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes. Twenty-eight out of 69 (40.6%) of E. coli and all 10 Salmonella isolates were exhibited multidrug resistance phenotypes. The most commonly reported resistance phenotypes were against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, and trimethoprim/sulfamethoxazole. PCR screening for integrons showed that 8 (11.6%) of the E. coli isolates were positive for the class 1 integrons and 1 isolate (1.4%) was positive for the class 2 integrons. Among the 10 Salmonella isolates, 9 were positive for class 1 integrons and none was positive for class 2 integrons. The identified antibiotic resistance gene cassettes within the class 1 integrons were dfrA1, dfrA7, aadA1, aadB, and catB3, while dfrA1, sat2, and aadA1 were identified within class 2 integron. The beta-lactamase resistance gene bla(TEM-1) was identified in 12 (17.3%) of E. coli isolates and in only one of the Salmonella isolates. The bla(CMY-2) gene, encoding AmpC beta-lactamase, was detected in 16 (23.2%) of the E. coli isolates only. Conjugation experiments demonstrated that there was plasmid-mediated transfer of bla(CMY-2) and bla(TEM-1). These results highlighted the role of retail chicken meat as a potential source for multidrug-resistant strains of E. coli and Salmonella. To the best of our knowledge, this is the 1st report of isolation and molecular characterization of multidrug-resistant strains of E. coli from retail chicken meat in Japan.

Molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Japan.

Aims:  To investigate the prevalence of integrons and antimicrobial resistance genes in Salmonella recovered from animals in Japan.

Emergence of Plasmid-Mediated Colistin Resistance Gene mcr-1 in a Clinical Escherichia coli Isolate from Egypt.

Colistin is considered a last resort for the treatment of infections caused by carbapenemase-producing members of the family Enterobacteriaceae. Recently, a transferable plasmid conferring resistance to colistin was discovered in Escherichia coli and Klebsiella pneumoniae from China ([1][1]) and was

Molecular characterization of antimicrobial resistance in Gram‐negative bacteria isolated from bovine mastitis in Egypt

The aim of this study was to characterize the genetic basis of multidrug resistance in Gram‐negative bacteria isolated from bovine mastitis cases in Egypt. Multidrug resistance phenotypes were found in 34 of 112 (30.4%) Gram‐negative bacterial isolates, which harbored at least one antimicrobial resistance gene. The most prevalent multidrug‐resistant (MDR) species were Enterobacter cloacae (8 isolates, 7.1%), Klebsiella pneumoniae (7 isolates, 6.3%), Klebsiella oxytoca (7 isolates, 6.3%), Escherichia coli (5 isolates, 4.5%), and Citrobacter freundii (3 isolates, 2.7%). The most commonly observed resistance phenotypes were against ampicillin (97.0%), streptomycin (94.1%), tetracycline (91.2%), trimethoprim–sulfamethoxazole (88.2%), nalidixic acid (85.3%), and chloramphenicol (76.5%). Class 1 integrons were detected in 28 (25.0%) isolates. The gene cassettes within class 1 integrons included those encoding resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA17, and dfrA25), aminoglycosides (aadA1, aadA2, aadA5, aadA7, aadA12, aadA22, and aac(3)‐Id), chloramphenicol (cmlA), erythromycin (ereA2), and rifampicin (arr‐3). Class 2 integrons were identified in 6 isolates (5.4%) with three different profiles. Furthermore, the β‐lactamase encoding genes, blaTEM, blaSHV, blaCTX‐M, and blaOXA, the plasmid‐mediated quinolone resistance genes, qnr and aac(6)‐Ib‐cr, and the florfenicol resistance gene, floR, were also identified. To the best of our knowledge, the results identified class 2 integrons, qnr and aac(6)‐Ib‐cr from cases of mastitis for the first time. This is the first report of molecular characterization for antimicrobial resistance in Gram‐negative bacteria isolated from bovine mastitis in Africa.

Molecular screening and risk factors of enterotoxigenic Escherichia coli and Salmonella spp. in diarrheic neonatal calves in Egypt

Abstract The aim of the present study was to carry out molecular epidemiological investigation on enterotoxigenic Escherichia coli (ETEC) K99 and Salmonella spp. in diarrheic neonatal calves. Fecal samples were obtained from 220 diarrheic calves at 9 farms related to four governorates in central and northern Egypt. E. coli and Salmonella spp. isolates were examined for E. coli K99 and Salmonella spp. using PCR. ETEC K99 was recovered from 20 (10.36 %) out of 193 isolates, whereas Salmonella spp. was recovered from nine calves (4.09%). Multivariable logistic regression was used to evaluate the risk factors associated with both infections. ETEC K99 was significantly affected by age (P <0.01; OR: 1.812; CI 95%: 0.566–1.769), colostrum feeding practice (P <0.01; OR: 5.525; CI 95%: 2.025–15.076), rotavirus infection (P <0.001; OR: 2.220; CI 95%: 0.273–1.251), vaccination of pregnant dams with combined vaccine against rotavirus, coronavirus and E. coli (K99) (P <0.001; OR: 4.753; CI 95%: 2.124–10.641), and vitamin E and selenium administration to the pregnant dam (P <0.01; OR: 3.933; CI 95%: 0.703–1.248). Infection with Salmonella spp. was found to be significantly affected by the animal age (P <0.05; OR: 0.376; CI 95%: 0.511–1.369), Hygiene (P <0.05; OR: 0.628; CI 95%: 1.729–5.612), and region (P <0. 01; OR: 0.970; CI 95%: 0.841–1.624). The results of the present study indicate the importance of PCR as rapid, effective and reliable tool for screening of ETEC and Salmonella spp. when confronted with cases of undifferentiated calf diarrhea. Moreover, identification of the risk factors associated with the spreading of bacteria causing diarrhea may be helpful for construction of suitable methods for prevention and control.

Genetic analysis of antimicrobial resistance in Escherichia coli isolated from diarrheic neonatal calves.

This study was carried out to screen and analyze the genetic basis of antimicrobial resistance in Escherichia coli strains isolated from neonatal calf diarrhea in Egypt. A total of 182 isolates of E. coli recovered from 91 diarrheic neonatal calves were analyzed for antimicrobial susceptibilities, the presence of class 1 and class 2 integrons and antimicrobial resistance genes. Nineteen isolates (10.4%) showed multidrug resistance phenotypes and harbored at least three antimicrobial resistance genes. PCR screening detected class 1 integrons in 19 isolates (10.4%) and class 2 integrons in 2 isolates (1.1%). The identified antimicrobial resistance genes within class 1 integrons were dihydrofolate reductase types: dfrA1, dfrA12, dfrA15 and dfrA17, which confer resistance to trimethoprim; aminoglycoside adenyltransferase types: aadA1, aadA2, aadA5, aadA7 and aadA23, which confer resistance to streptomycin and spectinomycin; and aminoglycoside acetyltransferase gene, aac(3)-Id, which confers resistance to gentamicin and sisomicin. Furthermore, many beta-lactamases encoding genes, plasmid-mediated quinolone resistance genes and florfenicol resistance gene were identified in this study. To the best of our knowledge, this is the first report for molecular characterization of antimicrobial resistance in E. coli isolated from diarrheic neonatal calves in Africa.

Characterization of integrons and antimicrobial resistance genes in clinical isolates of Gram-negative bacteria from Palestinian hospitals.

Sixty Gram‐negative bacterial isolates were collected from Palestinian hospitals in 2006. Thirty‐two (53.3%) isolates showed multidrug resistance phenotypes. PCR and DNA sequencing were used to characterize integrons and antimicrobial resistance genes. PCR screening showed that 19 (31.7%) and five (8.3%) isolates were positive for class 1 and class 2 integrons, respectively. DNA‐sequencing results for the captured antimicrobial resistance gene cassettes within class 1 integrons identified the following genes: dihydrofolate reductases, dfrA1, dfrA5, dfrA7, dfrA12, dfrA17 and dfrA25; aminoglycoside adenyltransferases, aadA1, aadA2, aadA5, aadA12 and aadB; aminoglycoside acetyltransferase, aac(6′)‐Ib; and chloramphenicol resistance gene, cmlA1. ESBL were identified in 25 (41.7%) isolates. The identified ESBL were blaCTX‐M‐15, blaCTX‐M‐56, blaOXA‐1, blaSHV‐1, blaSHV‐12, blaSHV‐32 and blaTEM‐1 genes. Moreover, we characterized the plasmid‐mediated quinolone resistance genes, aac(6′)‐Ib‐cr and qnrB2, which were detected in seven (11.7%) and two (3.3%) isolates, respectively. In this study various types of antibiotic resistance genes have been identified in Gram‐negative bacteria from Palestinian hospitals, many of which are reported in the Middle East area for the first time.

Emergence of a cefepime- and cefpirome-resistant Citrobacter freundii clinical isolate harbouring a novel chromosomally encoded AmpC β-lactamase, CMY-37

Citrobacter freundii strain 4306 was isolated from a urine specimen of a patient in March 2006 in Palestine. This strain showed a unique multidrug resistance phenotype, as it was resistant both to 7-alpha-methoxy- and oxyimino-cephalosporins, including cefepime, cefpirome and monobactams, in addition to quinolones, streptomycin and trimethoprim/sulfamethoxazole. Clavulanic acid did not act synergistically with cephalosporins by the double-disk synergy test. Molecular characterisation showed that the resistance to 7-alpha-methoxy- and oxyimino-cephalosporins was due to a novel AmpC beta-lactamase, designated CMY-37, with an isoelectric point of approximately 9.0. CMY-37 is a variant of C. freundii chromosomal AmpC enzymes with at least seven amino acid substitutions. One of these substitutions, L316I, is located within the R2 loop that is considered the hotspot region responsible for the extended substrate spectrum in class C beta-lactamases. The blaCMY-37 gene was cloned and expressed in Escherichia coli TG1. CMY-37 is chromosomally encoded and is not associated with ISEcp1-like element. Phylogenetic analysis suggested that CMY-37 is the origin of many plasmid-mediated AmpC beta-lactamases. This study highlights the emergence of cefepime and cefpirome resistance in C. freundii owing to a new type of AmpC beta-lactamase.

Molecular characterization of multidrug-resistant Shigella spp. of food origin.

Shigella spp. are the causative agents of food-borne shigellosis, an acute enteric infection. The emergence of multidrug-resistant clinical isolates of Shigella presents an increasing challenge for clinicians in the treatment of shigellosis. Several studies worldwide have characterized the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin, however, to date, no such characterization has been reported for Shigella spp. of food origin. In this study, we characterized the genetic basis of multidrug resistance in Shigella spp. isolated from 1600 food samples (800 meat products and 800 dairy products) collected from different street venders, butchers, retail markets, and slaughterhouses in Egypt. Twenty-four out of 27 Shigella isolates (88.9%) showed multidrug resistance phenotypes to at least three classes of antimicrobials. The multidrug-resistant Shigella spp. were as follows: Shigella flexneri (66.7%), Shigella sonnei (18.5%), and Shigella dysenteriae (3.7%). The highest resistance was to streptomycin (100.0%), then to kanamycin (95.8%), nalidixic acid (95.8%), tetracycline (95.8%), spectinomycin (93.6%), ampicillin (87.5%), and sulfamethoxazole/trimethoprim (87.5%). PCR and DNA sequencing were used to screen and characterize integrons and antibiotic resistance genes. Our results indicated that 11.1% and 74.1% of isolates were positive for class 1 and class 2 integrons, respectively. Beta-lactamase-encoding genes were identified in 77.8% of isolates, and plasmid-mediated quinolone resistance genes were identified in 44.4% of isolates. These data provide useful information to better understand the molecular basis of antimicrobial resistance in Shigella spp. To the best of our knowledge, this is the first report of the molecular characterization of antibiotic resistance in Shigella spp. isolated from food.