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123500.9849Characterization 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 bla(CTX-M-15), bla(CTX-M-56), bla(OXA-1), bla(SHV-1), bla(SHV-12), bla(SHV-32) and bla(TEM-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.200919903259
123610.9848Molecular 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, bla(TEM), bla(SHV), bla(CTX-M), and bla(OXA), 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.201121338385
123420.9847Isolation and Genetic Analysis of Multidrug Resistant Bacteria from Diabetic Foot Ulcers. Severe diabetic foot ulcers (DFUs) patients visiting Sir Sunderlal Hospital, Banaras Hindu University, Varanasi, were selected for this study. Bacteria were isolated from swab and deep tissue of 42 patients, for examining their prevalence and antibiotic sensitivity. DFUs of majority of the patients were found infected with Enterococcus spp. (47.61%), Escherichia coli (35.71%), Staphylococcus spp. (33.33%), Alcaligenes spp. (30.95%), Pseudomonas spp. (30.95%), and Stenotrophomonas spp. (30.95%). Antibiotic susceptibility assay of 142 bacteria with 16 antibiotics belonging to eight classes showed the presence of 38 (26.76%) isolates with multidrug resistance (MDR) phenotypes. MDR character appeared to be governed by integrons as class 1 integrons were detected in 26 (68.42%) isolates. Altogether six different arrays of genes (aadA1, aadB, aadAV, dhfrV, dhfrXII, and dhfrXVII) were found within class 1 integron. Gene cassette dhfrAXVII-aadAV (1.6 kb) was present in 12 (3 Gram positive and 9 Gram negative) isolates and was conserved across all the isolates as evident from RFLP analysis. In addition to the presence of class 1 integron, six β-lactamase resistance encoding genes namely bla TEM, bla SHV, bla OXA, bla CTX-M-gp1, bla CTX-M-gp2, and bla CTX-M-gp9 and two methicillin resistance genes namely mecA and femA and vancomycin resistance encoding genes (vanA and vanB) were identified in different isolates. Majority of the MDR isolates were positive for bla TEM (89.47%), bla OXA (52.63%), and bla CTX-M-gp1 (34.21%). To our knowledge, this is the first report of molecular characterization of antibiotic resistance in bacteria isolated from DFUs from North India. In conclusion, findings of this study suggest that class-1 integrons and β-lactamase genes contributed to the MDR in above bacteria.201526779134
138830.9846Snapshot Study of Whole Genome Sequences of Escherichia coli from Healthy Companion Animals, Livestock, Wildlife, Humans and Food in Italy. Animals, humans and food are all interconnected sources of antimicrobial resistance (AMR), allowing extensive and rapid exchange of AMR bacteria and genes. Whole genome sequencing (WGS) was used to characterize 279 Escherichia coli isolates obtained from animals (livestock, companion animals, wildlife), food and humans in Italy. E. coli predominantly belonged to commensal phylogroups B1 (46.6%) and A (29%) using the original Clermont criteria. One hundred and thirty-six sequence types (STs) were observed, including different pandemic (ST69, ST95, ST131) and emerging (ST10, ST23, ST58, ST117, ST405, ST648) extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Eight antimicrobial resistance genes (ARGs) and five chromosomal mutations conferring resistance to highest priority critically important antimicrobials (HP-CIAs) were identified (qnrS1, qnrB19, mcr-1, bla(CTX-M1,15,55), bla(CMY-2), gyrA/parC/parE, ampC and pmrB). Twenty-two class 1 integron arrangements in 34 strains were characterized and 11 ARGs were designated as intI1 related gene cassettes (aadA1, aadA2, aadA5, aad23, ant2_Ia, dfrA1, dfrA7, dfrA14, dfrA12, dfrA17, cmlA1). Notably, most intI1 positive strains belonged to rabbit (38%) and poultry (24%) sources. Three rabbit samples carried the mcr-1 colistin resistance gene in association with IS6 family insertion elements. Poultry meat harbored some of the most prominent ExPEC STs, including ST131, ST69, ST10, ST23, and ST117. Wildlife showed a high average number of virulence-associated genes (VAGs) (mean = 10), mostly associated with an ExPEC pathotype and some predominant ExPEC lineages (ST23, ST117, ST648) were identified.202033172096
137540.9841Characterization of integrons and their cassettes in Escherichia coli and Salmonella isolates from poultry in Korea. Ninety-nine Escherichia coli and 33 Salmonella isolates were assessed for antimicrobial susceptibility (disc diffusion test). Sulfonamide and tetracycline resistance genes were identified through PCR, and class 1 and class 2 integrons with resistance gene cassettes were identified with PCR followed by sequencing. Salmonella (63.6%) and E. coli (85.8%) isolates were multidrug resistant (resistance to 3 or more antimicrobials), and the highest incidences of resistance were observed for tetracycline, nalidixic acid, and sulfamethoxazole. The sul1, sul2, tetA, and tetB resistance determinant genes were predominant in E. coli, whereas only sul2 and tetA were identified in Salmonella isolates. In the E. coli isolates, 54 (54.5%) class 1 integrons, 6 (6.1%) class 2 integrons, and 5 (5.1%) class 1 and class 2 integrons together were detected, whereas only 3 (9.1%) integrons were found in the Salmonella serovars. Around 87% of the integrons in E. coli harbored resistance gene cassettes conferring resistance to streptomycin/spectinomycin (aadA, aminoglycoside resistance gene), trimethoprim (dfrA, dihydrofolate reductase gene), streptothricin [sat1 and sat2 (streptothricin acetyltransferase), and estX (putative esterases)]. The most common gene cassettes were aadA1+dfrA1 and dfrA1+sat2+aadA1 in class 1 and class 2 integrons, respectively. Other cassettes including aadA5+dfrA7, dfrA12+aadA2, aadA2+aadA1+dfrA12, and aadA5+aadA2/dfrA7 were also identified. Among the Salmonella serovars, Salmonella Malmoe harbored aadA1+dfrA1 and dfrA12+sat2+aadA1 genes. The aadA1, aadA2, sat2, and dfrA1 had wide variation in similarity among themselves and from previously reported genes worldwide. The diverse gene cassettes could be responsible for the prominent resistance profiles observed and a potential source for dissemination of antimicrobial resistance determinants to other bacteria.201324135609
82750.9840Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient. Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6 % G + C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66 % with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, bla(OXA-61), tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.202439208964
122360.9840Characterization of Escherichia coli virulence genes, pathotypes and antibiotic resistance properties in diarrheic calves in Iran. BACKGROUND: Calf diarrhea is a major economic concern in bovine industry all around the world. This study was carried out in order to investigate distribution of virulence genes, pathotypes, serogroups and antibiotic resistance properties of Escherichia coli isolated from diarrheic calves. RESULTS: Totally, 76.45% of 824 diarrheic fecal samples collected from Isfahan, Chaharmahal, Fars and Khuzestan provinces, Iran were positive for E. coli and all of them were also positive for cnf2, hlyA, cdtIII, f17c, lt, st, stx1, eae, ehly, stx2 and cnf1 virulence genes. Chaharmahal had the highest prevalence of STEC (84.61%), while Isfahan had the lowest (71.95%). E. coli serogroups had the highest frequency in 1-7 days old calves and winter season. Distribution of ETEC, EHEC, AEEC and NTEC pathotypes among E. coli isolates were 28.41%, 5.07%, 29.52% and 3.49%, respectively. Statistical analyses were significant for presence of bacteria between various seasons and ages. All isolates had the high resistance to penicillin (100%), streptomycin (98.25%) and tetracycline (98.09%) antibiotics. The most commonly detected resistance genes were aadA1, sul1, aac[3]-IV, CITM, and dfrA1. The most prevalent serogroup among STEC was O26. CONCLUSIONS: Our findings should raise awareness about antibiotic resistance in diarrheic calves in Iran. Clinicians should exercise caution when prescribing antibiotics.201425052999
129570.9840Phenotypic and genotypic characterisation of antimicrobial resistance in faecal bacteria from 30 Giant pandas. To study the prevalence of antimicrobial resistance in faecal bacteria from Giant pandas in China, 59 isolates were recovered from faecal pats of 30 Giant pandas. Antimicrobial susceptibility testing of the isolates was performed by the standardised disk diffusion method (Kirby-Bauer). Of the 59 study isolates, 32.20% were resistant to at least one antimicrobial and 16.95% showed multidrug-resistant phenotypes. Thirteen drug resistance genes [aph(3')-IIa, aac(6')-Ib, ant(3'')-Ia, aac(3)-IIa, sul1, sul2, sul3, tetA, tetC, tetM, cat1, floR and cmlA] were analysed using four primer sets by multiplex polymerase chain reaction (PCR). The detection frequency of the aph(3')-IIa gene was the highest (10.17%), followed by cmlA (8.47%). The genes aac(6')-Ib, sul2 and tetA were not detected. PCR products were confirmed by DNA sequence analysis. The results revealed that multidrug resistance was widely present in bacteria isolated from Giant pandas.200919168331
144080.9840High prevalence of carbapenem-resistant Escherichia coli ST410 from clinical isolates in Weifang, China. The objective of our work is to identify antimicrobial-resistance genes and to analyze clonality of carbapenem-resistant Escherichia coli. A total of 75 carbapenem-resistant E. coli (CREco) strains were isolated in a Chinese hospital from January 2021 to May 2023. The antibiotic susceptibility testing was conducted by BD PhoenixTM M50 System and Kirby-Bauer disk diffusion method. Whole-genome sequencing was performed on Illumina NovaSeq 6000 platform. Antimicrobial resistance genes were identified based on NCBI with ABRicate 0.8. Multilocus sequence typing (MLST) analysis for CREco was performed. Among the 75 CREco strains in this study, the most of them were isolated from urine samples (n = 20, 26.67%) at the intensive care unit (n = 14, 18.67%). Among the detected carbapenem resistance genes, blaNDM-5 was the most prevalent (n = 57, 76.00%), followed by blaNDM-4 (n = 3, 4.00%), blaNDM-9 (n = 3, 4.00%), and blaNDM-1 (n = 2, 2.67%). In addition, the colistin resistance gene mcr-1.1 (n = 11, 14.67%) and the tigecycline resistance gene tetX4 (n = 2, 2.67%) were also detected. The results of MLST revealed 25 sequence types (STs), and ST410 (n = 17) was the dominant clone. Other major STs included ST167 (n = 12), ST156 (n = 10), ST361 (n = 5), and ST101 (n = 4). Overall, CREco strains exhibited a high-level resistance rate to commonly used antimicrobial agents, and the most of them carried various NDM-coding genes, with blaNDM-5 being the predominant type. In this study, we demonstrated the diversity of carbapenem-resistant E. coli; however, the major clone was ST410. These results also show the dissemination of different clones of carbapenem-resistant E. coli.202540531574
83290.9837Development of antibiotic resistance in the ocular Pseudomonas aeruginosa clone ST308 over twenty years. Corneal infection caused by a bacteria Pseudomonas aeruginosa is common cause of ocular morbidity. Increasing antibiotic resistance by ocular P. aeruginosa is an emerging concern. In this study the resistome of ocular isolates of Pseudomonas aeruginosa clone ST308 isolated in India in 1997 (PA31, PA32, PA33, PA35 and PA37) and 2018 (PA198 and PA219) were investigated. All the isolates of ST308 had >95% nucleotide similarity. The isolates from 2018 had larger genomes, coding sequences, accessory and pan genes compared to the older isolates from 1997. The 2018 isolate PA219 was resistant to all antibiotics except polymyxin B, while the 2018 isolate PA198 was resistant to ciprofloxacin, levofloxacin, gentamicin and tobramycin. Among the isolates from 1997, five were resistant to gentamicin, tobramycin and ciprofloxacin, four were resistant to levofloxacin while two were resistant to polymyxin B. Twenty-four acquired resistance genes were present in the 2018 isolates compared to 11 in the historical isolates. All isolates contained genes encoding for aminoglycoside (aph(6)-Id, aph(3')-lIb, aph(3″)-Ib), beta-lactam (blaPAO), tetracycline (tet(G)), fosfomycin (fosA), chloramphenicol (catB7), sulphonamide (sul1), quaternary ammonium (qacEdelta1) and fluoroquinolone (crpP) resistance. Isolate PA198 possessed aph(3')-VI, rmtD2, qnrVC1, blaOXA-488, blaPME-1, while PA219 possessed aadA1, rmtB, qnrVC1, aac(6')-Ib-cr, blaTEM-1B, blaVIM-2, blaPAO-1, mph(E), mph(A), msr(E). In both recent isolates qnrVC1 was present in Tn3 transposon. In 219 blaTEM-1 was carried on a transposon and blaOXA-10 on a class 1 integron. There were no notable differences in the number of single nucleotide polymorphisms, but recent isolates carried more insertions and deletions in their genes. These findings suggest that genomes of P. aeruginosa ocular clonal strains with >95% nucleotide identity isolated twenty years apart had changed over time with the acquisition of resistance genes. The pattern of gene mutations also varied with more insertions and deletions in their chromosomal genes which confer resistance to antibiotics.202133610601
1409100.9836Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya. Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to β-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.202133617559
1111110.9836Molecular Characterization of Cotrimoxazole Resistance Genes and Their Associated Integrons in Clinical Isolates of Gram-Negative Bacteria from Tanzania. Cotrimoxazole is widely used, particularly as a prophylactic drug in HIV patients. We assessed resistance mechanisms among cotrimoxazole resistant-Gram negative bacterial isolates (n = 123) obtained from blood (n = 69) and urine (n = 54) from Tanzanian patients. sul genes were detected in 98% (121/123) of the isolates. Coexistence of sul1 and sul2 was common (49/123). The dfr genes were found in 63% (77/123) of all isolates. sul1, dfrA15, and dfrA5 genes predominated among Klebsiella pneumoniae, while sul2 and dfrA1 genes were frequent in Escherichia coli isolates. Two isolates, both K. pneumoniae, carried sul3. Integrons were detected in 81.3% (100/123) of all isolates. Class 1 integrons were found in 95% (42/44), 53% (23/43), and 80.6% (25/31) of K. pneumoniae, E. coli, and other Enterobacteriaceae isolates, respectively. Class 2 integrons were found in 14% of E. coli, but not in K. pneumoniae. All sul1 genes in K. pneumoniae were carried in class 1 integrons. Gene cassette arrays dfrA5 and dfrA15-aadA1 were most frequently associated with class 1 integrons, while class 2 integrons contained only dfrA1-sat2-aadA1 gene cassettes. This is the first report of sul3 gene in K. pneumoniae from human sources. The finding that mechanisms differ between E. coli and K. pneumoniae may broaden our understanding of cotrimoxazole resistance.201727533639
1233120.9835Prevalence, Antibiogram, and Resistance Profile of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates from Pig Farms in Luzon, Philippines. This cross-sectional study was conducted to determine the prevalence, antibiogram, and resistance profile of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) isolates from healthy pigs and pig farms in Luzon, Philippines. A total of 162 rectal samples from healthy finisher and breeder pigs and boot swab samples from pig houses were collected from 54 randomly selected pig farms. Bacteria were isolated and screened using MacConkey agar plate supplemented with 1 mg/L cefotaxime. Identification of bacteria and antimicrobial susceptibility test were carried out through Vitek(®) 2 and combined disk test. PCR amplifications were carried out in all isolates targeting bla(CTX-M) and its five major groupings, bla(TEM), and bla(SHV). The farm prevalence of ESBL-EC was 57.41% (95% confidence interval [CI] = 43.21-70.77). A total of 48 (29.63%) ESBL-EC isolates were isolated from samples that showed 14 different phenotypic multidrug resistance patterns. The prevalence of bla(CTX-M) gene was 91.67% (95% CI = 80.02-97.68). All major bla(CTX-M-groups) except bla(CTX-M-25group) were detected. The bla(CTX-M-1) was the most prevalent bla(CTX-M) gene, 75.0% (95% CI = 60.40-86.36). The prevalence of bla(TEM) and bla(SHV) genes was 91.67% (95% CI = 80.02-97.68) and 60.42% (95% CI = 45.27-74.23), respectively. Coexistence of different bla(CTX-M), bla(TEM), and bla(SHV) genes was observed in 44 isolates with 20 different genotypic patterns. High prevalence, diverse antibiogram profile, and genotypic resistance pattern of ESBL-EC isolates from healthy pigs and pig farms were observed in this study that could result in possible transmission to farm workers, susceptible bacteria, and the environment.202031532307
1311130.9835Prevalence and Molecular Characterization of Antimicrobial Resistance in Escherichia coli Isolated from Raw Milk and Raw Milk Cheese in Egypt. The goal of this study was to examine antimicrobial resistance and characterize the implicated genes in 222 isolates of Escherichia coli from 187 samples of raw milk and the two most popular cheeses in Egypt. E. coli isolates were tested for susceptibility to 12 antimicrobials by a disk diffusion method. Among the 222 E. coli isolates, 66 (29.7%) were resistant to one or more antimicrobials, and half of these resistant isolates showed a multidrug resistance phenotype (resistance to at least three different drug classes). The resistance traits were observed to tetracycline (27.5%), ampicillin (18.9%), streptomycin (18.5%), sulfamethoxazole-trimethoprim (11.3%), cefotaxime (4.5%), kanamycin (4.1%), ceftazidime (3.6%), chloramphenicol (2.3%), nalidixic acid (1.8%), and ciprofloxacin (1.4%). No resistance to fosfomycin and imipenem was observed. Tetracycline resistance genes tetA, tetB, and tetD were detected in 53 isolates, 9 isolates, and 1 isolate, respectively, but tetC was not detected. Aminoglycoside resistance genes strA, strB, aadA, and aphA1 were detected in 41, 41, 11, and 9 isolates, respectively. Sulfonamide resistance genes sul1, sul2, and sul3 were detected in 7, 25, and 3 isolates, respectively. Of 42 ampicillin-resistant isolates, bla(TEM), bla(CTX-M), and bla(SHV) were detected in 40, 9, and 3 isolates, respectively, and 10 (23.8%) ampicillin-resistant isolates were found to produce extended-spectrum β-lactamase. Each bla gene of extended-spectrum β-lactamase-producing E. coli was further subtyped to be bla(CTX-M-15), bla(CTX-M-104), bla(TEM-1), and bla(SHV-12). The class 1 integron was also detected in 28 resistant isolates, and three different patterns were obtained by PCR-restriction fragment length polymorphism. Sequencing analysis of the variable region revealed that four isolates had dfrA12/orfF/aadA2, two had aadA22, and one had dfrA1/aadA1. These data suggest that antimicrobial-resistant E. coli are widely distributed in the milk production and processing environment in Egypt and may play a role in dissemination of antimicrobial resistance to other pathogenic and commensal bacteria.201829323530
1232140.9835Monitoring of Non-β-Lactam Antibiotic Resistance-Associated Genes in ESBL Producing Enterobacterales Isolates. Genetic context of extended spectrum β-Lactamase (ESBL) producing Enterobacterales and its association with plasmid mediated quinolone resistance (PMQR), aminoglycoside modifying enzymes (AME) and Trimethoprim/Sulfamethoxazole (TMP-SMX) resistance is little known from North India. Therefore, the current study was aimed to investigate the frequency of Non-β-Lactam antibiotic resistance associated genes in extended spectrum β-Lactamase producing Enterobacterales. For this study, Non-Duplicate phenotypically confirmed ESBL producing Enterobacterales isolates (N = 186) were analyzed for ESBLs, PMQRs, AMEs and TMP-SMX resistance genes using polymerase chain reaction (PCR). PCR detected presence of PMQR genes in 81.29% (N = 139) of ESBL isolates (N = 171), AME genes in 60.82% and TMP-SMX resistance genes in 63.74% of the isolates. Molecular characterization of ESBL producing Enterobacterales showed 84.79% bla(TEM) followed by 73.68% bla(CTX-M), 43.86% bla(SHV), 19.88% bla(PER) and 9.94% bla(VEB), respectively. Analysis of PMQR genes revealed 77.7% aac(6')-lb-cr the most commonly detected gene followed by 67.63% oqxB, 62.59% oqxA, 43.17% qnrB, 19.42% qnrD, 18.7% qnrS, 9.35% qnrA, 3.6% qepA and 2.88% qnrC, respectively. Analysis of AMEs gene profile demonstrated 81.73% aac(6')-Ib, the most frequently encountered gene followed by 46.15% aph(3')-Ia, 44.23% ant(3")-Ia, respectively. A 100% prevalence of sul1, followed by dfrA (54.63%) and sul2 (15.74%) was observed. In summary, prevalence of ESBL-Producing genes (particularly bla(TEM) and bla(CTX-M)) along with PMQR, AMEs, and TMP-SMX resistant genes may potentially aid in the transfer of antimicrobial resistance among these strains.202033317078
833150.9835Diverse gene cassettes in class 1 integrons of facultative oligotrophic bacteria of River Mahananda,West Bengal, India. BACKGROUND: In this study a large random collection (n=2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007-2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products. METHODOLOGY/PRINCIPAL FINDINGS: Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ(2)) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6'-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae. CONCLUSIONS: Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.201323951238
834160.9835Molecular diversity of class 2 integrons in antibiotic-resistant gram-negative bacteria found in wastewater environments in China. The molecular architecture of class 2 integrons among gram-negative bacteria from wastewater environments was investigated in Jinan, China. Out of the 391 antibiotic-resistant bacteria found, 38 isolates harboring class 2 integrons encoding potentially transferrable genes that could confer antibiotic resistance were found. These isolates were classified into 19 REP-PCR types. These strains were identified using 16S rRNA gene sequencing and found to be as follows: Proteus mirabilis (16), Escherichia coli (7), Providencia spp. (7), Proteus spp. (2), P. vulgaris (3), Shigella sp. (1), Citrobacter freundii (1), and Acinetobacter sp. (1). Their class 2 integron cassette arrays were amplified and then either analyzed using PCR-RFLP or sequenced. The typical array dfrA1-sat2-aadA1 was detected in 27 isolates. Six atypical arrays were observed, including three kinds of novel arrangements (linF2(∆attC1)-dfrA1(∆attC2)-aadA1-orf441 or linF2(∆attC1)-dfrA1(∆attC2)-aadA1, dfrA1-catB2-sat2-aadA1, and estX(Vr)-sat2-aadA1) and a hybrid with the 3'CS of class 1 integrons (dfrA1-sat2-aadA1-qacH), and dfrA1-sat1. Twenty-four isolates were also found to carry class 1 integrons with 10 types of gene cassette arrays. Several non-integron-associated antibiotic resistance genes were found, and their transferability was investigated. Results showed that water sources in the Jinan region harbored a diverse community of both typical and atypical class 2 integrons, raising concerns about the overuse of antibiotics and their careless disposal into the environment.201323264021
1226170.9835Multi-drug resistant gram-negative enteric bacteria isolated from flies at Chengdu Airport, China. We collected flies from Chengdu Shuangliu International Airport to examine for the presence of bacteria and to determine the sensitivity patterns of those bacteria. A total of 1,228 flies were collected from 6 sites around Chengdu Shuangliu International Airport from April to September 2011. The predominant species was Chrysomya megacephala (n=276, 22.5%). Antimicrobial-resistant gram-negative enteric bacteria (n=48) were isolated from flies using MacConkey agar supplemented with cephalothin (20 microg/ml). These were identified as Escherichia coli (n=37), Klebsiella pneumoniae (n=6), Pseudomonas aeruginosa (n=3) and Aeromonas hydrophila (n=2). All isolated bacteria were tested for resistance to 21 commonly used antimicrobials: amoxicillin (100%), ticarcillin (100%), cephalothin (100%), cefuroxime (100%), ceftazidime 1 (93.8%), piperacillin (93.8%), cefotaxime (89.6%), ticarcillin-clavulanate (81.3%), trimethoprim-sulfamethoxazole (62.5%), ciprofloxacin (54.2%), gentamicin (45.8%), cefepime (39.6%), tobramycin (39.6%), ceftazidime (22.9%), cefoxitin (16.7%), amikacin (16.7%), netilmicin (14.6%), amoxicillin-clavulanate (6.3%) and piperacillin-tazobactam (2.1%). No resistance to meropenem or imipenem was observed. Antibiotic resistance genes among the isolated bacteria were analyzed for by polymerase chain reaction. Thirty of the 48 bacteria with resistance (62.5%) possessed the blaTEM gene.201324450236
1333180.9835Virulence-encoding genes related to extraintestinal pathogenic E. coli and multidrug resistant pattern of strains isolated from neonatal calves with different severity scores of umbilical infections. Umbilical infections in calves comprise a major cause of neonatal mortality and have been related to a variety of microorganisms. E. coli is an opportunistic enteropathogen characterized by a diversity of virulence factors (VF). Nonetheless, the gene profiles that encode VF associated with umbilical infections in calves and their effect on the clinical severity remains unclear. In this scenario, microbial identification (with an emphasis on E. coli), was carried out among 150 neonatal calves (≤30 days of age) with umbilical infections, where the omphalopathies were clinically scored as mild, moderate, or severe. Also, a panel of 16 virulence-encoding genes related to extraintestinal pathogenic E. coli (ExPEC) were investigated, i.e., fimbriae/adhesins (sfa/focDEa, papA, papC, afaBC), toxins (hlyA, sat, cnf1, cdt), siderophores (iroN, irp2, iucD, ireA), invasins (ibeA), and serum resistance (ompT, traT, kpsMT II). Bacteria and yeasts isolates were identified using mass spectrometry. Bacteria, yeasts, and fungi were isolated in 94.7% (142/150) of neonatal calves sampled. E. coli was the agent most frequently isolated (59/150 = 39.3%), in pure culture (27/59 = 45.8%) and combined infections (32/59 = 54.2%), although a great variety (n = 83) of other species of microorganisms were identified. Clinical severity scores of 1, 2, and 3 were observed in 32.2% (19/59), 23.7% (14/59), and 44.1% (26/59) of E. coli infections, respectively. The ExPEC genes detected were related to serum resistance (traT, 42/59 = 72.2%; ompT, 35/59 = 59.3%, kpsMTII, 10/59 = 17%), invasins (ibeA, 11/59 = 18.6%), siderophores (iucD, 9/59 = 15.3%; iroN, 8/59 = 13.6%), and adhesins/fimbriae (papA, 8/59 = 13.6%; papC, 15/59 = 9.6%). The presence of each virulence gene was not associated with the case's clinical score. Among all isolates, 89.8% (53/59) showed in vitro resistance to sulfamethoxazole/trimethoprim and 59.3% to ampicillin (35/59), while 94.1% (55/59) revealed a multidrug resistant profile. Great complexity of bacteria, yeast, and fungi species was identified, reinforcing the umbilical infections of neonatal calves as a polymicrobial disorder. The high occurrence of E. coli (39.3%) highlights the role of this pathogen in the etiology of umbilical infections in calves. Furthermore, a panel of ExPEC genes was investigated for the first time among calves that were clinically scored for case severity. The high prevalence of traT and ompT indicates that these serum resistance-related genes could be used as biomarkers for further investigations of ExPEC isolates from umbilical infections. Our results contribute to the etiological investigation, clinical severity scoring, antimicrobial resistance pattern, and virulence-related to ExPEC genes involved in umbilical infections of neonatal calves.202336427660
1238190.9834Lineages, Virulence Gene Associated and Integrons among Extended Spectrum β-Lactamase (ESBL) and CMY-2 Producing Enterobacteriaceae from Bovine Mastitis, in Tunisia. Extended Spectrum Beta-Lactamase (ESBL) Enterobacteriaceae are becoming widespread enzymes in food-producing animals worldwide. Escherichia coli and Klebseilla pneumoniae are two of the most significant pathogens causing mastitis. Our study focused on the characterization of the genetic support of ESBL/pAmpC and antibiotic resistance mechanisms in cefotaxime-resistant (CTXR) and susceptible (CTXS) Enterobacteriaceae isolates, recovered from bovine mastitis in Tunisia, as well as the analyses of their clonal lineage and virulence-associated genes. The study was carried out on 17 ESBL/pAmpC E. coli and K. pneumoniae and 50 CTXS E. coli. Detection of resistance genes and clonal diversity was performed by PCR amplification and sequencing. The following β-lactamase genes were detected: blaCTX-M-15 (n = 6), blaCTX-M-15 + blaOXA-1 (2), bla CTX-M-15 + blaOXA-1 + blaTEM-1b (2), blaCTX-M-15 + blaTEM-1b (4), blaCMY-2 (3). The MLST showed the following STs: ST405 (n = 4 strains); ST58 (n = 3); ST155 (n = 3); ST471 (n = 2); and ST101 (n = 2). ST399 (n = 1) and ST617 (n = 1) were identified in p(AmpC) E. coli producer strains. The phylogroups A and B1 were the most detected ones, followed by the pathogenic phylogroup B2 that harbored the shigatoxin genes stx1/stx2, associated with the cnf, fimA, and aer virulence factors. The qnrA/qnrB, aac(6′)-Ib-cr genes and integrons class 1 with different gene cassettes were detected amongst these CTXR/S isolated strains. The presence of different genetic lineages, associated with resistance and virulence genes in pathogenic bacteria in dairy farms, may complicate antibiotic therapies and pose a potential risk to public health.202236015067