Dissemination Of Class Research Articles

Two non-active site residues W165 and L166 prominently influence the beta-lactam hydrolytic ability of OXA-23 beta-lactamase.

Dissemination of class D OXA-type carbapenemases is one of the significant causes of beta-lactam resistance in Gram-negative bacteria. The amino acid residues present near the active site are involved in hydrolytic mechanism of class D carbapenemases, though it is not identified in OXA-23. Here, with the help of site-directed mutagenesis, we aimed to explicate the importance of the residues W165, L166 and V167 of the possible omega loop and residue D222 in the short β5-β6 loop on the activity of OXA-23. All the residues were substituted with alanine. The resultant proteins were assayed for the changes in activity in E. coli cells and purified for in vitro activity, and stability assessment. E. coli cells harboring OXA-23_W165A and OXA-23_L166A, individually, exhibited a significant decrease in resistance towards beta-lactam antibiotics as compared to OXA-23. Further, purified OXA-23_W165A and OXA-23_L166A imparted about >4-fold decrease in catalytic efficiency and displayed reduced thermal stability as compared to OXA-23. Bocillin-FL binding assay revealed that W165A substitution results in improper N-carboxylation of K82, leading to deacylation deficient OXA-23. Therefore, we infer that the residue W165 maintains the integrity of N-carboxylated lysine (K82) of OXA-23 and the residue L166 might be responsible for properly orientating the antibiotic molecules.

High incidence of multidrug resistance and class 1 and 2 integrons in Escherichia coli isolated from broiler chickens in South of Iran.

The objective was to investigate the multidrug resistance and presence of class 1 and 2 integrons in 300 Escherichia coli isolates obtained from 20 broiler farms during three rearing periods (one-day-old chicks, thirty-day-old chickens, and one day before slaughter) in Fars, South Iran. Results showed that 81.00%, 82.00%, and 85.00% of isolates were multidrug-resistant on the first day, thirty-day-old chickens, and one day before slaughter, respectively. Multidrug-resistant E. coli isolates were further examined for the presence of class 1 and 2 integrons using PCR assay. The existence of class 1 integron-integrase gene (intI1) was confirmed in 68.40%, 72.70%, and 60.90% of multidrug-resistant isolates from stage 1, stage 2, and stage 3 of the rearing period, respectively. The frequency of class 2 integron-integrase gene (intI2) during the first to the third stage of sampling was 2.60%, 25.50%, and 30.40%. Also, sequence analysis of the cassette arrays within class 1 integron revealed the presence of the genes associated with resistance for trimethoprim (dfrA), streptomycin (aadA), erythromycin (ereA), and orfF genes. The results revealed that percentages of antimicrobial resistance in E. coli isolates were significantly higher in the middle and end stages of the rearing period. In conclusion, widespread dissemination of class 1 integrons in all three stages and rising trends of class 2 integrons existence in E. coli isolates during the rearing period of broiler chickens could exacerbate the spread of resistance factors among bacteria in the poultry industry. Future research is needed to clarify its implication for human health.

Tn6603, a Carrier of Tn5053 Family Transposons, Occurs in the Chromosome and in a Genomic Island of Pseudomonas aeruginosa Clinical Strains.

Transposons of the Pseudomonas aeruginosa accessory gene pool contribute to phenotype and to genome plasticity. We studied local P. aeruginosa strains to ascertain the encroachment of mer-type res site hunter transposons into clinical settings and their associations with other functional modules. Five different Tn5053 family transposons were detected, all chromosomal. Some were solitary elements; one was in res of Tn1013#, a relative of a reported carrier of int-type res site hunters (class 1 integrons), but most were in res of Tn6603, a new Tn501-related transposon of unknown phenotype. Most of the Tn6603::Tn elements, and some Tn6603 and Tn6603::Tn elements found in GenBank sequences, were at identical sites in an hypothetical gene of P. aeruginosa genomic island PAGI-5v. The island in clonally differing strains was at either of two tRNALys loci, suggesting lateral transfer to these sites. This observation is consistent with the membership of the prototype PAGI-5 island to the ICE family of mobile genetic elements. Additionally, the res site hunters in the nested transposons occupied different positions in the Tn6603 carrier. This suggested independent insertion events on five occasions at least. Tn5053 family members that were mer-/tni-defective were found in Tn6603- and Tn501-like carriers in GenBank sequences of non-clinical Pseudomonas spp. The transposition events in these cases presumably utilized tni functions in trans, as can occur with class 1 integrons. We suggest that in the clinical context, P. aeruginosa strains that carry Tn6603 alone or in PAGI-5v can serve to disseminate functional res site hunters; these in turn can provide the requisite trans-acting tni functions to assist in the dissemination of class 1 integrons, and hence of their associated antibiotic resistance determinants.

The Role of Agriculture in the Dissemination of Class 1 Integrons, Antimicrobial Resistance, and Diversity of Their Gene Cassettes in Southern China.

Integrons are hot spots for acquiring gene cassettes from the environment and play a major role in the bacterial evolution and dissemination of antimicrobial resistance (AMR), thus posing a serious threat. There are currently studies on integrons and antibiotic resistance genes; however, the presence and association of integrons in different agricultural crops and their subsequent dissemination and role in AMR have not been reported previously. This study examines the abundance of integrons, their gene cassette diversity in various crop soils, and their role in the dissemination of AMR in the southern region of China. Samples from different agri-crop soil, such as rice (R.S), sugarcane (S.S), citrus (C.S), banana (B.S), agricultural runoff (the point where the runoff of all sites meet (R.O)), and wild (non-agricultural) soil (W.S), were collected. Quantitative PCR was used to determine the abundance of integrons, and clone libraries were constructed to examine the gene cassette arrays. All the tested samples were found positive for Class-I (CL1) integrons and revealed a higher concentration and higher relative abundance of R.S than the others, with the least found at the W.S site. The W.S CL1 cassette arrays were found empty, and no putative conserved domains were found. The R.O was found to contain a high number of gene cassettes with various functions, while the smallest number of gene cassettes was found in the S.S among the crop soils. Most of the gene cassettes presented by the R.O were primarily shared with other sites, and the antibiotic-resistant genes were consistently observed to be dominant. The constructed clone libraries represented a diverse gene cassette array with 16% novel gene cassettes that play a vital role in pathogenesis, transportation, biosynthesis, and AMR. Most resistance-related gene cassettes were associated with the genes encoding resistance to quaternary ammonium compound (QAC) and aminoglycosides. This study highlights the significant differences in the abundance of integrons among various agricultural soils and offers deep insight into the pools of gene cassettes that play a key role in the dissemination of integrons and AMR.

Dissemination of Class 1, 2 Genes in Extensive Drug Resistant (XDR) Acinetobacterbaumannii Isolated from Clinical Speciemens in Babylon Province, Iraq

Dissemination of Class 1, 2 Genes in Extensive Drug Resistant (XDR) Acinetobacterbaumannii Isolated from Clinical Speciemens in Babylon Province, Iraq

The widespread dissemination of integrons throughout bacterial communities in a riverine system

Anthropogenic inputs increase levels of antimicrobial resistance (AMR) in the environment, however, it is unknown how these inputs create this observed increase, and if anthropogenic sources impact AMR in environmental bacteria. The aim of this study was to characterise the role of waste water treatment plants (WWTPs) in the dissemination of class 1 integrons (CL1s) in the riverine environment. Using sample sites from upstream and downstream of a WWTP, we demonstrate through isolation and culture-independent analysis that WWTP effluent significantly increases both CL1 abundance and antibiotic resistance in the riverine environment. Characterisation of CL1-bearing isolates revealed that CL1s were distributed across a diverse range of bacteria, with identical complex genetic resistance determinants isolated from both human-associated and common environmental bacteria across connected sites. Over half of sequenced CL1s lacked the 3′-conserved sequence ('atypical’ CL1s); surprisingly, bacteria carrying atypical CL1s were on average resistant to more antibiotics than bacteria carrying 3′-CS CL1s. Quaternary ammonium compound (QAC) resistance genes were observed across 75% of sequenced CL1 gene cassette arrays. Chemical data analysis indicated high levels of boron (a detergent marker) downstream of the WWTP. Subsequent phenotypic screening of CL1-bearing isolates demonstrated that ~90% were resistant to QAC detergents, with in vitro experiments demonstrating that QACs could solely select for the transfer of clinical antibiotic resistance genes to a naive Escherichia coli recipient. In conclusion, this study highlights the significant impact of WWTPs on environmental AMR, and demonstrates the widespread carriage of clinically important resistance determinants by environmentally associated bacteria.

Dissemination of Class 1, 2 and 3 Integrons among Different Multidrug Resistant Isolates of Acinetobacter baumannii in Tehran Hospitals, Iran

A total of 100 non-duplicate Acinetobacter baumannii isolates were collected from different hospitals in Tehran and were confirmed as A. baumannii by conventional biochemical and API testing. Antimicrobial susceptibility of these isolates was checked by a disk diffusion method in accordance with CLSI guidelines. The isolates were then detected as carrying class 1 and 2 integron gene cassettes by PCR evaluation and then genotyped by REP-PCR. More than 50% (n = 50) of the isolates were multidrug resistant. The results showed that more than 80% of all multidrug resistant A. baumannii strains carry a class 1 integron. Distribution of IntI 1 and IntI2 among A. baumannii isolates was 58% and 14%, respectively. Analysis of a conserved segment of class 1 integron showed a range from 100 bp to 2.5 kb. REP-PCR fingerprinting showed more than 20 genotypes among A. baumannii strains. TIhere was no relationship between REP genotypes and the distribution of different classes of integrons. This is a comprehensive study on the distribution of different classes of integrons among A. baumannii in Iran. Considering the exact role of integrons in coding drug resistance in bacteria, the findings of this study could help us find antimicrobial resistant mechanisms among A. baumannii isolates in Iran.

Identification and characterization of class 1 integrons among Pseudomonas aeruginosa isolates from patients in Zhenjiang, China

The role of integrons in the spread of antibiotic resistance has been well established. The aim of this study was to investigate the resistance profiles of Pseudomonas aeruginosa isolated from patients in Zhenjiang to 13 antibiotics, and to identify the structure and dissemination of class 1 integrons. The Kirby-Bauer disk diffusion assay was used to determine the rate of P. aeruginosa resistance. Class 1 integrons from multidrug-resistant isolates were amplified by PCR, and their PCR products were sequenced. We also analyzed the integron structures containing the same gene cassettes by restriction fragment length polymorphism (RFLP). Isolates were genotyped by pulsed-field gel electrophoresis (PFGE). The resistance rates were between 29.6% and 90.1%. The prevalence of class 1 integrons was 38.0%. These integrons included five gene cassettes (aadB, aac6-II, blaPSE-1, dfrA17, and aadA5). The dfrA17 and aadA5 gene cassettes were found most often. Class 1 integrons were found to be widespread in P. aeruginosa isolated from clinical samples in the Zhenjiang area of China. The antibiotic resistance rates in class 1 integron-positive strains of P. aeruginosa were noticeably higher than those in class 1 integron-negative strains. PFGE showed that particular clones were circulating among patients.

The distribution and structure of class I integron in the multidrug-reisistant Pseudomonas aeruginosa

Objective To investigate the antimicrobial susceptibility of Pseudomonas aeruginosa (P. aeruginosa) isolated from Zhenjiang area to 13 routinely used antibiotics and identify the structure and dissemination of class Ⅰ integron. Methods K-B test was used to determine the resistant rate of 71 strains of P. aeruginosa. DNA template was extracted by boiling method, PCR method was utilized to detect class Ⅰintegron, and subsequently gene cassettes were analyzed by sequencing. Results The resistant rates to 13 routinely used antibiotics were quite different from 18. 3 to 77.5% among 71 strains of P. aeruginosa. The prevalence of class Ⅰ integron was 38%. These integrons include 5 gene cassettes ( aadB, aac (6) - Ⅱ , PSE-Ⅰ , dfrA17 and aadAS), in which dfrA17 and aadA5 gene cassette were frequently found. Comparing with the negative strains of integron, the positive strains of integron has obviously higher resistance to ten the antibiotics including piporacillin, piperacillin-tazobactam, ceftriaxone, cefepime, ceftazidime, gentamicin,amikacin, tobmmycin, levofloxacin, and ciprofloxacin. Conclusions The resistant rates of P. aeruginosa to 13 drugs were different, and the resistant rates of integron positive strains were obviously higher than integron negative strains, which indicates that integron may play an important role in multidrug reisistance of P. aeruginoosa. Key words: Pseudomonas aeruginosa; Drug-resistance,multiple, bacterial; Integrons