Characterisation of two new gene cassettes, aadA5 and dfrA17

Escherichia coli INS33 was isolated from the urinary tract of an infected patient. It was resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfafurazole, tetracycline and trimethoprim. PCR screening revealed the presence of a class 1 integron that harboured two new gene cassettes, designated dfrA17 and aadA5. The new dfrA17 cassette was 91% identical to the known dfrA7 cassette. The aadA5 cassette was 95% identical over the first 830 bp to aadA4, but lacked the IS26 element found at the 3' end of this truncated cassette. Cloning and expression of the cassette region demonstrated that dfrA17 conferred high level resistance to trimethoprim but aadA5 conferred resistance to spectinomycin but not to streptomycin.     

Identification and molecular characterization of class 1 integrons in multiresistant Escherichia coli isolates from poultry litter

This study describes the prevalence of arrays of class 1 integron cassettes and Qnr determinants (A, B, and S) in 19 fluoroquinolone-resistant Escherichia coli isolates from chicken litter. qnrS and qnrA were the predominant genes in these fluoroquinolone-resistant isolates, and an uncommon array of aacA4-catB3-dfrA1 gene cassettes from a class1 integron was found. Additionally, aadA1 and dfrA1 gene cassettes, encoding resistance to streptomycin and trimethoprim, constituted the most common genes identified and was located on megaplasmids as well on the chromosome. Antibiotic resistance, pulsed-field gel electrophoresis (PFGE), and plasmid data suggest a genetically diverse origin of poultry E. coli isolates.     

Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.     

Plasmid pB8 is closely related to the prototype IncP-1beta plasmid R751 but transfers poorly to Escherichia coli and carries a new transposon encoding a small multidrug resistance efflux protein

The IncP-1beta plasmid pB8, which confers resistance to amoxicillin, spectinomycin, streptomycin, and sulfonamides, was previously isolated from a sewage treatment plant. It was found to possess abnormal conjugative transfer properties, i.e., transfer to Escherichia coli by conjugation or electroporation could not be detected. We showed in this study that plasmid pB8 is transferable to E. coli by conjugation, but only at low frequencies and under specific experimental conditions, a phenomenon that is very unusual for IncP-1 plasmids. Determination of the complete 57,198bp pB8 nucleotide sequence revealed that the backbone of the plasmid consists of a complete set of IncP-1beta-specific genes for replication initiation, conjugative plasmid transfer, stable inheritance, and plasmid control with an organisation identical to that of the prototype IncP-1beta plasmid R751. All of the minor differences in the pB8 backbone sequence compared to that of R751 were also found in other IncP-1beta plasmids known to transfer to and replicate in E. coli. Plasmids pB8 and R751 can be distinguished with respect to their accessory genetic elements. First, the pB8 region downstream of the replication initiation gene trfA contains two transposable elements one of which is similar to Tn5501. The latter transposon encodes a putative post-segregational-killing system and the small multidrug resistance (SMR) protein QacF, mediating quaternary ammonium compound resistance. The accessory genes in this region are not responsible for the poor plasmid transfer to E. coli since a pB8 deletion derivative devoid of all genes in that region showed the same conjugative transfer properties as pB8. A Tn5090/Tn402 derivative carrying a class 1 integron is located between the conjugative transfer modules. The Tn5090/Tn402 integration-sites are exactly identical on pB8 and R751 but in contrast to R751 the pB8 element carries the resistance gene cassettes oxa-2 for amoxicillin resistance and aadA4 for streptomycin/spectinomycin resistance, the integron-specific conserved segment consisting of the genes qacEDelta1, sul1, and orf5, and a truncated tni transposition module (tniAB). Although future work will have to determine the molecular basis for the poor transfer of pB8 to E. coli, our findings demonstrate that the host-range of typical IncP-1 plasmids may be less broad than expected.     

Characterization of the sat 4 gene encoding a streptothricin acetyltransferase in Campylobacter coli BE/G4

Abstract The sat 4 streptothricin resistance gene from Campylobacter coli BE/G4 was cloned into pUC18, and its nucleotide sequence was determined. Streptothricin acetyltransferase activity was detected in Escherichia coli cells containing recombinant plasmid pAT132 which carries the sat4 gene as an insert. The deduced amino acid sequence displayed 21–27% amino acid identity with streptothricin acetyltransferases from E. coli and streptothricin producers Streptomyces lavendulae and Streptomyces noursei . The sat 4 gene was detected by hybridization in clinical and environmental isolates of Campylobacter spp.     

Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates

A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coli O157:H7.     

Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in E. coli. A Tn5* mutant able to express streptomycin resistance in E. coli was isolated. With this mutant, it was demonstrated that in E. coli the expression of the three resistance genes is coordinated in a single operon.     

Molecular characterisation of In51, a class 1 integron containing a novel aminoglycoside adenylyltransferase gene cassette, aadA6, in Pseudomonas aeruginosa

Polymerase chain reaction-amplification and subsequent sequencing of the variable region of a novel integron, In51, from Pseudomonas aeruginosa revealed the presence of a novel aminoglycoside adenylyltransferase gene, aadA6, together with an open reading frame of unknown function, orfD. AADA6 enzyme has only 75% amino acid identity with AADA1 and is able to confer high level resistance to streptomycin and spectinomycin in Escherichia coli.     

An integron of class 1 is present on the plasmid pCG4 from Gram-positive bacterium Corynebacterium glutamicum

The streptomycin/spectinomycin resistance determinant of the 29-kb plasmid pCG4 from Corynebacterium glutamicum was found to be a part of a typical class 1 integron. The sequence analysis revealed that the integron (designated InCg) identified in this Gram-positive bacterium is almost identical to the integron InC present on the plasmid pSA1700 from the Gram-negative bacterium Pseudomonas aeruginosa. Differences in only two base pairs were found in the 3.8-kb sequence. One base substitution (G→C) is present in the streptomycin/spectinomycin resistance determinant which is thus identical to the aadA2a gene from the integron In6 of the broad-host-range plasmid pSa. The other one (C→G) is present in the extended −10 region of the integron promoter involved in expression of the antibiotic resistance gene. It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one.     

Effect of mutation to streptomycin resistance on amber suppressor genes

Three classes of nonidentical streptomycin-resistant mutations were distinguished in Escherichia coli by their effect on the efficiency of suppression by an amber suppressor gene, sup E. The first class of mutation caused a strong restriction in efficiency of suppression of an amber codon in various cistrons of phage lambda and in an alkaline phosphatase structural gene of E. coli. The second class caused weak restriction, and the third class caused no restriction. The restrictive effect of the streptomycin resistance mutation of the first class on the sup E gene was reduced by addition of streptomycin. This mutation had little effect on efficiencies of suppression by amber suppressor genes sup D and sup F. Analyses on the alkaline phosphatase formed in the suppressor strain indicated that mutation to restrictive streptomycin resistance causes a reduction in translation of the amber codon in the alkaline phosphatase structural gene.     

产超广谱β-内酰胺酶大肠埃希菌Ⅰ类整合子检测与耐药性关系

目的了解I类整合子在产ESBLs和非产ESBLs大肠埃希菌中分布状况,分析I类整合子在细菌多重耐药中的作用。方法用PCR方法扩增I类整合酶基因,经电泳后检测扩增产物。用2χ检验进行统计学分析,P<0.05为差异有显著性。结果105株大肠埃希菌检出I类整合子46株,检出率为43.8%。I类整合子在产ESBLs菌的检出率为53.4%,明显高于非产ESBLs菌(31.9%),2χ检验,P<0.01。I类整合子阳性菌株多重耐药率为68.8%(33/48),明显高于阴性菌株(33.3%),P<0.05。I类整合子阳性菌株和产ESBLs菌均对青霉素类、喹诺酮类、磺胺类抗生素表现出较高的耐药率。所有菌株均对亚胺培南敏感。结论I类整合子携带与产ESBLs菌株耐药有关,I类整合子阳性菌株对多种抗生素的耐药率大于整合子阴性菌株。     

临床分离肺炎克雷伯菌Ⅰ类整合子的结构与功能

为了探索细菌多重耐药性的产生和播散的分子机制, 文章对2002～2007年间179株临床分离的肺炎克雷伯菌进行耐药性、I类整合子可变区基因盒结构以及基因盒携带的耐药性基因进行分段克隆和耐药性功能测定。结果显示：65.9%(118/179)的肺炎克雷伯菌表现出对至少两种以上的抗生素(主要为β-内酰胺类、氨基糖苷类和喹诺酮类抗菌药物)的耐药性; 36.3%(65/179)的菌株检出单条或者双条I类整合子基因盒条带; 对整合子阳性组与阴性组的耐药率进行比较发现, 除氨基糖苷类、喹诺酮类和复方新诺明等药物的耐药性存在显著性差异(P<0.01)外, 其余药物的差异不显著; 共发现15种耐药基因构成形式的整合子基因盒, 其中以dfrA17-aadA5最为多见, 实验证明整合子可由接合转移耐药性质粒携带; 对整合子基因盒(dhfr17-orfF-aadA2)分段克隆的耐药性功能研究发现, 3个克隆重组子(pET28a-dhfr17、pET28a-dhfr17-orfF和pET28a-dhfr17-orfF-aadA2)对复方新诺明的抗性(MIC值)均为256 µg/mL, 重组子pET28a-dhfr17-orfF与重组子pET28a-dhfr17对链霉素的抗性无明显区别, 和受体菌一样MIC值均为8 µg/mL, 而pET28a-dhfr17-orfF-aadA2对链霉素的抗性则明显提高, MIC值为256 µg/mL。结果表明, I类整合子在肺炎克雷伯菌中较常见, 携带氨基糖苷类和甲氧苄啶类的耐药基因盒在数量上占优势, 且整合子携带的耐药基因具有耐药性功能, 位于可水平转移耐药性质粒的耐药性基因相关的整合子对病原菌耐药性播散具有重要意义。 目的基因     

Antimicrobial resistance patterns and characterization of integrons of <Emphasis Type="Italic">Shigella sonnei</Emphasis> isolates in Seoul, 1999–2008

A total of 66 Shigella sonnei isolates from 1999 to 2008 in Seoul was analyzed for their antimicrobial resistance, carriage of integron, and the patterns of Pulsed-field gel electrophoresis (PFGE). A high level of antimicrobial resistance to streptomycin (100%), trimethoprim/sulfamethoxazole (95%), tetracycline (94%), nalidixic acid (65%), and ampicillin (41%) was observed among S. sonnei isolates. Fourteen profiles of antimicrobial resistance were identified with the most common resistance profile being nalidixic acid, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole (35%). PCR and DNA sequencing analysis revealed the presence of class 2 integron in all isolates, and class 1 and 2 integrons in 7 isolates. The class 2 integron carried two types of gene cassettes. One cassette array was dfrI, sat2, and aadA1 (91%), and the other was dfr1 and sat1 (8%). dfrA12 and aadA2 gene cassette was found in one isolate containing class 1 integron. PFGE was carried out to examine the genetic relatedness among isolates. All isolates except for one showed similar PFGE patterns (similarity of 80.1%). These results suggest that the S. sonnei isolated during 1999–2008 in Seoul have similar lineages that have not undergone evolutionary changes with time.     

Investigation of a global collection of nontyphoidal Salmonella of various serotypes cultured between 1953 and 2004 for the presence of class 1 integrons

In this study, antibiotic resistance profiles, and the presence of class 1 integrons were determined for 108 Salmonella isolates comprising 37 serotypes cultured from a variety of sources between 1953 and 2004. Antibiogram analyses showed that all isolates were resistant to streptomycin/spectinomycin. Molecular analysis revealed that 50% of the collection contained an integrase-encoding gene (int1) and 25% contained class 1 integrons. A Salmonella Wien isolate possessing a complete class 1 integron with a dfrA5-ereA2 gene arrangement within the variable region was characterized.     

Impact of antimicrobial usage on antimicrobial resistance in commensal Escherichia coli strains colonizing broiler chickens

Escherichia coli strains isolated from commercial broilers and an experimental flock of chickens were screened to determine phenotypic expression of antimicrobial resistance and carriage of drug resistance determinants. The goal of this study was to investigate the influence of oxytetracycline, sarafloxacin, and enrofloxacin administration on the distribution of resistance determinants and strain types among intestinal commensal E. coli strains isolated from broiler chickens. We detected a high prevalence of resistance to drugs such as tetracycline (36 to 97%), sulfonamides (50 to 100%), and streptomycin (53 to 100%) in E. coli isolates from treated and untreated flocks. These isolates also had a high prevalence of class 1 integron carriage, and most of them possessed the streptomycin resistance cassette, aadA1. In order to investigate the contribution of E. coli strain distribution to the prevalence of antimicrobial resistance and the resistance determinants, isolates from each flock were DNA fingerprinted by enterobacterial repetitive intergenic consensus sequence (ERIC) PCR. Although very diverse E. coli strain types were detected, four ERIC strain types were present on all of the commercial broiler farms, and two of the strains were also found in the experimental flocks. Each E. coli strain consisted of both susceptible and antimicrobial agent-resistant isolates. In some instances, isolates of the same E. coli strain expressed the same drug resistance patterns although they harbored different tet determinants or streptomycin resistance genes. Therefore, drug resistance patterns could not be explained solely by strain prevalence, indicating that mobile elements contributed significantly to the prevalence of resistance.     

Gene lmrB of Corynebacterium glutamicum confers efflux-mediated resistance to lincomycin

The lmrB gene of Corynebacterium glutamicum, which confers specific resistance to lincosamides, such as lincomycin and clindamycin, was isolated. C. glutamicum cells, carrying the lmrB gene in a multicopy plasmid, showed increased resistance to lincomycin with a MIC of 230 microg/ml, which is a 9-fold increase compared to that of the wild type. The lmrB-disrupted mutant became sensitive to the compound. No difference in sensitivity to erythromycin, penicillin G, tetracycline, chloramphenicol, spectinomycin, nalidixic acid, gentamicin, streptomycin, ethidium bromide, and sodium dodecyl sulfate was observed. The protonophore carbonyl cyanide m-chlorophenylhydrazone abolished the lincomycin-resistance of lmrB-carrying cells. The putative protein product of the gene contained 14-transmembrane regions and showed high amino acid-sequence homology to the drug efflux pumps of other organisms. In addition, the putative protein contained a motif for major facilitators, suggesting a role in efflux-mediated resistance to lincomycin.     

Characterization of antibiotic resistance genes linked to class 1 and 2 integrons in strains of Salmonella spp. isolated from swine

The aim of this study was to characterize the antibiotic resistance profiles, the integron-associated resistance determinants, and the potential ability of transferring these determinants by conjugation in Salmonella enterica isolated from swine. Fifty-four strains of Salmonella spp. were isolated from healthy swine. The percentages of resistance, determined by the plate dilution method were as follows: oxytetracycline (41%), streptomycin (39%), sulphamethoxazol+trimethoprim (19%), enrofloxacin-ciprofloxacin (13%), and amoxicillin (0%). The most important resistance serovars were Salmonella Branderburg, Salmonella Derby, Salmonella Typhimurium, and Salmonella Heidelberg. The oxytetracycline-resistant strains amplified the genes tetA (36%), tetB (64%); and the strains resistant to streptomycin and trimethoprim amplified the genes aadA1 (100%) and dfrA1 (100%), respectively. None of the fluoroquinolone-resistant strains amplified the gene qnr. Ten strains amplified the class 1 integron harboring the cassette aadA1. Six strains amplified the class 2 integron harboring the cassettes dfrA1, sat1, and aadA1. The conjugation assays showed that 2 strains transferred the tetA and aadA1 genes and the class 1 integron to a recipient strain. Taken together, the results obtained in this study show a high percentage of resistance in and the presence of integrons in strains of S. enterica isolated from swine. This information should support the implementation of regulations for the prudent use of antimicrobial agents in food-producing animals.     

Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa

Abstract A new integron, located on the R plasmid of Pseudomonas aeruginosa , was isolated in Japan. This integron was made up of two conserved segments (5'- and 3'-conserved segments) and a single streptomycin resistance gene as a gene cassette. The structure of this integron resembles that of integron InC, the existence of which was postulated by Bissonnette and Roy (J. Bacteriol. 174, 1248–1257, 1992).