Variable gene cassette patterns of class 1 integron-associated drug-resistant Escherichia coli in Taiwan

This study characterized class 1 integrons in Escherichia coli in Taiwan. The stability and changes in gene cassettes inserted into integrons were also evaluated. The study included 436 clinical strains of E. coli isolated in 2002. Class 1 integrons were characterized by polymerase chain reaction and direct sequencing. Genetic localization of class 1 integrons was determined by conjugal transfer and Southern hybridization. The results indicated that 64% of E. coli isolates carried class 1 integrons. Molecular analysis revealed that the class 1 integrons harbored 13 different antimicrobial resistance gene cassettes and two unknown gene cassettes; the predominant cassettes were aadA and dfrA. Novel gene cassettes first recovered from E. coli were aacA4 and linF. Cassette arrays orfD-aacA4-catB8 and aadA1-linF were also observed. Gene cassette dfrA12-orfF-aadA2 was stable. The class 1 integron and dfrA17-aadA5 gene cassette were located on the same transferable plasmids and were capable of transmission. Therefore, the increased drug resistance of clinical isolates may be explained by antibiotic selective pressure and widespread presence of integrons. Under antibiotic selective pressure, gene cassette-mediated resistance may not be easily lost. The potential role of integrons in the uptake and dissemination of resistance genes by plasmid between species of bacteria may decrease the therapeutic effectiveness of antibiotics.     

Characterization of class 1 integrons and antimicrobial resistance in CTX-M-3-producing Serratia marcescens isolates from southern Taiwan

The objective of this study was to characterize the gene cassettes of class 1 integrons and antimicrobial resistance among CTX-M-3-producing Serratia marcescens isolates from different specimens in southern Taiwan. One hundred and twenty-two isolates (70.5%) of 173 CTX-M-3-producing S. marcescens isolates were positive for class 1 integrons, including 53.3% of blood isolates, 94.1% of urine isolates, and 87.2% of sputum isolates. No class 2 or class 3 integrons were detected in this study. By PCR with primers 5'-CS and 3'-CS for the amplification of gene cassettes regions, amplicons ranging from 0.7 to 3.0 kb in length were found in 108 (88.5%) of the 122 class 1 integron-containing isolates of CTX-M-3-producing S. marcescens isolates. Ten different types by pattern of amplicons for class 1 integrons were obtained. The Type I amplicon (46.3%) harbors two different class 1 integrons containing the gene cassette of aadA2 and aadB-catB3, respectively, and was most prevalent in the gene cassette region-positive S. marcescens isolates, followed by the Type II amplicon, which harbors one class 1 integron containing the gene cassette dfrA12-orfF-aadA2 (28.7%). Most of the S. marcescens isolates (66.7%, 8/12) harboring three different class 1 integrons (Type IV amplicon) were found in blood isolates. Class 1 integrons were conjugally transferred to recipients in 92.0% of S. marcescens harboring two different class 1 integrons containing the gene cassettes aadA2 and aadB-catB3, respectively. The transfer rate of class 1 integron carrying dfrA12-orfF-aadA2 was detected in 77.4% of S. marcescens isolates. The results showed that all those isolates with conjugative transfer of integrons carried their class 1 integrons on the conjugative plasmids.     

Genetic basis of multidrug resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves in Egypt

Up to this date, nothing is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals in Africa. Therefore, this study was carried out to screen the incidence of multidrug-resistant (MDR) strains of Salmonella from neonatal calf diarrhea in Egypt and also to characterize the molecular basis of this resistance. Nine unique Salmonella isolates were obtained from 220 fecal samples, and six of these showed multidrug resistance phenotypes and harbored at least two antimicrobial resistance genes. Four were Salmonellaenterica serovar Typhimurium and two were S.enterica serovar Enteritidis. Class 1 integrons were identified in all MDR Salmonella isolates. The identified gene cassettes within class 1 integrons were as follows; aminoglycoside adenyltransferase type A (aadA1, aadA2 and aadA5), which confer resistance to streptomycin and spectinomycin, and dihydrofolate reductase gene cassettes (dfrA1, dfrA15 and dfrA15), which confer resistance to trimethoprim. A class 2 integron containing dfrA1-sat2-aadA1 gene cassettes was identified in only one isolate of S. enterica serovar Enteritidis. The β-lactamase-encoding gene, bla_TEM-1, was identified in five isolates and the extended-spectrum β-lactamase-encoding genes, bla_CMY-2 and bla_SHV-12, were identified in S. enterica serovar Typhimurium. Furthermore, the plasmid-mediated quinolone resistance genes, qnrB, qnrS and aac(6′)-Ib-cr, were also identified. To the best of our knowledge, this is the first report of qnrS in S. enterica serovar Enteritidis, qnrB in S. enterica serovar Typhimurium, and aac(6′)-Ib-cr in Salmonella of animal origin. Also, this is the first report of the molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Africa.     

临床痢疾志贺菌整合子检测及耐药基因盒序列分析

目的 检测印度东部1988、1995和2002年部分临床分离痢疾志贺菌中细菌耐药关系密切的1、2、3类整合酶基因及整合子携带的耐药基因盒的分布,分析整合子系统对志贺菌耐药的影响.方法 纸片扩散法检测实验菌株对药物的敏感性;应用PCR方法对16株临床耐药菌株进行1、2、3类整合酶基因(intI)筛选,对阳性样本可变区基因盒序列进行鉴定分析.结果 所有16株菌均耐4种及4种以上药物,包括β-内酰胺类、氨基糖苷类、四环素类、磺胺类、氯霉素类和喹诺酮类.13株菌检出1类整合酶基因,全部菌株含2类整合酶基因,即发现同时存在两种整合子结构菌株,未检测到3类整合酶基因.1类整合酶插入基因盒以blaara30-aadAl基因家族为主,分别对β-内酰胺类抗生素、链霉素、壮观霉素耐药;2类整合酶插人基因盒以dfrAl-satl组合为主,分别对甲氧苄氨嘧啶、链丝菌素耐药,同时在4株菌中发现dfrAl-satl-aadAl基因盒组合.结论 2类整合子普遍存在于临床志贺菌中.整合子与志贺菌的多重耐药具有密切相关性.     

Molecular diversity of class 1 integrons in human isolates of Aeromonas spp. from southern Taiwan

This work studies antimicrobial resistance and class 1 integrons of Aeromonas spp. in human isolates from southern Taiwan. PCR amplification and DNA sequence analyses were performed to characterize the gene cassette regions of the class 1 integron in 204 isolates of Aeromonas hydrophila, 36 isolates of A. sobria, 23 isolates of A. veronii, and 4 isolates of A. caviae. By using Southern hybridization with an intI1 probe to determine the presence of class 1 integrons in the 9 isolates of Aeromonas spp. harboring plasmid DNA, only 2 isolates, one A. veronii AV69 harboring 176-kb plasmid DNA, and one A. hydrophila AH207 harboring 149-kb plasmid DNA were identified. A conjugation experiment was carried out with 2 isolates of A. veronii AV69 and A. hydrophila AH207. Only one transconjugant of Escherichia coli AH207, containing 149-kb plasmids obtained from A. hydrophila AH207, was identified. ERIC-PCR analysis was performed to analyze the genetic relatedness in all isolates of Aeromonas spp. that carry class 1 integrons. The results of cluster analysis in this experiment revealed that none of these isolates were clonal, which may indicate that they were not related to the outbreak. Among the 267 isolates tested, class 1 integrons were detected in 37 isolates (13.9%) of Aeromonas spp. from humans. No class 2 or class 3 integrons were detected in this study. Gene cassette structures were identified in 30 (81.0%) of 37 isolates of Aeromonas spp. containing class 1 integrons. The gene cassette of dfrA12-orfF-aadA2 was the most prevalent in the gene cassette array (16.0%), followed by arr3-aacA4 (13.3%) and dfr2d-catB3-aadA1 (10.0%). Four novel arrays of gene cassettes were also identified, namely, dfr2d-catB3-aadA1, aadA1-aac(6')-II, aadA4a, and aac(6')-II-blaOXA-21-catB3. This is the first report of Aeromonas spp. isolates from humans.     

Widespread presence of dfrA12 and its association with dfrA12-aadA2 cassette in Salmonella enterica isolates from swine

One hundred and eighty-nine Salmonella isolates from swine were tested for susceptibility to nine antimicrobial agents, presence of dfrA12 and class 1 integrons containing dfrA12-orfF-aadA2 cassette. All isolates were multidrug resistant and exhibited highest resistance prevalence to trimethoprim (93%). Most isolates (89%) were intll-positive and 107 isolates (57%) carried dfrA12, all of which were resistant to trimethoprim. Forty-eight dfrA12-harboring strains (45%) were intl1-positive together with dfrA12-aadA2 gene cassette. Fifteen isolates contained dfrA12 but not intl1 and dfrA12-aadA2 cassette. The results indicated a wide distribution of dfrA12 and its role in dissemination of trimethoprim resistance among Salmonella isolates from fattening pigs.     

肉鸡源多重耐药空肠、结肠弯曲菌的耐药分子特征

目的了解肉鸡源空肠和结肠弯曲菌的耐药谱特征,检测多重耐药菌株Ⅰ类整合子/基因盒、gyrA基因突变位点、tetO基因、23S rRNA突变位点的分子特征。方法利用PCR检测弯曲菌Ⅰ类整合子/基因盒的存在情况;利用MAMA PCR技术检测弯曲菌gyrA基因第257位碱基的突变情况;针对弯曲菌23S rRNA的V区2075突变位点检测突变菌株。结果多重耐药菌株占分离株的94.5%。146株多重耐药空肠和结肠弯曲菌中Ⅰ类整合子检出率为98.6%,有78株菌株检出3种基因盒,1 000 bp+750 bp+500 bp+250 bp为主要谱型,所占比例为92.3%;有131株在gyrA喹诺酮类耐药决定区发生突变,突变率为92.9%。127株四环素耐药弯曲菌tetO基因的检出率为95.3%。81株红霉素耐药菌株中,23S rRNA的V区2075处突变发生率为96.3%。结论空肠和结肠弯曲菌分离株携带aadA2耐药基因盒,与氨基糖苷类药物的耐药性相关;gyrA基因突变、tetO基因的携带以及23S rRNA突变,与弯曲菌对喹诺酮、四环素和大环内酯类耐药密切相关。     

产超广谱β内酰胺酶临床分离株可转移多重耐药分子机制的研究

目的 研究产超广谱β内酰胺酶(ESBLs)临床分离株可转移多重耐药性的分子机制。方法 采用E-试验条进行药敏检测，电转化试验筛选、分离耐药质粒，聚合酶链反应(PCR)扩增Ⅰ型整合子基因盒插入序列及其分子克隆和序列分析。结果 9个产ESBLs耐药质粒中有8个检测出了插入序列，其中7个携带了1～2种抗药性基因盒。包括氨基糖苷类钝化酶aacA4、aadA2和aadA5；甲氧苄啶钝化酶dhfrA12和dfrA17；利福平钝化酶arr-3；氯霉素外排蛋白cmlA6。基因盒功能与转化子耐药表型一致。结论 质粒定位和整合子介导的抗药性基因盒可能是导致ESBLs产酶株多重耐药性产生和(或)播散的重要原因。     

Phenotypic and genotypic characterization of Salmonella enterica recovered from poultry meat in Tunisia and identification of new genetic traits

Thirty-seven Salmonella enterica isolates obtained from poultry meat in Tunisia were included in this study for characterization of antibiotic resistance mechanisms. High percentages of resistance were detected to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%-89.2%), and lower percentages to amoxicillin-clavulanic acid, kanamycin, amikacin, trimethoprim-sulfamethoxazol, and chloramphenicol (2.7%-18.9%). All strains showed susceptibility to ceftazidime, cefotaxime, gentamicin, and ciprofloxacin. Class 1 integrons were detected in 30% of Salmonella isolates, and four different gene cassette arrangements were detected, including genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1). Four different Pc variants (PcW, PcH1, PcH1(TTN-10), PcW(TGN-10)) with inactive P2 have been found among these isolates. Integron-positive isolates were ascribed to eight different serotypes. A Salmonella Schwarzengrund isolate harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement, with the very unusual PcH1(TTN-10) promoter, which has been registered in GenBank (accession no. HQ874651). Different plasmid replicon types were demonstrated among integron-positive isolates: IncI1 (8 isolates), IncN (8), IncP (2), IncFIB (2), and IncFII (2). Ten different pulsed-field gel electrophoresis profiles were detected among the 11 integron-positive isolates and 8 different sequence types were identified by multilocus sequence typing, one of them (registered as ST867) was new, detected in 3 Salmonella Zanzibar isolates. A high diversity of clones is observed among poultry Salmonella isolates and a high proportion of them show a multiresistant phenotype with very diverse mobile genetic structures that could be implicated in bacterial dissemination in different environments.     

大肠埃希菌中新的耐药基因盒aadA23的变异

目的基于整合子-细菌耐药系统在细菌耐药机制中的重要作用,对成人腹泻患者的大肠埃希菌Ⅰ类整合子阳性菌株携带的耐药基因盒的基因特征进行分析.方法应用聚合酶链反应（PCR）检测Ⅰ类整合酶基因intⅠ阳性菌株并对其整合的耐药基因进行测序及用生物信息软件对序列进行分析.结果5株Ⅰ类整合酶基因阳性菌株的耐药基因盒PCR扩增得到1009 bp的产物.序列分析结果表明,1009 bp序列含有780 bp的开放阅读框,与已知的aadA23和aadA21分别有99.6%和99.5%的相似性,与aadA5只有66.4%相似,为对氨基糖苷类抗菌药物壮观霉素、链霉素产生耐药的基因盒,建议命名为aadA23b.结论随着选择环境不同,整合子整合的基因盒会发生变异,提示我们要用分子生物学的手段从基因水平分析耐药基因的遗传与变异.     

Characterization of class 1 integrons with unusual 3' conserved region from Salmonella enterica isolates

The unusual 3' conserved sequence region of class 1 integrons was characterized in seven Salmonella isolates from swine and poultry. Three types of gene cassette arrays, aadA2-cmlA1-aadA1, sat-psp-aadA2-cm1A1-aadA1 and drfA12-orf-aadA2-cmlA1-aadA1, were found to be linked to a genetic organization qacH-IS440-sul3. All class 1 integrons were located on a conjugative plasmid that could be transferred to Escherichia coli. The results support the notion that the use of an antibiotic can select for resistance not only to that specific agent, but also to other unrelated antimicrobials including those that are no longer approved for use in food animal production.     

Characterization of antimicrobial resistance and integrons among Escherichia coli isolated from animal farms in Eastern China

A total of 182 Escherichia coli isolates from animals, environment and workers of dairy cattle, swine and chicken farms in Shandong which locates in Eastern China, were investigated for antimicrobial resistance as well as prevalence and the transfer mechanisms of integrons. The results revealed isolates from swine and chicken farm exhibited high levels of resistance to antimicrobial agents. The positive rate of gene cassette of class 1 integron in dairy cattle, swine and chicken farms was 5%, 20% and 41.94%, respectively. Only four isolates possessed class 2 integron, all of which were from chicken farm. Nine distinct cassette arrays were detected and two novel gene cassette arrays yheSΔ-yheR-kefBΔ and chrAΔ-sul1-qacEΔ1-orf5-aadA5-dfrA17 were identified in class 1 integron for the first time. Class 1 integrons were found to be located mostly in both chromosomal and conjugative plasmid through southern hybridization and conjugation. PFGE revealed clonal relatedness among the isolates from different sources, especially within the same farm. The results confirmed the antimicrobial resistance and prevalence of integrons were strongly associated with the selection pressure of antimicrobial agents, and resistance genes in animal farms were probably spread by both vertical and horizontal transfer.     

多重抗药大肠杆菌中Ⅰ型整合子的分布与抗药关系

目的检测多重抗药大肠杆菌中检测Ⅰ型整合子的携带率及其对细菌抗药性的影响。方法用大肠杆菌显色．培养基分离奶牛场、猪场和肉鸡场的565株大肠杆菌,测定其对15种抗生素的MIC;用PCR扩增其中91株多重抗药性（最少耐3种抗生素）菌株的Ⅰ型整合酶基因及抗药基因盒。结果565株大肠杆菌仅对头孢噻呋、阿米卡星和硫酸粘菌素的抗药率低于50％,其余药物均表现出较高抗药率,且几乎所有细菌都对3种以上抗生素具有抗性。91株多重抗药大肠杆菌Ⅰ型整合子的平均检出率为83．52％,大多数整合aadA基因和dhfr基因,抗药性基因携带率为43．42％。结论Ⅰ型整合子对细菌多重抗药性的产生和传播起着重要作用,是临床细菌多重抗药性监测在基因水平的重要指标。     

天津地区福氏志贺菌整合子携带及耐药性研究

目的了解天津地区不同年份福氏志贺菌血清型分布,整合子携带情况,耐药性及其变化趋势。方法采用血清学方法对天津地区不同年份分离的56株福氏志贺菌进行分型;PCR扩增整合子整合酶及可变区,并测序;采用K-B法测定其对16种抗菌药物的敏感性。结果 1981~1983年分离株福氏志贺菌1类整合酶阳性率为87.50%(28/32),其中27株可变区含氨基糖苷类药物耐药基因aadA;2009~2011年分离株福氏志贺菌1类整合酶阳性率为91.67%(22/24),可变区及3’末端扩增均阴性。1981~1983年分离株福氏志贺菌2类整合酶均阴性;2009~2011年分离株福氏志贺菌2类整合酶阳性率79.17%(19/24),可变区含dfrA1、sat1、aadA1,介导对甲氧苄啶、链丝菌素和链霉素耐药。有17株福氏志贺菌1、2类整合酶均阳性。1981~1983年分离株福氏志贺菌对四环素、链霉素、氯霉素及甲氧苄啶/磺胺甲噁唑耐药率高,多重耐药率为65.63%;2009~2011年分离株福氏志贺菌对氨苄西林、链霉素、甲氧苄啶/磺胺甲噁唑、哌拉西林和四环素耐药率高,多重耐药率为83.33%。结论 1、2类整合子广泛存在于天津地区福氏志贺菌中,其中1类整合子3’保守末端可能缺失或存在变异。2009~2011年分离株福氏志贺菌对抗菌药物耐药性较1981~1983年分离株增强,多重耐药率增高。福氏志贺菌优势血清型发生转变,血清型分布呈现多样化。     

High prevalence of integron-mediated resistance in clinical isolates of Salmonella enterica

Salmonella enterica has become progressively resistant to antimicrobial agents worldwide as a result of genes carried on different classes of integrons. The aim of the current study was to investigate the molecular diversity of these integrons and their association with antimicrobial resistance in clinical S. enterica isolates from Tehran, Iran. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute. The presence of integrons was investigated by PCR using specific primers. Integrons were detected in 65 (47.1%) strains, with classes 1 and 2 being observed in 54 (39%) and 11 (8%) strains, respectively. Integron-positive isolates belonged to seven different S. enterica serovars, and all showed a multidrug-resistant (MDR) phenotype. Our findings show that integrons are widely disseminated among S. enterica strains from Tehran. Furthermore, the results that class 1 integrons were more prevalent than class 2 in Salmonella isolates, and that a statistical association with MDR patterns was observed, suggest that they are more likely to be important in conferring a resistant phenotype to Salmonella strains.     

Multidrug-resistant human and animal Salmonella typhimurium isolates in France belong predominantly to a DT104 clone with the chromosome- and integron-encoded beta-lactamase PSE-1

Epidemiologic relationships were investigated in 187 ampicillin-resistant Salmonella typhimurium strains (86 human, 101 animal) from >2000 strains isolated in 1994. Of 23 resistance patterns, the most frequent (ampicillin [Am], chloramphenicol [Cm], tetracycline [Tc], streptomycin and spectinomycin [Sm], and sulfonamides [Su]) was found in 69.5% of human and 64.8% of animal isolates. Four beta-lactamase genes were identified, blaTEM (24%), blaPSE-1 (78%), and blaSHV and oxa-2 (each <3%). blaPSE-1 and the integrase gene, intI1, but not blaTEM, blaSHV or oxa-2, were chromosomeborne and found almost exclusively in the AmCmTcSmSu strains. In these, polymerase chain reaction mapping revealed two distinct integrons carrying blaPSE-1 or aadA2. Lysotypes, plasmid profiles, and restriction fragment length polymorphisms (IS200) were determined for 50 representative isolates and for 3 DT104 strains from the United Kingdom (UK). The phage type of the PSE-1-producing AmCmTcSmSu strains was 12 atypic, indistinguishable from that of the DT104 strains. The combined data indicate that the same multiresistant clone has spread through human and animal ecosystems in the UK and France.     

Role of integrons in antimicrobial susceptibility patterns of Acinetobacter baumannii

The relationship between the presence and types of integrons and the antimicrobial susceptibility patterns of Acinetobacter baumannii was investigated. A total of 134 non-duplicated A. baumannii isolates, 54.5% (n=73) of which were subsequently found to carry class 1 integrons, were collected from a regional hospital in Taiwan between March and September 2007. Only two types of gene cassette array, aacA4-catB8-aadA1 and aacC1-orfP-orfP-orfQ-aadA1, were identified. Susceptibility data showed that those strains carrying integrons were significantly more resistant to all antibiotics tested except ampicillin/sulbactam and imipenem. An epidemiological study revealed that the same integron could be found in different unrelated strains. These findings suggest that the presence of integrons in A. baumannii is responsible for both the horizontal transfer of antibiotic-resistance genes related to aminoglycosides and chloramphenicol and also represents a marker of multidrug resistance and epidemic potential.     

Resistencia a antibióticos y factores de virulencia en aislados clínicos de Salmonella enterica

Introduction

The increase of Salmonella enterica isolates multi-resistant to different antibiotics, including β-lactams and fluoroquinolones, is a problem of clinical importance. The dissemination of Salmonella Typhimurium resistant to ampicillin (AMP)-chloramphenicol (CHL)-streptomycin (STR)-sulphonamides and (SUL)-tetracycline (TET), that harbour the Salmonella Genomic Island type 1 (SGI1), and the acquisition of transferable genetic material have favoured the multi-resistance in this genus.

Methods

A total of 114 clinical S. enterica isolates were studied (period 2009-2010). The susceptibility to 20 antibiotics was determined by disc diffusion and microdilution. The antimicrobial resistance mechanisms and the integrons were analysed by PCR, and sequencing in the AMP^R isolates. In all the bla_PSE-1-positive isolates, the clonal relationship was determined by PFGE, as well as the presence of SGI1 and 29 virulence genes by PCR.

Results

Eighteen different serotypes were found among the 114 isolates studied, Typhimurium (61%) and Enteritidis (16%) being the most prevalent. High percentages of resistance to SUL (68%), TET (58%), AMP (55%) and STR (46%) were observed. The great majority (92%) of 63 AMP^R isolates were multi-resistant, with the AMP-STR-TET-SUL phenotype (19 isolates) being the most frequent one and associated with the bla_TEM-1b + strA-strB + tet(B) + sul2 genotype. Class 1 integrons (7 different structures) were observed in 48% AMP^R isolates, highlighting the bla_OXA-1 + aadA1 structure (8 isolates), one empty integron and non-classical integrons (5 isolates). The bla_PSE-1 gene was detected inside the classical SGI1 structure in 13 clonally-related isolates that showed the same virulence profile.

Conclusions

The high percentage of multi-resistant S. enterica isolates, especially associated to S. Typhimurium, to the AMP, STR, TET and SUL phenotype, and to the bla_TEM-1b + strA-strB + tet(B) + sul2 genotype, shows an important risk of possible failures in the treatment of serious infections caused by this serotype.     

Complex clonal and plasmid epidemiology in the first outbreak of Enterobacteriaceae infection involving VIM-1 metallo-beta-lactamase in Spain: toward endemicity?

BACKGROUND: We report the emergence and spread of metallo-beta-lactamases (MBLs) among enterobacterial isolates at Ramón y Cajal University Hospital (Madrid, Spain). METHODS AND RESULTS: During the period from March 2005 through September 2006, 25 patients (52% of whom were in the intensive care unit) were infected and/or colonized with single or different MBL-producing Enterobacteriaceae isolates (Klebsiella pneumoniae, 14 patients; Enterobacter cloacae, 12 patients; Escherichia coli, 1 patient; and/or Klebsiella oxytoca, 1 patient). Clonal analysis (XbaI pulsed-field gel electrophoresis) revealed that all K. pneumoniae isolates belonged to the same clone, but 6 patterns were found among the E. cloacae isolates. Carbapenems were affected to different degrees (minimum inhibitory concentration, < or = 1 to > 8 microg/mL), as were aminoglycosides and ciprofloxacin. The bla(VIM-1) MBL gene was present in all isolates; in addition, the bla(SHV-12) extended-spectrum beta-lactamase gene was detected in K. pneumoniae and E. coli isolates. The bla(VIM-1) gene was detected within a 4.0-kb class 1 integron (bla(VIM-1)-aacA4-dfrII-aadA1-catB2) in K. pneumoniae and E. coli and in a 2.5-kb class 1 integron (bla(VIM-1)-aacA4-aadA1) in E. cloacae and K. oxytoca isolates. The bla(VIM-1) gene was transferable (filter-mating) in 14 of 14 K. pneumoniae isolates, 4 of 11 E. cloacae isolates, and 1 of 1 E. coli isolate. A 60-kb plasmid belonging to the IncI1 group was detected in the epidemic VIM-1-K. pneumoniae clone. Plasmids of 300- or 435-kb belonging to IncH12 group were found among E. cloacae isolates. CONCLUSIONS: K. pneumoniae-MBL monoclonal epidemics coexisted with E. cloacae-MBL multiclonal epidemics in our hospital. The spread of the bla(VIM-1) gene among Enterobacteriaceae was driven by clonal spread associated with intergeneric plasmid transfer with different class I integron platforms. Such complex epidemiology might anticipate endemicity and should be considered for the design of containment epidemiology strategies.     

The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons

Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant. The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria. Yet the origin of MRIs remains unknown. Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome. Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1. We have also identified equivalent integron superstructures in nine distinct genera throughout the gamma-proteobacterial radiation. Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures. The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable. Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here. Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs. Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes. However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general gene-capture system for bacterial innovation.