Class 1 integrons and virulence genes in Salmonella enterica isolates from pork and humans

In this study, 183 Salmonella enterica isolates were characterised for integrons and virulence genes. Among the isolates, 46% were positive for intI1, but no isolates carried intI2 or intI3. Eighteen class 1 integrons (21%) contained resistance gene cassettes (i.e. dfrA1-orfC, dfrA12-aadA2, bla(PSE-1) and aadA2) and five class 1 integrons with the dfrA12-aadA2 array were conjugally transferable. Two Salmonella pork isolates of serotypes Albany and Kedougou possessed Salmonella genomic island 1 variants SGI1-G and SGI1-F, respectively. Four class 1 integrons contained an atypical 3'-CS linked to the qacH-sul3 domain, and three were not a sul type. Two novel GyrA mutations (Pro-45→Ser and Met-48→Ile) and three novel ParC mutations (Ser-5→Arg, Thr-31→Met and Leu-77→Arg) were identified in ciprofloxacin-resistant isolates. At least 90% of the Salmonella isolates contained pagC, prgH, sitC, sipB or spaN, whereas all isolates harboured invA, msgA, spiA and tolC.     

多重耐药临床菌株中整合子结构的检测与分析

目的研究广州暨南大学附属第一医院2004年部分临床菌株样本的整合子及其基因盒的分布特性。方法多重PCR检测与细菌耐药关系密切的1、2、3类整合酶基因,进一步对阳性样本可变区的基因盒序列鉴定分析。结果随机抽取109株临床菌株,整合酶阳性检出率为97.2%(106/109),其中1类整合酶阳性菌100株(91.7%),2类整合酶阳性菌1株(0.92%),此外有5株(4.6%)同时检出1、2类整合酶,没有检测到3类整合酶;基因盒鉴定结果显示,插入基因盒以dfrA(甲氧苄氨嘧啶耐药相关)和aadA(氨基糖苷类耐药相关)基因家族为主,也在少数菌株中发现了aacA4、cmlA1、catB3以及sat1基因盒。其中又以dfrA12、orfF和aadA2组合最为常见,耐药基因盒PCR扩增片段为1913bp(64.6%);此外,还发现了同时存在两种整合子结构的菌株。结论整合子普遍存在于临床菌株中,可通过基因水平转移在不同菌属间传播,提示各医药单位必须加强耐药监测及合理选择抗菌药物,以减少多重耐药细菌的发生和发展。     

Molecular analysis of multiresistant porcine Salmonella enterica subsp. enterica serovar Bredeney isolates from Southern Brazil: identification of resistance genes, integrons and a group II intron

The relationships of 83 porcine Salmonella enterica subsp. enterica serovar Bredeney isolates obtained at two slaughterhouses in Southern Brazil were analysed by XbaI and BlnI macrorestriction analysis, plasmid profiling and determination of antimicrobial resistance patterns. Twenty-nine XbaI and 30 BlnI macrorestriction patterns were identified. The 72 plasmid-bearing isolates exhibited 20 different plasmid profiles. Multiresistance was detected in 49 isolates (59%), of which 39 isolates showed at least resistance to sulfonamides, tetracyclines, chloramphenicol, streptomycin, kanamycin and/or ampicillin. A representative subset of 12 isolates was chosen for identification of resistance genes, their localisation and transferability. The sulfonamide resistance genes sul1, sul2 and sul3, the tetracycline resistance genes tet(A) and tet(B), the phenicol resistance genes catA1 and floR, the streptomycin resistance gene strA, the kanamycin resistance gene aphA1 and the ampicillin resistance gene bla_TEM were detected and found to be located most frequently on plasmids. In addition, class 1 and 2 integrons with the cassette arrangements dfrA21/bla_OXA-129/aadA1 and dfrA1/sat1/aadA1, respectively, were detected. A group II intron was found to be inserted into the 59-base element of an aadA1 gene cassette in a class 1 integron. This study revealed a wide genomic variety among the S. Bredeney isolates, and the high number of multiresistant isolates may point towards the risks that these S. Bredeney isolates can represent to human health.     

Molecular 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.     

Class 1 integron gene cassettes in multidrug-resistant Gram-negative bacteria in southern China

Non-duplicate multidrug-resistant Gram-negative bacteria (n=1447) isolated from January 2008 to December 2009 were investigated for the presence of integrons as well as characterisation of gene cassettes. Among 825 strains carrying the class 1 integrase gene intI1, 461 gene cassette-positive isolates were found. Thirty-eight distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP) and DNA sequencing analyses. In addition, several novel gene cassette arrays detected in this study were reported for the first time in some species: one in Escherichia coli, six in Klebsiella pneumoniae, six in Enterobacter cloacae, three in Enterobacter aerogenes, one in Proteus mirabilis, one in Acinetobacter spp., one in Stenotrophomonas maltophilia and one in Pseudomonas putida. Among them, three cassettes, including HAD-like, ΔMFS-1 and qnrVC-like genes, were originally detected in integrons.     

Novel cassette arrays of integrons in clinical strains of Enterobacteriaceae in China

One hundred and forty-two Enterobacteriaceae isolates randomly selected from four general hospitals in Nanjing region of China were investigated for antibiotic susceptibility, the presence of integrons and the characterisation of gene cassettes. Eighty-five (59.9%) Enterobacteriaceae strains contained class 1 integrons, and 17 (65.4%) of 26 Shigella contained class 2 integrons. Class 3 integrons were not detected in this study. Restriction fragment length polymorphism (RFLP) and DNA sequencing analysis revealed seven different cassette arrays of class 1 integrons; additionally, four cassette arrays identified in this study were reported for the first time in some species. The genes most commonly found in these class 1 integrons were those for aminoglycoside and trimethoprim resistance. In conclusion, class 1 and 2 integrons were widespread in Enterobacteriaceae and Shigella, respectively, in Nanjing area of China and it was likely that integrons played an important role in antibiotic resistance. Characterisation of cassette arrays of integrons could be a useful epidemiological tool to study the evolution of multidrug resistance and the dissemination of antibiotic resistance genes.     

Molecular epidemiology and resistance mechanisms involved in reduced susceptibility to amoxicillin/clavulanic acid in Klebsiella pneumoniae isolates from a chronic care centre

The aim of this work was to investigate the molecular epidemiology and mechanisms responsible for reduced susceptibility to amoxicillin/clavulanic acid (AMC) amongst cefazolin-susceptible Klebsiella pneumoniae isolates from patients admitted to a chronic care institution. In total, 51 (29.8%) of 171 K. pneumoniae isolates recovered between 2006 and 2008 were non-susceptible to AMC, of which 45 were susceptible to cefazolin. Nucleotide sequencing analysis revealed that 19 produced IRT-11 and the remaining 26 were OXA-1-producers. All of the OXA-1-producing isolates harboured the aac(6')-Ib-cr-bla(OXA-1) cassette array, which in 23 isolates was located together with catB3 and arr3 within a class 1 integron and associated with qnrS2 (in 3 cases the integron lacked the qacEΔ1 and sul1 or sul3 genes). Genotyping analysis performed by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) identified three different patterns amongst IRT-11-producing isolates (E1 to E3), with E1 being the most prevalent (63.2%), whilst the OXA-1-producing isolates were assigned to patterns E3 and E3a (isolates carrying typical class 1 integrons), E4 (isolates carrying defective integrons) and E5 (isolates without integrons). Genes encoding IRT-11 and OXA-1 were transferred by conjugation, and aac(6')-Ib-cr and qnrS2 were systematically co-transferred with bla(OXA-1). These results demonstrate that the high prevalence of decreased susceptibility to AMC amongst K. pneumoniae isolates from a chronic care hospital was mainly due to the simultaneous spread of two different clones, one of which comprised isolates producing IRT-11 and the other one comprised isolates that had acquired either the bla(OXA-1) gene located in a class 1 integron and linked to qnrS2 or the bla(IRT-11) gene.     

Multiclonal spread of VIM-1-producing Enterobacter cloacae isolates associated with In624 and In488 integrons located in an IncHI2 plasmid

Over a 6-year period (2007–2012), the emergence of Enterobacter cloacae isolates resistant to β-lactams and with reduced susceptibility to carbapenems was observed in Hospital Universitario 12 de Octubre (Madrid, Spain). To determine the possible role of metallo-β-lactamases (MBLs) in the resistance profile of these isolates, a molecular and clinical epidemiological study was performed, including determination of patients’ clinical characteristics, genetic diversity of strains, resistance mechanisms to carbapenems, and the genetic environment of VIM-1. A total of 73 E. cloacae isolates showed resistance to extended-spectrum cephalosporins and reduced susceptibility to at least one carbapenem during 2007–2012. PCR amplification revealed the presence of bla_VIM-1 gene in 37 isolates, bla_VIM-2 in 1 isolate and bla_KPC in 5 isolates. Molecular typing showed high clonal diversity of E. cloacae isolates carrying bla_VIM-1. The genetic environment of bla_VIM-1 was investigated and two integron structures were found: intI–bla_VIM-1–aacA4–dfrB1–aadA1–catB2–qacEΔ1/sul1 (In624); and intI–bla_VIM-1–aacA4–aadA1–qacEΔ1/sul1 (In488). Isolates belonging to three clones (A, F and G) harboured different types of integron (In624 or In488) despite belonging to the same clone. Conjugal experiments showed an association with a conjugative plasmid of ca. 300 kb belonging to IncHI2 group, which is common in Spanish hospitals, suggesting that the widespread dissemination of bla_VIM-1 may be due to horizontal transfer of mobile genetic determinants rather than the result of spreading of a few clones. These results have implications for infection control programmes in the hospital.     

Stenotrophomonas maltophilia resistance to trimethoprim/sulfamethoxazole mediated by acquisition of sul and dfrA genes in a plasmid-mediated class 1 integron

Stenotrophomonas maltophilia is becoming a more and more common cause of infections. In this study, the minimal inhibitory concentrations of trimethoprim/sulfamethoxazole (SXT), ceftazidime, minocycline, levofloxacin, chloramphenicol and ticarcillin/clavulanic acid were determined and the distribution of integrons and sul1, sul2 and dfrA genes was investigated in 102 S. maltophilia isolates collected from patients treated in 31 hospitals in Anhui, China, in the month of September in 2006-2008. The rate of resistance to SXT was up to 30.4%, and 64.7% of isolates were class 1 integron-positive. Sequencing data revealed the following novel gene cassettes embedded in class 1 integrons: dfrA17-aadA5; dfrA12-aadA2; aacA4-catB8-aadA1; aadB-aac(6')-II-bla(CARB-8); and arr-3-aacA4. This is the first report of the gene cassettes dfrA17-aadA5 and dfrA12-aadA2 and of sul2 genes in SXT-resistant S. maltophilia isolates in China. None of the SXT-susceptible S. maltophilia isolates were positive for sul2 or dfrA gene products by polymerase chain reaction (PCR), but PCR products for sul1 were detected in 27 SXT-susceptible and 25 SXT-resistant isolates. The findings from this study indicate that the sul1 gene, in combination with dfrA17 and dfrA12 gene cassettes and sul2 genes located within a 7.3kb plasmid, lead to a high rate of SXT resistance and also confirm the need for ongoing resistance surveillance.     

细菌耐药整合子intI分类的多重PCR方法的建立及应用

目的建立快速筛选细菌耐药整合子的分类方法,并对21株来自临床的菌株进行整合子筛选和分类。方法根据G enB ank/EM BL内的第一、第二和第三类的整合酶序列,通过软件C lustalW的多重比对分析设计出各类整合子的特异性引物,用对照菌株建立多重PCR方法并对21株临床菌株进行PCR扩增,通过其PCR扩增产物片段大小的不同进行整合子分类。结果对照菌株实验结果显示,21株临床分离菌株中,15株在565bp处有扩增片段,即为含有第一类整合酶基因;4株在403bp处有扩增片段,即含有第二类整合酶基因;1株在565bp和403bp处均有扩增片段,提示其同时含有第一、二类整合酶基因;1株没有得到任何扩增片段,即不含有这三类整合酶基因;在被测菌株中未发现第三类整合酶基因的阳性菌株。结论本文报道的筛选细菌耐药整合酶基因分类的多重PCR方法效果良好,具有可行性,为更加全面细致研究整合子类型以及整合子介导的细菌耐药机制提供了一种简单易行、快捷有效的方法。     

鲍曼不动杆菌Ⅰ类整合子与多重耐药相关性研究

目的 了解临床分离鲍曼不动杆菌的耐药状况、Ⅰ类整合子的分布情况,探讨Ⅰ类整合子与多重耐药的关系.方法 检测20种临床常用抗菌药物对鲍曼不动杆菌临床分离株的最低抑菌浓度(MIC).PCR扩增Ⅰ类整合酶基因.对部分Ⅰ类整合酶阳性菌株进行耐药基因盒序列分析.结果 鲍曼不动杆菌呈现多重耐药,鲍曼不动杆菌对IMP和MRP耐药率分别为0.9%和1.8%,对CPZ/SB的耐药率为35.7%,对其它抗菌药物的耐药率均大于60%,多重耐药率为76.8%(86/112),但对COL和MIN均敏感.80.4%(90/112)的菌株检测出Ⅰ类整合子.Ⅰ类整合子阳性株对多种药物的耐药率均高于阴性株,且Ⅰ类整合子阳性株多重耐药率(90%)明显高于阴性株(22.7%)(P<0.01).Ⅰ类整合子基因盒序列分析显示,Ⅰ类整合子携带aacA4,catB8和aadA13种耐药基因.结论 Ⅰ类整合子在鲍曼不动杆菌中检出率很高并与其多重耐药性关系密切.     

Occurrence,abundance and elimination of class 1 integrons in one municipal sewage treatment plant

Integrons are elements that encode a site-specific recombination system that recognizes and captures mobile gene cassettes and are closely related to multiple resistances of environmental microorganisms. This study was undertaken to determine the efficiency of an activated sludge process to remove integrons. The prevalence and characteristics of class 1 integrons were investigated for bacterial species isolated from the activated sludge of Nanjing Jiangxinzhou sewage treatment plant (STP, China). A total of 189 bacterial strains were isolated from influent water, activated sludge and effluent water, and PCR–RFLP (Polymerase chain reaction—restriction fragment length polymorphism) of 16S rRNA gene showed that the isolated bacteria were Escherichia coli, Aeromonas veronii, Klebsiella spp., Aeromonas salmonicida and Aeromonas media. PCRs showed that 57 isolates contained class 1 integronase gene intI1. The integron detection frequency in the isolated strains was 20.4% for influent, 30.9% for activated sludge and 38.9% for effluent. Quantitative real-time PCR assay showed that the abundance of integrons in effluent was higher than that in influent. This study indicates that class 1 integrons are wide-spread in STPs which might be involved in multiple resistances in the activated sludge characterized by high biomass and biodiversity.     

Antibiotic resistance genes in surface water of eutrophic urban lakes are related to heavy metals,antibiotics, lake morphology and anthropic impact

Urban lakes are impacted by heavy human activities and represent potential reservoirs for antibiotic resistance genes. In this study, six urban lakes in Wuhan, central China were selected to analyze the distribution of sulfonamide resistance (sul) genes, tetracycline resistance (tet) genes and quinolone resistance (qnr) genes and their relationship with heavy metals, antibiotics, lake morphology and anthropic impact. sul1 and sul2 were detected in all six lakes and dominated the types of antibiotic resistance genes, which accounted for 86.28–97.79% of the total antibiotic resistance gene abundance. For eight tested tet genes, antibiotic efflux pumps (tetA, tetB, tetC, and tetG) genes were all observed in six lakes and had higher relative abundance than ribosomal protection protein genes (tetM and tetQ). For 4 plasmid mediated quinolone resistance genes, only qnrD is found in all six lakes. The class I integron (intI1) is also found to be a very important media for antibiotic resistance gene propagation in urban lakes. The results of redundancy analysis and variation partitioning analysis showed that antibiotic and co-selection with heavy metals were the major factors driving the propagation of antibiotic resistance genes in six urban lakes. The heavily eutrophic Nanhu Lake and Shahu Lake which located in a high density building area with heavy human activities had the higher relative abundance of total antibiotic resistance genes. Our study could provide a useful reference for antibiotic resistance gene abundance in urban lakes with high anthropic impact.     

Emergence of multidrug-resistant Salmonella enterica serovar Typhi associated with a class 1 integron carrying the dfrA7 gene cassette in Nepal

A total of 121 Salmonella enterica serovars Typhi and Paratyphi A isolated from enteric fever patients at a university hospital in Nepal between February 2004 and January 2006 were tested for their antimicrobial susceptibility. The occurrence and cassette content of integrons as well as the molecular mechanisms of resistance among the multidrug-resistant (MDR) S. Typhi were evaluated. Thirty-nine percent of the isolates were susceptible to all the antimicrobial agents tested. Seven of the S. Typhi strains were MDR. None of the 121 S. enterica isolates were resistant to ciprofloxacin, cefazolin, rifampicin or kanamycin. All MDR S. Typhi isolates contained a class 1 integron with a single cassette, dfrA7, conferring resistance to trimethoprim. Pulsed-field gel electrophoresis (PFGE) of XbaI-generated genomic restriction fragments yielded 12 different patterns. Five of the seven MDR isolates containing class 1 integrons had an identical PFGE pattern. Resistance to sulfamethoxazole, streptomycin, ampicillin, tetracycline and chloramphenicol was mediated by sul1, strA-strB, blaTEM-like, tetB and catA genes, respectively. To the best of our knowledge, this is the first report of integron-associated multidrug resistance as well as the first molecular characterisation of the mechanism of resistance of S. Typhi isolated from Nepal. This study indicates the spread of integron-associated multidrug resistance in S. Typhi in Nepal.     

Integrons in Enterobacteriaceae: diversity,distribution and epidemiology

Integrons are versatile gene acquisition systems that allow efficient capturing of exogenous genes and ensure their expression. Various classes of integrons possessing a wide variety of gene cassettes are ubiquitously distributed in enteric bacteria worldwide. The epidemiology of integrons associated multidrug resistance in Enterobacteriaceae is rapidly evolving. In the past two decades, the incidence of integrons in enteric bacteria has increased drastically with evolution of multiple gene cassettes, novel gene arrangements and complex chromosomal integrons such as Salmonella genomic islands. This review focuses on the distribution, versatility, spread and global trends of integrons among important members of the Enterobacteriaceae, including Escherichia coli, Klebsiella, Shigella and Salmonella, which are known to cause infections globally. Such a comprehensive understanding of integron-associated antibiotic resistance, their role in the spread of such resistance traits and their clinical relevance especially with regard to each genus individually is paramount to contain the global spread of antibiotic resistance.     

Integron gene cassettes in Acinetobacter spp. strains from South China

The epidemiology of emerging antibiotic resistance genes in Asia is inadequately defined and studies within the major pools of transmissible genes such as integron gene cassettes are important. One hundred and twenty-two non-repetitive Acinetobacter spp. isolates were obtained from inpatients of a major hospital in South China. Fifty-three of these isolates contained class 1 integrons, and there is evidence of horizontal gene transfer between unrelated clones. The common pool of gene cassettes was dominated by four cassette arrays: arr3-aacA4 (24 isolates of several unrelated strains); aacC1-orfP-orfQ-aadA1a (11 isolates, probably all the same strain); aacA4-catB8-aadA1 (2 isolates); and dfrVII (1 isolate). We developed a simple restriction fragment length polymorphism (RFLP)-based identification of these and other cassettes reported in China, using readily available enzymes, to facilitate further studies of this type.     

Characterisation of integrons containing the plasmid-mediated quinolone resistance gene qnrA1 in Klebsiella pneumoniae

The aim of this study was to determine the structural relationships of qnrA1 and other resistance genes in four integrons contained in four clinical isolates of Klebsiella pneumoniae. In the four integrons, the sequences surrounding qnrA1 were similar to those described for pMG252 (accession no. AY070235). The four integrons carried a class 1 integrase gene belonging to the complex class 1 integron. Three of the strains contained an identical integron coding for resistance to β-lactams, aminoglycosides, chloramphenicol and trimethoprim. The fourth strain contained a different integron coding for resistance to β-lactams, aminoglycosides and chloramphenicol. Downstream of the last integron, copies of IS6100 and IS26 were present. We describe two new and different integrons containing qnrA1. These integrons code for resistance to different groups of antimicrobial agents from K. pneumoniae clinical strains isolated in the USA.     

Class 1 integrons in environmental and clinical isolates of Pseudomonas aeruginosa

The aims of this study were to ascertain the presence and spread of class 1 integrons amongst environmental and clinical isolates of Pseudomonas aeruginosa and to characterise their variable regions. A total of 76 isolates (56 clinical and 20 environmental) were studied. The presence of plasmids was explored, and polymerase chain reaction (PCR) was used for integron detection. All amplicons were sequenced. PCR detected class 1 integrons in 26 of the 56 clinical isolates; environmental isolates were integron-free. No plasmids were found, thus all the integrons found are possibly on the chromosome. Most isolates presented one amplicon, except PA110514 and PA116136, which showed two PCR products each. Variable regions revealed that 18 strains carried only one gene involved in aminoglycoside resistance, whereas in 3 strains gene cassettes were not found. The most prevalent cassettes amongst isolates were those encoding aminoglycoside adenyltransferase B (aadB). Several of the strains had acquired the same or a highly similar cassette array as those detected in geographically distant P. aeruginosa. This finding suggests that contact with bacterial reservoirs contributes to the evolution of this pathogen towards multiresistance. Empty structures found may represent a reservoir increasing the capacity to adapt to the environment. However, these integrons are not retained when the selective pressure disappears. It is hypothesised that integrons containing gene cassettes are crucial vehicles for the rapid horizontal transfer of resistance. If this is so, reduced use of antibiotics may lead to a significant decrease in the carriage of integrons amongst P. aeruginosa strains.