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.     

100株住院儿童大肠埃希菌Ⅰ类整合子研究

目的:了解Ⅰ类整合子在住院儿童分离大肠埃希菌中分布流行情况。方法:根据NCCLS推荐的纸片扩散法进行药敏试验;PCR扩增临床大肠埃希菌Ⅰ类整合子整合酶基因和可变区基因盒。结果:患者整合酶检出率68%,Ⅰ类整合子基因盒检出率为65%。Ⅰ类整合子的大小约为772 bp～2360 bp,100株细菌各含1～2个Ⅰ类整合子,整合子中最常见的基因盒排列为dfrA17+aadA5和dfrA12+aadA2。结论:Ⅰ类整合子在多重耐药大肠埃希菌中广泛流行,是介导细菌多重耐药性的重要分子机制,应加强基因水平耐药监测。     

辽宁地区沙门菌整合子与耐药相关性研究

摘要：目的 及时掌握辽宁地区沙门菌整合子的分布以及对常用抗菌药物的耐药情况,获取沙门菌整合子与耐药性的相关性,从而为临床用药及治疗提供指导。方法 本文对来源于食品以及病患的149株沙门菌,利用PCR扩增的方法,进行整合子类别的筛选,并将扩增的整合子基因盒进行基因测序,同时通过药敏板测定(耐药性实验)对沙门菌与15种临床常用抗菌药物的耐药相关性进行研究。结果 149株沙门菌中未发现第II类、第III类整合子,检出第I类整合子的菌株50株,I类整合子阳性率为33.6%。50株整合子阳性菌株中25株携带耐药基因盒,片段范围从1500~1800 bp。测序结果表明,其中22株整合子携带dfrA17-aadA5基因盒,2株携带dfrA12-aadA2基因盒,1株首次检出罕见耐药基因盒linG。根据整合子携带情况不同,对15种抗菌药物的耐药率进行对比分析,结果显示整合子阳性菌对9种抗菌药物的耐药率显著高于整合子阴性菌(P<0.01),分别为氨苄西林、四环素、氯霉素、头孢噻肟、头孢唑林、庆大霉素、复方磺胺甲恶唑、阿奇霉素和环丙沙星。辽宁地区沙门菌多重耐药率达50.3%。结论 I类整合子在沙门菌中分布广泛,抗性基因表型与耐药结果相一致,整合子的携带与沙门菌多重耐药率高度相关。沙门菌对亚胺培南和头孢西丁保持高敏感率,可以用于对常规抗菌药物耐药的沙门菌的治疗。     

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。     

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.     

Antibiotic resistance and class 1 integron patterns of non-typhoidal human Salmonella serotypes isolated in Hungary in 2002 and 2003

The antibiotic resistance profiles of 5178 Salmonella strains representing 19 non-typhoidal serotypes isolated from human salmonellosis cases in Hungary in 2002 and 2003 were analysed for resistance to 10 antibiotic agents. The most frequent resistances were to nalidixic acid (Nx), streptomycin (S), tetracycline (Tc), ampicillin (Amp) and chloramphenicol (Cm) (ranging from 27% to 13%). Forty-five percent of the Salmonella Typhimurium strains were multiple resistant and belonged mainly to the definitive phage types 104 and U302. A prevalence of 83-94% of strains of serotypes S. Infantis, S. Hadar and S. Virchow was observed with the NxSTc resistance pattern, sometimes complemented with other resistances. Multiple resistance was uncommon in S. Enteritidis; nevertheless, 20% of the strains, most of which belonged to phage type 4, were nalidixic acid resistant. One strain of S. Typhimurium was found to be resistant to ciprofloxacin. Four S. Typhimurium strains were resistant to cefotaxime and produced extended-spectrum beta-lactamase. Selected isolates were screened for the presence of class 1 integrons by polymerase chain reaction (PCR). Nucleotide sequencing of the PCR products revealed nine different variable regions. One resistance gene was identified in five variable regions (aadA1, aadA2, aadA23, dfrV and pse-1), and four variable regions carried two resistance gene cassettes (aadB-catB3, dhfrI-aadA, dfrA17-aadA5 and oxa-1-aadA1).     

Detection of integrons and antibiotic-resistance genes in Salmonella enterica serovar Typhimurium isolates with resistance to ampicillin and variable susceptibility to amoxicillin-clavulanate

We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:-, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990-2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla(TEM) gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded beta-lactamase as TEM-1; all of these strains carried a second gene encoding beta-lactamase production, either pse-1 or oxa1. However, the association of bla(TEM) plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.     

第一类整合子整合酶基因intI1的定位分析

目的　整合子 ( integrons)介导的细菌耐药特性已成为研究细菌耐药机制的热点 ,在研究了来自正常人携带沙门氏菌中整合子的分布和特性的基础上 ,进一步探讨整合子的基因定位。方法　从已鉴定的整合子阳性菌株出发 ,分别提取其质粒和染色体 DNA,进行质粒的接合转移试验。对染色体 DNA进行限制性酶切 ,以第一类整合酶基因 int I1( DIG标记 )为探针 ,进行 Southern杂交。结果　 4株整合酶阳性菌株不存在含有第一类整合子的接合性质粒 ,确定 4株整合子阳性菌株的整合酶基因 int I1基因位于染色体上。结论　本文发现的整合子阳性菌株对耐药基因的捕获是通过染色体 DNA介导的。     

AP-PCR法对沙门菌第一类整合子鉴定指纹图谱分析

目的在研究沙门菌携带的第一类整合子基因结构的基础上，对第一类整合子的阳性菌株进行基因指纹图谱分析。方法任意引物聚合酶链反应（AP-PCR）方法对两种不同血清型、整合子阴性与整合子阳性菌株研究基因指纹图谱，利用遗传距离矩阵，采用非加权配对法（UPGMA法）做遗传分析的树状图，进行指纹图谱分析。结果在最佳AP-PCR方法的反应条件下，可以将第一类整合子阴性、第一类整合子阳性菌株进行鉴定，同时对两种不同的血清型也可以加以区分，得到5个基因型。结论AP-PCR可以作为第一类整合子阳性菌株及其相关血清型进行尝试性的鉴定方法，对研究整合子介导细菌耐药机制的特性具有重要意义。     

Characterisation of antimicrobial resistance patterns and class 1 integrons among Escherichia coli and Salmonella enterica serovar Choleraesuis strains isolated from humans and swine in Taiwan

Escherichia coli isolates from humans (n=110) and swine (n=61) and Salmonella enterica serovar Choleraesuis isolates (n=95) from swine in southern Taiwan were characterised for antimicrobial resistance patterns and class 1 integrons. All E. coli isolates and S. Choleraesuis isolates were multidrug resistant and demonstrated high resistance to beta-lactams, aminoglycosides, tetracycline, sulfonamides, spectinomycin, chloramphenicol and nalidixic acid. By polymerase chain reaction and DNA sequencing, 104 (61%) E. coli isolates and 31 (33%) S. Choleraesuis isolates were found to carry class 1 integrons. The gene cassette array dfrA12-orfF-aadA2 was the most prevalent (24%) among the human and swine E. coli isolates, whilst the gene cassette array dfrA12-orfF-aadA2-sul1 was the most prevalent (24%) among S. Choleraesuis strains. For E. coil isolates, all class 1 integrons were located on conjugated plasmids. Meanwhile, human and swine E. coli isolates carrying identical gene cassettes were genetically unrelated. Our results revealed that multidrug resistance and class 1 integrons were widely present in E. coli and S. Choleraesuis isolates obtained in Taiwan and that class 1 integrons might play an important role in contributing to the horizontal transfer of antimicrobial resistance.     

Antimicrobial susceptibility phenotypes, resistance determinants and DNA fingerprints of Salmonella enterica serotype Typhimurium isolated from bovine in Southern Japan

A longitudinal study was conducted in cattle to determine the antimicrobial resistance phenotypes, integron elements, resistance genes and pulsed-field gel electrophoresis fingerprints among Salmonella enterica serotype Typhimurium isolates. A total of 33 strains were isolated and categorised into Groups A, B and C during the period 1989-2004. Thirty-one strains (93.9%) showed resistance to ampicillin (A) encoded by bla(OXA-1), bla(TEM) and bla(PSE-1) genes; 84.8% showed resistance to chloramphenicol (C) encoded by floR and catA1; 97.0% were resistant both to streptomycin (S) and sulfamethoxazole (Su), the former encoded by aadA1 and aadA2; 100% were resistant to oxytetracycline (T) encoded by tetA, tetB and tetG; and 42.4% were resistant to kanamycin (Km) encoded by aphA1-Iab. Multidrug resistance types observed were ACSSuT-Km (n=13), ACSSuT (n=15), ASSuT (n=3) and SSuT (n=2). Class 1 integrons ranging from 1.0 kb to 1.9 kb were detected from 54.5% of isolates (18/33). Integrons were not detected initially (1989-1992), then during the 1993-1996 interval a high frequency of 1.0 kb and 1.2kb amplicons were detected and during 2000-2004 the amplicon size increased to 1.7 kb and 1.9 kb. We report evidence of additional integration of resistance gene cassettes as shown by integrons with increased size. Finally, group B strains showed banding patterns indistinguishable from S. Typhimurium DT104 reference strain, indicating that the DT104 lineage existed on the island since 1993.     

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.     

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.     

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.     

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.     

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.     

不同地区铜绿假单胞菌第一类整合子和整合子相关基因盒的分布

目的对石家庄、昆明两地分离的铜绿假单胞菌临床菌株进行第一类整合子的分布及整合子相关基因盒携带率的比较分析，探讨第一类整合子在两地菌株分布的差别。方法采用PCR方法筛选检测64株石家庄、昆明两地临床分离的铜绿假单胞菌第一类整合子和及其所携带的整合子相关基因盒。结果64株铜绿假单胞菌临床菌中共有43株为第一类整合子阳性菌（阳性率67．2％）；其中石家庄菌株和昆明菌株阳性率分别为71．4％和64．3％，两地铜绿假单胞菌第一类整合子阳性率无显著性差异。第一类整合子阳性铜绿假单胞菌中，石家庄菌株和昆明菌株携带一类整合子相关基因盒的阳性率分别为80．0％和38．％，两地第一类整合子阳性菌中整合子相关基因盒的携带率具有显著性差异（P〈0．01）；两地菌株携带的基因盒大小不同。结论第一类整合子在铜绿假单胞菌临床菌株中广泛分布，环境的选择压力影响细菌整合子相关基因盒的分布。     

Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons.

Resistance of gram-negative organisms to antibiotics such as beta-lactams, aminoglycosides, trimethoprim and chloramphenicol is caused by many different acquired genes, and a substantial proportion of these are part of small mobile elements known as gene cassettes. A gene cassette consists of the gene and a downstream sequence, known as a 59-base element (59-be), that acts as a specific recombination site. Gene cassettes can move into or out of a specific receptor site (attl site) in a companion element called an integron, and integration or excision of the cassettes is catalysed by a site-specific recombinase (Intl) that is encoded by the integron. At present count there are 40 different cassette-associated resistance genes and three distinct classes of integron, each encoding a distinct Intl integrase. The same cassettes are found in all three classes of integron, indicating that cassettes can move freely between different integrons. Integrons belonging to class I often contain a further antibiotic resistance gene, sull, conferring resistance to sulphonamides. The sull gene is found in a conserved region (3'-CS) that is not present in all members of this class. Class I integrons of the sull type are most prevalent in clinical isolates and have been found in many different organisms. Even though most of them are defective transposon derivatives, having lost at least one of the transposition genes, they are none the less translocatable and consequently found in many different locations. The transposon Tn7 is the best known representative of class 2 integrons, and Tn7 and relatives are also found in many different species.