Molecular characterization of class 1 integrons from Irish thermophilic Campylobacter spp

OBJECTIVES: In this study a large random collection (n = 378) of Irish thermophilic Campylobacter isolates were investigated for the presence of integrons, genetic elements associated with the dissemination of antimicrobial resistance. METHODS: Purified genomic DNA from each isolate was analysed by PCR for the presence of class 1 integrons. Four gene cassette-associated amplicons were completely characterized. RESULTS: Sixty-two of the isolates possessed a complete class 1 integron with a recombined gene cassette located within a 1.0 kb amplicon containing an aadA2 gene. This cassette was present in both Campylobacter jejuni and Campylobacter coli isolates and following sequence analysis was shown to be similar to sequences recently reported in Salmonella enterica Hadar and on an 85 kb plasmid conferring quinolone resistance in Escherichia coli. CONCLUSIONS: Aminoglycoside aadA2-encoding class 1 integrons were identified among unrelated Campylobacter spp. Amino acid sequence comparisons revealed identical structures in both Salmonella and E. coli. The presence of class 1 integrons in Campylobacter spp. may be significant should these organisms enter the food chain and especially when antimicrobial treatment for severe infections is being considered.     

Diverse class 2 integrons in bacteria from beef cattle sources

OBJECTIVES: The purpose of this study was to determine the diversity of class 2 integrons in bacteria isolated from beef cattle sources. METHODS: The variable regions of a subset of 11 class 2 integron-containing bacteria were analysed by PCR and DNA sequencing for the presence of novel rearrangements. RESULTS: A total of six different class 2 integron arrays were identified and four of these were fully characterized. Three of the four arrays characterized have been previously described; however the remaining array is unlike previously described class 2 integrons. The novel class 2 integron was found in Providencia stuartii and contains an apparently functional class 2 integrase. Examination of the variable region of the P. stuartii integron identified nine open reading frames, mostly of unknown function, and represents the first report of a class 2 integron without inserted antibiotic resistance gene cassettes. CONCLUSIONS: This study has identified a novel class 2 integron found in P. stuartii that contains an apparently functional naturally occurring class 2 integrase. Further investigation of this novel class 2 integron is required to determine the impact of a functional class 2 integrase upon the evolution of class 2 integrons.     

Incidence of class 1 integrons in a quaternary ammonium compound-polluted environment

Samples of effluent and soil were collected from a reed bed system used to remediate liquid waste from a wool finishing mill with a high use of quaternary ammonium compounds (QACs) and were compared with samples of agricultural soils. Resistance quotients of aerobic gram-negative and gram-positive bacteria to ditallowdimethylammomium chloride (DTDMAC) and cetyltrimethylammonium bromide (CTAB) were established by plating onto nutrient agar containing 5 microg/ml or 50 microg/ml DTDMAC or CTAB. Approximately 500 isolates were obtained and screened for the presence of the intI1 (class 1 integrase), qacE (multidrug efflux), and qacE Delta1 (attenuated qacE) genes. QAC resistance was higher in isolates from reed bed samples, and class 1 integron incidence was significantly higher for populations that were preexposed to QACs. This is the first study to demonstrate that QAC selection in the natural environment has the potential to coselect for antibiotic resistance, as class 1 integrons are well-established vectors for cassette genes encoding antibiotic resistance.     

739株革兰阴性杆菌中I类整合子的分布及其基因盒结构分析

目的 了解I类整合子在合肥地区革兰阴性杆菌中的分布状况,研究其整合的基因盒结构。 方法 PCR扩增I类整合子整合酶基因及其基因盒可变区和3′保守区,可变区产物进行测序分析。 结果 739株临床分离革兰阴性杆菌中,I类整合酶基因（IntI1）阳性菌株为399株（54.0%）;随机抽取的80株IntI1阳性菌中64株扩增出长度不等的9种基因盒产物,它们在所测的细菌中大致有17种不同的组合模式;77株扩增出3′保守区预期产物。出现频率较高的2种I类整合子基因盒可变区产物分别为dfrA15和aadA2,片段最长的基因盒可变区产物为aadB、aadA1和cmlA6。 结论 I类整合子在合肥地区临床分离革兰阴性杆菌中广泛分布,其整合的基因盒种类多样且在不同菌株中呈多种组合模式。     

耐亚胺培南铜绿假单胞菌整合子的鉴定与分析

目的 鉴定并分析耐亚胺培南铜绿假单胞菌(IRPa)中整合子及其携带耐药基因盒的特点。 方法 收集2003～2007年临床分离IRPa共74株,用PCR-RFLP筛查携带整合酶的菌株,并对整合子进行分类。整合酶阳性菌株用普通PCR扩增检测整合子的qacE△1sul1基因、整合子可变区及膜微孔蛋白基因oprD2,并对整合子可变区扩增产物测序分析。 结果 74株菌中有16株菌（21.6%）携带整合酶基因,均为Ⅰ类整合子;有14株检出整合子可变区,经测序共得到7种整合子耐药基因盒的组合形式,其中有4种为首次发现,在GenBank上的登录号分别为FJ917747、FJ817422、FJ655384和FJ817423;另携带IMP-9基因2株。16株IRPa中有8株oprD2基因检测为阳性,其余为阴性。 结论 在本地区临床分离的IRPa中,仅2株菌的Ⅰ类整合子中携带的IMP-9与亚胺培南的耐药有关,其他菌株的整合子中携带的基因盒主要是介导对氨基糖苷类与β内酰胺类抗生素的耐药。金属酶的产生并不是其对亚胺培南耐药的主要原因。     

Prevalence and characterization of integrons from bacteria isolated from a slaughterhouse wastewater treatment plant

OBJECTIVES: To investigate the presence and distribution of integron-carrying bacteria from a slaughterhouse wastewater treatment plant (WWTP). METHODS: Enterobacteriaceae and aeromonads were isolated at different stages of the wastewater treatment process and screened for the presence of integrase genes by dot-blot hybridization. Integrase-positive strains were characterized in terms of phylogenetic affiliation, genetic content of integrons and antimicrobial resistance profiles. Plasmid location of some integrons was established by Southern-blot hybridization. Strains containing integron-carrying plasmids were selected for mating experiments. RESULTS: Integrase genes were present in all samples, including the final effluent. The global prevalence was determined to be 35%, higher than in other aquatic environments. Forty-two integrase-positive isolates were further characterized. Nine distinct cassette arrays were found, containing genes encoding resistance to beta-lactams (bla(OXA-30)), aminoglycosides (aadA1, aadA2, aadA13, aadB), streptothricin (sat1, sat2), trimethoprim (dfrA1, dfrA12), a putative esterase (estX) and a protein with unknown function (orfF). Gene cassette arrays aadA1, dfrAI-aadA1 and estX-sat2-aadA1 were common to aeromonads and Enterobacteriaceae. The class 2 integron containing an estX-sat2-aadA1 cassette array was detected for the first time in Aeromonas sp. Nearly 12% (5 out of 43) of intI genes were located in plasmids. intI genes from isolates MM.1.3 and MM.1.5 were successfully conjugated into Escherichia coli at frequencies of 3.79 x 10(-5) and 5.46 x 10(-5) per recipient cell, respectively. CONCLUSIONS: Our data support the hypothesis that WWTPs constitute a potential hot spot for horizontal gene transfer and for selection of antimicrobial resistance genes among aquatic bacteria. Moreover, water discharges represent a possible risk for dissemination of undesirable genetic traits.     

Genetic context and structural diversity of class 1 integrons from human commensal bacteria in a hospital intensive care unit

Most surveys for class 1 integrons are at least partly predicated on PCR screening that targets integron conserved regions. However, class 1 integrons are structurally diverse, so dependence on conserved regions may lead to missing clinically relevant examples of class 1 integrons. Here, we surveyed a commensal population of bacteria from patients in an intensive care unit to identify class 1 integrons irrespective of their structure or genetic context. We identified several examples of class 1 integrons linked to complete Tn402-like or Tn402 hybrid transposition modules and diverse insertion points with respect to the inverted repeat IRi boundary. The diversity and abundance of class 1 integrons identified are such that many novel elements seen here would not have been identified by commonly used methods, and they revealed an additional level of complexity.     

Prevalence and characterization of class 1 and class 2 integrons in Escherichia coli isolated from meat and meat products of Norwegian origin

OBJECTIVES: The aim of the study was to investigate the prevalence of integrons and to characterize inserted gene cassettes in Escherichia coli isolated from meat and meat products of Norwegian origin. METHODS: The strains investigated (n = 241 resistant out of 944 investigated) were collected within the frame of the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET) during the years 2000-2003. PCR and DNA sequencing were used for detection of the integrase genes and gene cassettes within the integrons. RESULTS: Integrons were detected in 43 (18%) of the 241 resistant isolates. Class 1 integrons were detected in 29 (12%) strains and class 2 integrons were detected in 14 (6%) strains. Ten different gene cassettes were detected: dfrA1, dfr2a, dfrA12, aadA1, aadA2, catB2, oxa-30, sat, sat1 and orfF. The dfrA1 + aadA1 combination was the most prevalent cassette combination, detected in 12 of 29 class 1 integrons. Twelve (of 14) class 2 integrons contained a cassette area consistent with that on Tn7, the remaining two contained the cassettes sat + sat1 + aadA1. Nearly one-third of the class 1 integrons (9 of 29) lacked the sul1 gene. Ten gene cassettes (one dfr2a, two catB2 and seven aadA1) were expressed at levels below breakpoint values normally used to classify strains as resistant. CONCLUSIONS: Integrons of class 1 or 2 were present in approximately 18% of the resistant E. coli strains investigated. Certain cassette combinations in class 1 integrons seem to be more widespread than others, like the dfrA1 + aadA1. Low-level expression of antimicrobial resistance, caused by the expression of certain gene cassettes in some integrons represents an obstacle in classifying strains as susceptible or resistant.     

Identification of antimicrobial resistance and class 1 integrons in Shiga toxin-producing Escherichia coli recovered from humans and food animals

OBJECTIVES: The objective of this study was to identify antimicrobial resistance and class 1 integrons among Shiga toxin-producing Escherichia coli (STEC). METHODS: Two-hundred and seventy-four STEC recovered from poultry, cattle, swine and humans were characterized by antimicrobial susceptibility testing, screened for the presence of class 1 integrons by PCR, and assayed for integron transfer by conjugation. RESULTS: Ninety-three (34%) of the isolates were resistant to streptomycin, followed by 89 (32%) to sulfamethoxazole, 83 (30%) to tetracycline, 48 (18%) to ampicillin, 29 (11%) to cefalothin, 22 (8%) to trimethoprim/sulfamethoxazole, 18 (7%) to gentamicin, 13 (5%) to chloramphenicol and 10 (4%) to cefoxitin. Class 1 integrons were detected in 43 (16%) of the 274 isolates. The adenyl acetyltransferase gene, aadA, which confers resistance to streptomycin, was identified in integrons from 41 (95%) of these 43 isolates, and the dfrA12 gene, which confers resistance to trimethoprim, was identified in integrons from eight (19%) of the isolates. The sat1 gene, which confers resistance to streptothricin, an antimicrobial that has never been approved for use in the United States, was identified in integrons from three (7%) of the isolates. Transfer of integrons by conjugation between strains of E. coli resulted in transfer of antimicrobial-resistant phenotypes for ampicillin, chloramphenicol, cefalothin, gentamicin, tetracycline, trimethoprim, sulfamethoxazole and streptomycin. CONCLUSIONS: Antimicrobial resistance is common in STEC. Class 1 integrons located on mobile plasmids have facilitated the emergence and dissemination of antimicrobial resistance among STEC in humans and food animals.     

Molecular characterization of class 1 integrons and antimicrobial resistance in Aeromonas strains from foodborne outbreak-suspect samples and environmental sources in Taiwan

One hundred thirty-three Aeromonas spp. isolates were examined for multiple antibiotic resistance phenotypes and prevalence of class 1 integron sequences. Twenty-four (18.0%) of these isolates contained class 1 integron. Seven different class 1 integrons were found among 24strains, with a total of 10 different gene cassettes encoding for resistance to trimethoprim (dfr12 and dfr2d), aminoglycosides (aadA1 and aadA2), beta-lactam antibiotics (oxa2), chloramphenicol (catB3 and catB8), quaternary ammonium amines (qacE2), and 2 ORFs (orfD and orfF) with unknown function. Rate of antibiotic resistance was different between integron-positive and integron-negative strains. Trimethoprim and trimethoprim-sulphamethoxazole resistances were commonly associated with integron, and all of integron-positive isolates were multiple resistant to more than 3 agents. Resistance to as many as 10 antimicrobial agents were observed in integron-positive strains. Several cassette arrays of class 1 integrons identified in this study were not previously reported in Aeromonas strains. This study demonstrates the wide distribution of class 1 integron in Aeromonas spp. isolated from foodborne outbreak-suspect samples and environmental sources in Taiwan.     

Prevalence and types of class 1 integrons in aminoglycoside-resistant Enterobacteriaceae from several Chilean hospitals

Members of the family Enterobacteriaceae are responsible for a variety of nosocomial infections, treatment of which is limited due to their increasing resistance to antibiotics. Some bacterial genes encoding antibiotic resistance comprise the major part of gene cassettes, most of which are associated with integrons. In this work, the carriage of class 1, 2 and 3 integrons was investigated in 191 Enterobacteriaceae isolates from clinical specimens of hospitalized patients. Class 1 integrons were found to be the most common, whereas no class 3 integrons were detected. The variable regions of 13 class 1 integrons were characterized and four types were found. Type 1 harbours only ant(3")I, type 2 harbours ant(2")I and ant(3")I, type 3 harbours aac(6')Ib and ant(3")I and type 4 lacks inserted gene cassettes.     

Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology

A screening technique for integrons in members of the family Enterobacteriaceae and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5' ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.     

Family of class 1 integrons related to In4 from Tn1696

The class 1 integron In28, found in the multidrug resistance transposon Tn1403, was found to be located in the res site of the backbone transposon and is flanked by a 5-bp direct duplication, indicating that it reached this position by transposition. In28 has a backbone structure related to that of In4, but has lost internal sequences, including the sul1 gene, due to an IS6100-mediated deletion. In28 also lacks the partial copy of IS6100 found in In4 and contains different gene cassettes, blaP1, cmlA1, and aadA1. In1, the class 1 integron found in the multidrug resistance plasmid R46, is also located in a putative res site and belongs to the In4 group. In1 has a shorter internal deletion than In28 and has also lost one end. Additional integrons with structures related to In4 were also found in databases, and most of them had also lost either one end or internal regions or both. Tn610 belongs to this group.     

Molecular characterization of integrons in non-typhoid Salmonella serovars isolated in Japan: description of an unusual class 2 integron

OBJECTIVES: To characterize class 1 and class 2 integrons among non-typhoid Salmonella enterica serovars in Japan, and also to monitor the spread of the multidrug-resistant S. enterica serovar Typhimurium DT104. METHODS: A total of 105 Salmonella isolates were included in this study. The broth microdilution method was used to determine the MIC values of a range of antibiotics for these isolates. PCR and DNA sequencing were used for screening and characterization of class 1 and class 2 integrons. RESULTS: PCR sequencing analysis revealed the presence of seven profiles of class 1 integrons in addition to a new type of class 2 integron. The identified gene cassettes within class 1 integrons were as follows; aadA1, aadA2 and aadA5, which confer resistance to streptomycin and spectinomycin; aadB, which confers resistance to gentamicin, kanamycin and tobramycin; dfrA1 and dfrA17, which confer resistance to trimethoprim; bla(PSE-1), which confers resistance to ampicillin; catB3, which confers resistance to chloramphenicol; and sat1, which confers resistance to streptothricin. Two strains of the multidrug-resistant S. Typhimurium DT104 were characterized in this study. DNA sequencing of class 2 integrons identified one with an unusual array of gene cassettes, sat, sat1 and aadA1. CONCLUSIONS: In this study, we characterized the antibiotic resistance gene cassettes within class 1 integrons in different isolates of non-typhoid Salmonella serovars, and we also identified a new type of class 2 integron.     

鲍曼不动杆菌Ⅰ类整合子系统与其耐药性关系的研究

目的了解Ⅰ类整合子系统在鲍曼不动杆菌中的分布状况,探讨其与该菌耐药性之间的关系。方法用琼脂稀释法测定鲍曼不动杆菌对14种抗生素的敏感性。用PCR法检测Ⅰ类整合子系统。结果29.2%(21/72)的菌株检测出Ⅰ类整合子系统。含与不含Ⅰ类整合子系统的菌株在耐药性上存在显著性差异(P<0.05)。不同类型Ⅰ类整合子的耐药表型存在一定的差异。结论Ⅰ类整合子系统与鲍曼不动杆菌多重耐药性以及耐药表型之间有密切的关系。     

Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes

The resistance profiles, for 15 antimicrobial agents, of 333 Salmonella strains representing the most frequent nontyphoidal serotypes, isolated between 1989 and 1998 in a Spanish region, and 9 reference strains were analyzed. All strains were susceptible to amikacin, ceftazidime, ciprofloxacin, and imipenem, and 31% were susceptible to all antimicrobials tested. The most frequent types of resistance were to sulfadiazine, tetracycline, streptomycin, spectinomycin, ampicillin, and chloramphenicol (ranging from 46 to 22%); 13% were resistant to these six drugs. This multidrug resistance pattern was found alone or together with other resistance types within serotypes Typhimurium (45%), Panama (23%), and Virchow (4%). Each isolate was also screened for the presence of class 1 integrons and selected resistance genes therein; seven variable regions which carried one (aadA1a, aadA2, or pse-1) or two (dfrA14-aadA1a, dfrA1-aadA1a, oxa1-aadA1a, or sat1-aadA1a) resistance genes were found in integrons.     

不同年代铜绿假单胞菌1型整合子结构比较

目的研究1992-1996年和2003-2005年两个时间段90份铜绿假单胞菌1型整合子结构，比较其结构差异，以了解不同年代1型整合子变化趋势。方法用常规和长片段PCR方法扩增1型整合子及耐药基因，并对PCR产物进行测序和GenBank比对分析。结果41份1992—1996年间样本有13份检出1型整合子结构，长片段PCR扩增1型整合子DNA平均长度为1868bp，平均包含2个耐药基因，包括qaeE△1（n=6）、sul1（n=14）、aadA1（n=2）、aadB（n=1）、PSE-1（n=2）和tetA（n=1）等6种耐药基因，形成5种耐药基因盒组合。49份2003-2005年间样本中有19份检出1型整合子结构，长片段PCR扩增产物平均长度为3383bp，平均包含3．6个耐药基因，包括qaeE△1（n=18）、sul1（n：25）、aadA1（n=6）、aadB（n=7）、aaeA4（n=2）、PSE-1（n=3）、VEB-1（n=4）、OXA10（n=1）、cm1A（n=1）和tetA（n=2）10种耐药基因，形成9种耐药基因盒组合。结论根据整合子碱基数量和所携带耐药基因盒数量，发现2003--2005年间检出的1型整合子结构比1992—1996间检出的复杂程度增加。     

Ⅰ类整合子在多重耐药鲍曼不动杆菌中的作用研究

目的了解我院多重耐药鲍曼不动杆菌临床分离株中Ⅰ类整合子的流行情况,建立一种快速简便的Ⅰ类整合子聚合酶链反应（PCR）检测方法。方法收集我院2008年9月至2010年9月临床分离的鲍曼不动杆菌63株,用MIC法检测耐药性,PCR技术检测Ⅰ类整合子基因。结果 63株鲍曼不动杆菌中Ⅰ类整合子基因阳性率56%（35株）。Ⅰ类整合子阳性和阴性的鲍曼不动杆菌对各种抗生素的耐药性存在明显差异。结论我院多重耐药鲍曼不动杆菌中Ⅰ类整合子携带率高,携带的耐药基因为aaeA4、catB8和aadA1。PCR技术可以方便快速地检出Ⅰ类整合子。     

Molecular epidemiology of orf513-bearing class 1 integrons in multiresistant clinical isolates from Argentinean hospitals

The spread of orf513-bearing class 1 integrons is associated with bla(CTX-M-2) in gram-negative clinical isolates in Argentina, with In35 being the most frequently found integron (74%). Among 65 isolates without bla(CTX-M-2), only one harbored a novel orf513-bearing class 1 integron with the dfrA3b gene. The finding of orf513 not associated with class 1 integrons in two gram-positive strains indicates the widespread occurrence of this putative site-specific recombinase.