Prevalence and diversity of class 1 integrons and resistance genes in antimicrobial-resistant Escherichia coli originating from beef cattle administered subtherapeutic antimicrobials

Aims: To characterize class 1 integrons and resistance genes in tetracycline‐resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control). Methods and Results: Tetracycline‐resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher (P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 (r = 0·44), aadA1 (r = 0·61), cat (r = 0·58) and intI1(r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays. Conclusions: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline‐resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline‐resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons. Significance and Impact of the Study: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline‐resistant E. coli isolated from feedlot cattle.     

Identification and characterization of class 1 integron resistance gene cassettes among Salmonella strains isolated from healthy humans in China

Twenty-three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in-F and in-B primers showed the existence of amplicons of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1 -positive isolates, respectively. Sequence analysis revealed that the 1,009-bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664-bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all-round surveillance of the antibiotic resistance of pathogens.     

环境中抗生素抗性基因与I型整合子的研究进展

抗生素抗性基因(Antibiotic resistance genes,ARGs)作为一种新型污染物在不同环境中广泛分布、来源复杂,对生态环境和人类健康造成了很大的潜在风险。同时,Ⅰ型整合子(Int Ⅰ)介导的ARGs水平转移是环境中微生物产生耐药性的重要途径,Ⅰ型整合子整合酶基因(intI1)与ARGs丰度在环境中表现出了较高的正相关性,Int Ⅰ可以作为标记物在一定程度上反映ARGs在环境中的迁移转化规律和人类活动影响程度。本文介绍ARGs与Int Ⅰ在环境中的来源与分布,总结Int Ⅰ介导的ARGs迁移转化机制以及相关研究方法,并展望未来的研究发展趋势。     

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.     

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     

水产养殖环境耐药细菌中复合1型整合子的流行特征

【目的】了解复合1型整合子在水产养殖环境中的分布和流行特征。【方法】对108株分离自福建水产养殖场的耐药细菌,通过PCR和序列分析,检测其复合1型整合子上下游保守区和可变区的携带情况。【结果】有86株(79.6%)和47株(43.5%)分别携带1型整合子和ISCR1元件,这两种上下游保守区均携带的耐药细菌则有26株(24.1%),其中16株(14.8%)耐药细菌成功地扩增出上下游可变区,分布于8属9种。进一步对ISCR1上下游序列的拼接和分析,表明这16株细菌携带两种类型的复合1型整合子:(1)int I1-aac(6')-Ib-cr-arr-3-dfr A27-aad A16-qac E 1-sul1-ISCR1-sdr-qnr B6-qac E 1-sul1(15株);(2)int I1-aac(6')-Ib-cr-arr-3-dfr A27-aad A16-qac E 1-sul1-ISCR1-sap A-like-qnr B2-qac E 1(truncated)-sul1(1株,肺炎克雷伯菌C12),该阵列为新发现的复合1型整合子结构。【结论】复合1型整合子在水产养殖环境中并不少见,且存在于多种细菌中,但其基因阵列结构缺乏多样性。     

Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria

Individual merRTΔP regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one ‘pristine’ (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (= 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria. Forty-five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRTΔP probe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Detection of Pseudomonas aeruginosa carried a new array of gene cassettes within class 1 integron isolated from a teaching hospital in Nanjing, China

We report here novel array of gene cassettes found in single variable region of class 1 integron disseminated in Pseudomonas aeruginosa isolated from a teaching hospital in Nanjing, Jiangsu Province, China. 29 of 47 (61%) P. aeruginosa strains were confirmed haboured class 1 integron, and all the positive strains have the same variable region confirmed by PCR and RFLP methods. The variable region contained an unreported order of four gene cassettes aac(6′)-II-aadA13-cmlA8-oxa-10. Of those, cmlA8 gene was a variant of cmlA5 encoding non-enzymatic protein which putatively confer resistance to chloramphenicol. Susceptibility testing revealed multidrug-resistant mechanisms were involved in the class 1 integron positive clinical isolates. And the class 1 integron located on an about 15 kb transferable plasmid was certified by conjugation experiment and plasmid DNA analysis. The macro restriction profile indicated those clinical strains were clonally related. These authors contributed equally to this work.     

Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey

Aims: To isolate Bacillus thuringiensis strains from different olive‐related habitats (olive groves and olive oil factories) in Turkey and to characterize these strains by molecular methods. Methods and Results: A total of 150 samples, consisting of olive grove soil, green olive leaves, olive leaf residues, animal faeces, olive pomace and dust, were examined for the presence of B. thuringiensis. One hundred B. thuringiensis strains were isolated from 54 environmental samples (36%) and characterized in terms of crystal morphology, cry and cyt gene content by polymerase chain reaction, plasmid profiles and 16S‐internal transcribed spacer ribosomal DNA restriction fragment length polymorphism (16S‐ITS rDNA RFLP). The highest percentage of samples containing B. thuringiensis was found in 38 out of 54 total soil samples (70%). Of the 100 B. thuringiensis isolates, the most frequent crystal shapes were irregularly shaped (24%), spherical‐irregular pointed (19%), cuboidal (17%) and spherical (16%). The cry1 plus cry4 genotype was the most abundant genotype in our collection (21%). RFLP analysis of the amplified 16S‐ITS rDNA revealed 11 distinct patterns for the isolates and 10 reference strains. Conclusions: Bacillus thuringiensis isolates showed a great genetic diversity and crystal shape heterogeneity. Significance and Impact of the Study: This is the first study on the isolation and characterization of B. thuringiensis from olive‐related habitats in Turkey. No correlation was observed between the cry genotypes and insecticidal crystal shapes of the isolates. Restriction profiles of 23% of the isolates were found to be different from those of the 10 reference strains used.     

Comparing the frequencies of restriction fragment length polymorphisms for dystrophin gene in Chinese with those from Japanese and Caucasian populations

The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuⅡ/la, Taq Ⅰ/2b-3, Taq Ⅰ/5b-7, and Xba Ⅰ/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuⅡ/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq Ⅰ/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq Ⅰ/2b-3 and Xba Ⅰ/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq Ⅰ/8 and A2 (10.Tkb) in EcoR Ⅴ/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq Ⅰ/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu Ⅱ/la, there was no significant difference between Chinese and Japanese.     

Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 10¹⁹ bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.     

Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.     

Genetic diversity of Fusarium oxysporum populations isolated from different soils in France

The genetic diversity of soil-borne populations of Fusarium oxysporum was assessed using 350 isolates collected from six different French soils. All isolates were characterised by restriction fragment analysis of the PCR-amplified ribosomal intergenic spacer (IGS). Twenty-six IGS types were identified among the 350 isolates analysed. Five to nine different IGS types were detected in each soil. None of the IGS types was common to all of the soils. An analysis of the molecular variance based on IGS type relationships and frequency revealed that the genetic structure of the populations of F. oxysporum varied widely among the soils. Some populations were both highly diverse within the soils and differentiated between the soils. A possible relationship between the intrapopulation or interpopulation level of diversity and some external factors such as the soil type or the crop history was evaluated. A subsample representative of the diversity of the six populations was further characterised by analysing the genomic distribution of two transposable elements, impala and Fot1. One to 10 copies of the impala element were present in most of the isolates, irrespective of their soil of origin. The Fot1 element was only detected in 40% of the isolates originating from the three populations less diverse in terms of IGS types, but in 82.6% of the isolates originating from the three more diverse populations.     

Genetic variability between two breeds based on restriction fragment length polymorphisms (RFLPs) of major histocompatibility complex class I genes in the pig

Summary Restriction fragment length polymorphism analyses of SLA class I genes were performed on 55 Duroc and 24 Hampshire boars from the 1986–87 national performance tests of each breed. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA isolated from white blood cells by using Pvu II, Bam HI, and Eco RI endonucleases and a swine MHC class I probe. Genetic variability within and between the two breeds was estimated in terms of nucleotide diversity, by using a mathematical analysis based on the different RFLP patterns. The nucleotide diversity calculated within each breed was less than that between the two breeds. The results from the nucleotide diversity analysis suggested that genetic variability was greater in the Duroc breed than in the Hampshire breed. A relatively high level of genetic variability was shown in the class I major histocompatibility complex genes in the pig.     

Distribution and molecular profiling of class 1 integrons in MDR Acinetobacter baumannii isolates and whole genome-based analysis of antibiotic resistance mechanisms in a representative strain

The class 1 integron is an important driver of the nosocomial dissemination of multidrug-resistant (MDR) bacteria, such as Acinetobacters. In this study, we characterized the gene cassette arrays of class 1 integrons in Acinetobacter baumannii, where the detailed structure of these integrons for 38 clinical strains was analyzed. The results showed that there are three types of gene cassette arrays that are carried by different class 1 integrons, among them the aac(6′)-IId-catB8-aadA1 array was the most prevalent. For detailed analysis of the integron structure, whole genome sequencing was carried out on strain AB16, and it was found that a single integron on its chromosome has a partial Tn21 transposon in its 5′ flanking region and two complete copies of the insertion element IS26 in both the 5′ and 3′ flanking regions, indicating that the integron could be acquired by horizontal gene transfer. Furthermore, there is one resistance island AbaR22, one bla gene containing a transposon, four intrinsic resistant genes and one efflux pump that together confer six types of antibiotic resistance.     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

Genetic and phenotypic diversity of autochthonous Saccharomyces spp. strains associated to natural fermentation of 'Malvasia delle Lipari'

AIMS: Characterize from both genetic and phenotypic standpoints the indigenous strains of Saccharomyces spp. associated with natural fermentation of 'Malvasia delle Lipari'. METHODS AND RESULTS: A total of 192 yeast isolates were obtained from completed fermentation of a mix of 'Malvasia delle Lipari' (92%) and 'Corinto nero' (8%) grapes in two wineries in Salina Island (Sicily, Italy). Fifty-one Saccharomyces spp. isolates were characterized using ITS-PCR, random amplified polymorphic DNA-PCR and mitochondrial DNA restriction fragment length polymorphism and 12 biotypes were identified. Representative strains of each biotype, tested for their physiological traits, exhibit different killer activity, fermentation vigour, production of hydrogen sulphide and show similar beta-glucosidase and proteolytic activity. CONCLUSIONS: It is possible to cluster in different groups naturally occurring indigenous biotypes of Saccharomyces cerevisiae from 'Malvasia delle Lipari' on the basis of molecular profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Deeper insight on indigenous wine yeast of a conserved environment. The knowledge gained might offer a contribution to the selection of autochthonous wine yeast as starters for controlled fermentations.