Gene Cassette Arrays In Class Research Articles

Characterization of class 1 integrons harboring blaVEB-1 in Vibrio parahaemolyticus isolated from ready-to-eat foods in China

The aim of this study is to investigate the prevalence of integrons and integron-associated antibiotic resistance in V. parahaemolyticus strains collected from RTE foods in China, and to carry out a comprehensive analysis on the molecular characterization of V. parahaemolyticus strains carrying blaVEB-1-positive class 1 integron. Of the 51 V. parahaemolyticus strains isolated from RTE food samples, none of the isolates was found to carry integrase genes intI2 and IntI3. However, all 51 strains were positive to integrase gene intI1, and only 2 of 51 (3.92%) intI1-positive isolates yielded polymerase chain reaction (PCR) products of gene cassette amplification. Sequence data and BLAST analysis indicated the gene cassette arrays of class 1 integron in VP007 is dfrA14-blaVEB-1-aadB, while the gene cassette arrays of class 1 integron in V187 is blaVEB-1-aadB-arr2-cmlA-blaOXA-10-aadA1. Antimicrobial susceptibility testing showed that the two V. parahaemolyticus isolates harboring class 1 integrons exhibited multi-drug resistance to various antibiotics. S1-PFGE and Southern blot analysis confirmed the class 1 integron harboring blaVEB-1 gene in V187 was located on the plasmid of ~175 kb and transferrable to the recipient strain by conjugation. This is the first detection of class 1 integrons harboring the ESBL gene blaVEB-1 in V. parahaemolyticus. To the best of our knowledge, this is also the first report of VEB-producing V. parahaemolyticus from RTE foods. Our findings revealed that class 1 integron on conjugative plasmid contributes significantly to the dissemination of VEB-producing V. parahaemolyticus, which warrants further investigation because of the public health threat it poses.

Proteae: a reservoir of class 2 integrons?

Our aim was to confirm with a large panel of clinical isolates that class 2 integrons are highly prevalent in Proteae and to analyse their genetic characteristics. Proteae (Proteus spp., Morganella spp. and Providencia spp.) isolates were collected from clinical samples during 2013 at Limoges University Hospital, France. The presence of class 1, 2 and 3 integrons was investigated by quantitative PCR. The presence of a stop codon in the intI2 gene was determined by Sanger sequencing. The gene cassette arrays of class 2 integrons were determined by PCR-RFLP and Sanger sequencing or next-generation sequencing when needed. Of the 327 Proteae collected, 103 (31.5%) harboured a class 2 integron and 45 (13.8%) a class 1 integron. No class 3 integrons were detected. One functional IntI2 integrase was detected in a Morganella morganii isolate. Six different gene cassette arrays were detected. Four had already been described in the literature: dfrA1-sat2-aadA1 (72 isolates), dfrA1-catB2-sat2-aadA1 (17), sat2-aadA1 (6) and lnu(F), dfrA1, aadA1 (1). We identified two new gene cassette arrays: (i) a new variant of the dfrA1 gene cassette (one isolate; the one with the functional IntI2); and (ii) the array dfrA1-gcu115-sat2 harbouring the new gcu115 gene cassette with two ORFs encoding proteins of unknown functions (five isolates). We showed a high frequency of class 2 integrons, as well as a diversity of gene cassette arrays, among Proteae. This work highlights that the Proteae tribe plays an important role as a reservoir of class 2 integrons.

Class 1 and 2 Integrons in Hospital Strains of Gram-Negative Bacteria Isolated in Moscow and in Regions of the Russian Federation

Natural systems of cloning and expression of mobile gene cassettes caught by site-specific recombination, class 1 and 2 integrons, play an important role in mobilization and spread of genetic determinants of antibiotic resistance in gram-negative bacterial human pathogens, especially in a hospital environment. The gene cassettes localized in variable parts of integrons determine resistance to antibacterial drugs (AD) of different functional classes. The aim of the work is the detection and characteristic of class 1 and 2 integrons in gram-negative bacteria isolated in multidisciplinary hospitals of Moscow and other regions of the Russian Federation in 2003–2015. Clinical strains of gram-negative bacteria (n = 1248) mainly had multidrug resistance phenotype (94%). An amount of 10% of strains were resistant to AD of three functional groups; 19%, four; 42%, five; 17%, six; and 7%, seven. A high level of resistance of the studied strains to beta-lactams is associated with the presence of beta-lactamase genes of blaTEM (35% strains), blaSHV (25%), blaCTX-M (38%), blaOXA (31%), blaVIM (3%), and blaNDM (2%) types; to AD of other functional groups, with the presence of class 1 integrons (59%) and class 2 integrons (8%). Most class 1 integrons (54%) and class 2 integrons (88%) contained in its variable part 22 variants of gene cassette arrays in class 1 integrons and 4 variants in class 2 integrons. During the study, 31 types of gene cassettes were identified (including the most widespread, aadB, aacA4, aacC1, aadA1, aadA2, aadA5, blaVIM-2, dfrA1, dfrA7, dfrA12, orfC, orfE, orfY, and sat1) associated with the resistance of strains to aminoglycosides, chloramphenicol, sulfonamides, and beta-lactams, as well as orf cassettes encoding the proteins with unknown functions. New gene cassette arrays were identified: dfrA12s-orfF-aadA2 (In1249) and dfrA1-IS911-sat1-aadA1 (not numbered).

Incidence of antibiogram, antibiotic resistance genes and class 1 and 2 integrons in tribe Proteeae with IMP27 gene for the first time in Providencia sp. isolated from pet turtles.

Proteeae is a tribe which consists of three genera: Proteus, Providencia and Morganella. The objective of this study was to determine antimicrobial resistance profile, virulence genotype and class 1 and 2 integrons in Proteeae isolated from pet turtles and to determine the impact of antibiotic resistance on virulence and antimicrobial resistance genes. Integron-positive isolates were used to detect their gene cassette array. Sixty four Proteeae were isolated and all were resistant to macrolides (100%). Among 64 isolates 56, 52, 36 and 25 were resistant to nitrofurans, β-lactams, tetracycline and aminoglycoside respectively. Sixteen (25%) isolates were positive for intI1 while 14 (21·87%) were positive for integrase 2 (intI2). Eleven (17·18%) isolates were positive for class 1 variable region while 7 (10·93%) were positive for class 2 variable region. IMP27, a novel metallo β-lactamase gene was found in Providencia isolates. Proteus sp. were positive for every tested virulence genes and UreC gene was detected in 48·44% followed by zapA (17·19%), mrpA (17·19%) and hlyA (14·06%) genes. In this study, integron associated-antibiotic resistance genes have been identified in Proteeae isolates in a considerable range representing clear threats to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, multidrug-resistant Proteeae isolates had several antibiotic resistance genes. Integrons are important contributors to the development and dissemination of antibiotic resistance. We could detect both class 1 and 2 integrons and several gene cassette arrays in class 1 integron. The gene cassette arrays of the Class 2 integrons contained IMP27-dfrA1-aadA1-catB2-ybeA-ybgA in two isolates. To the best of our knowledge this is the first study to report detection of IMP27 in Providencia rettgeri isolates. All results indicate that healthy pet turtles act as potential reservoirs for Proteeae species with zoonotic potential.

Extended-spectrum β-lactamase-producing Escherichia coli isolated from healthy humans in Mexico, including subclone ST131-B2-O25:H4-H30-Rx.

The resistance mechanisms, molecular type and plasmid content of cefotaxime-resistant Escherichia coli isolated from faecal samples of healthy volunteers in Puebla, Mexico, were characterised. Cefotaxime-resistant E. coli were recovered from 11 (18%) of 60 healthy volunteers. The isolates (one per sample) were characterised as multidrug-resistant and phenotypically extended-spectrum β-lactamase (ESBL)-producing strains. Genes encoding resistance to β-lactams (blaCTX-M-15, blaCTX-M-14a, blaCTX-M-14b, blaOXA-1, blaTEM-1b), quinolones [aac(6')-Ib-cr, qnrB19], aminoglycosides [aac(3')-II] and tetracycline [tet(A), tet(B)] were detected among the 11 ESBL-producing E. coli by PCR and sequencing, as well as gene cassette arrays in class 1 (dfrA17-aadA5) and class 2 (dfrA1-sat2-aadA1) integrons. Seven pulsotypes were identified by XbaI PFGE and the strains were distributed into phylogroups (number of isolates) A (2), B2 (4) and D (5). Seven sequence types were identified, four of them novel (ST5060, ST5079, ST5080 and ST5081), associated with phylogroups A-D. Transfer of a 140-kb IncFIA plasmid carrying the blaCTX-M-15 gene was evidenced in the ST5060 strain. Four CTX-M-15-producing E. coli strains of phylogroup B2 belonged to the ST131 complex, and IncFIB plasmids of 130kb and 155kb were detected in two of them. Multiple plasmid addiction systems were also found. Serotyping and fimH subtyping of ST131-B2 strains identified the ST131-B2-O25:H4-H30-Rx subclone. Additionally, this subclone and CTX-M-14-producing isolates were detected among residents living in the same household, suggesting clonal dissemination. This study reports the detection of E. coli ST131-B2-O25:H4-H30-Rx subclone in healthy humans in Mexico, highlighting its dissemination in the community setting.

Antimicrobial resistance and integron gene cassette arrays in commensal Escherichia coli from human and animal sources in IRI.

BackgroundThe human and animal intestinal tract harbors a complex community of microbes which enables bacteria to inherit antibiotic resistance genes. The aims of this study were to investigate clonality, antimicrobial resistance, prevalence and gene cassette arrays of class I and II integrons among commensal Escherichia coli from human and animals.MethodsA total of 200 E. coli isolates from human, chicken, cattle, and sheep were isolated followed by phenotypic antibiotic susceptibility testing and detection of class I and II integrons gene cassettes arrays. The clonal relationship of the isolates were analyzed by (GTG)5-PCR.ResultsOf 200 isolates, 136 isolates were multi drug resistance (MDR) including 47, 40, 31 and 18 isolates from chicken, human, cattle and sheep, respectively. Class I integron was detected in 50, 38, 6 and 16 %, while class II was detected in 26, 8, 0 and 4 % of chicken, human, cattle and sheep isolates, respectively. Variable regions were amplified and sequenced. Cassette arrays in class I integrons were: dfrA1, dfrA5, dfrA7, dfrA12, aadA1, dfrA17 aadA1, aadA22, aadB–aadA2 and dfrA12–orfF–aadA2, and for class II, dfrA1-sat-aadA1, and sat-sat1-aadA1 were detected. Six class I and three class II positive strains did not produce any amplicons for variable region. Integron-positive isolates showed higher rate of resistance to streptomycin and trimethoprim–sulphamethoxazole, especially in chicken isolates which were fed antibiotics. Low similarity and great genetic diversity of class I and II integrons carrying isolates indicated no clonal relation.ConclusionsIntegrons encoding for antibiotic resistance are significantly present among non-pathogenic commensal E. coli, especially from the hosts medicated by antibiotics. Uncontrolled use of antibiotics will increase the numbers of multiple drug resistant isolates and integrons prevalence.

Coliform bacteria isolated from recreational lakes carry class 1 and class 2 integrons and virulence-associated genes.

To characterize the integron-harbouring Gram-negative bacteria in recreational lakes, with focus on the genetic content of integrons, antimicrobial resistance profiles and virulence-associated genes. The presence and structure of integrons in coliform bacteria isolated from the water of four recreational lakes located in Poznań, Poland, was determined by PCR method. Antimicrobial resistance testing was done by disc diffusion method. Virulence-associated genes in integron-bearing Escherichia coli isolates were detected by PCR. A total of 155 integron-bearing strains of coliform bacteria were cultured. Sequence analysis showed the presence of dfrA7, aadA1, dfrA1-aadA1, dfrA17-aadA5 and dfrA12-orfF-aadA2 gene cassette arrays in class 1 integrons and dfrA1-sat2-aadA1 in class 2 integrons. Higher frequency of integron-positive bacteria and higher antimicrobial resistance ranges were noted in colder months (January and November) compared with spring and summer months. The integron-harbouring E. coli carried up to nine virulence-associated genes, with the highest frequency of kpsMT (84.6%) and traT (783%), coding for group 2 capsule and determining human serum resistance respectively. Integron-bearing multidrug resistant coliform bacteria carrying virulence genes are present in waters of recreational lakes. This study presents antimicrobial resistance and virulence-associated genes in integron-bearing coliform bacteria present in the waters of recreational lakes, which showed that multidrug resistant bacteria with virulence traits might pose a threat to public health. Moreover, the presence of genes typical for enterotoxigenic and Shiga toxin-producing E. coli is a concern.

Global dissemination patterns of common gene cassette arrays in class 1 integrons.

Integrons are genetic elements that contain a site-specific recombination system able to capture, express and exchange gene cassettes. Mobile integrons are widespread and often confer resistance to multiple antibiotics, due to the expression of the arrays of gene cassettes they carry. Although >300 cassette arrays have been described, < 10 array compositions prevail in the reports related to class 1 integrons. These common arrays are found in a broad variety of hosts and environments, highlighting the high level of horizontal dissemination of these elements amongst bacterial populations and species. Clonal expansion also contributes to the current prevalence and inter-regional spread of integron-carrying bacterial species. Here, we review the dissemination pattern of common cassette arrays with a focus on the bacterial species, the geographical dispersal pattern and the environments in which they reside. Conserved arrays of gene cassettes are found in at least 74 countries and 72 species present in different environments. The factors governing the further spread and population dynamics of these cassette arrays remain to be determined.

Epidemiological characterizations of class 1 integrons from multidrug-resistant acinetobacter isolates in Daejeon, Korea.

BackgroundMultidrug-resistant (MDR) Acinetobacter spp. acquire antimicrobial agent-resistance genes via class 1 integrons. In this study, integrons were characterized to investigate the antimicrobial resistance mechanisms of MDR Acinetobacter isolates. In addition, the relationship between the integron type and integron-harboring bacterial species was analyzed by using epidemiological typing methods.MethodsFifty-six MDR Acinetobacter spp.-A. baumannii (N=30), A. bereziniae (N=4), A. nosocomialis (N=5), and A. pittii (N=17)-were isolated. The minimum inhibitory concentrations (MICs) were determined on the basis of the results of the Epsilometer test (Etest). PCR and DNA sequencing was performed to characterize the gene cassette arrays of class 1 integrons. Multilocus sequence typing (MLST) and repetitive extragenic palindromic sequence (REP)-PCR were performed for epidemiological typing.ResultsClass 1 integrons were detected in 50 (89.3%) of the 56 isolates, but no class 2 or 3 integron was found within the cohorts. The class 1 integrons were classified into 4 types: 2.3-kb type A (aacA4-catB8-aadA1), 3.0-kb type B (aacA4-blaIMP-1-blaOXA-2), 3.0-kb type C (blaVIM-2-aacA7-aadA1), and 1.8-kb type D (aac3-1-blaOXA-2-orfD). Type A was most prevalent and was detected only in A. baumannii isolates, except for one A. bereziniae isolate; however, type B was amplified in all Acinetobacter isolates except for A. baumannii isolates, regardless of clone and separation time of the bacteria.ConclusionsAlthough class 1 integron can be transferred horizontally between unrelated isolates belonging to different species, certain types of class 1 integrons tend to transfer horizontally and vertically among A. baumannii or non-baumannii Acinetobacter isolates.

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.

Response of activated sludge to the treatment of oxytetracycline production waste stream

To investigate how the microbial community in activated sludge responded to high antibiotic levels, a bench-scale aerobic wastewater treatment system was used to treat oxytetracycline (OTC) mother liquor (OTC-ML). Removal efficiency of chemical oxygen demand decreased from 64.9 to 51.0% when the OTC level increased from 191.6 to 620.5mg/L, respectively. According to the cloning results, Psychrobacter and Cryptophyta were the dominant bacterium and eukaryote in the inoculated sludge, respectively, both of which related to low temperature. After OTC exposure, Alphaproteobacteria and Betaproteobacteria became the dominant bacteria, with a small proportion of Firmicutes, Actinobacteria appeared, and fungi (mainly Saccharomycotina) became the dominant eukaryotes, indicating the possible functions of these microorganisms in the wastewater treatment of OTC-ML. The relative abundance of nine tetracycline resistance genes and four mobile elements (class 1 integron, class 2 integron, transposon Tn916/1545, and pattern 1 insertion sequence common region) significantly increased from undetectable to 2.1 × 10(-3) in the inoculated sludge to 1.7 × 10(-4)-9.8 × 10(-1) in sludge exposed to 620.5mg/L OTC by using real-time PCR. The variety of gene cassette arrays of class 1 integron in the sludge samples increased with increasing OTC exposure concentration.