Occurrence of antibiotic resistance gene cassettes aac(6')-Ib, dfrA5, dfrA12, and ereA2 in class I integrons in non-O1, non-O139 Vibrio cholerae strains in India.
ABSTRACT: Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6')-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6')-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.
Project description:Twenty Acinetobacter baumannii strains resistant to various antibiotics were analyzed for integron content and sequences of the amplification products. Sixteen clinical isolates had a class 1 integron, 2 contained an additional class 1 or class 2 integron, but no class 3 integron was detected. Thirteen strains had integrons with a single cassette: aac(3)-Ia (9 strains), ant(2")-Ia (2 strains), or aac(6')-Ib (2 strains); 1 had aac(6')-Ib and oxa20 cassettes and an unknown gene; and 1 had an integron containing ant(2")-Ia and an oxa3 cassette truncated by IS6100. The remaining strains harbored class 1 integrons with gene cassettes previously found in Enterobacteriaceae. One integron had a hybrid structure composed of intI2 and the 3' conserved segment of class 1 integrons. These data indicate that integrons play a major role in multidrug resistance in Acinetobacter.
Project description:<h4>Purpose</h4> Gram-negative rods of the genus Serratia play an increasing role as etiological agents of healthcare-associated infections (HAI) in humans. These bacteria are characterized by natural and acquired resistance to several groups of antibacterial agents. The aim of the study was to characterize class 1, 2 and 3 integrons in the clinical isolates of Serratia spp. in Poland. <h4>Methods</h4> The study comprised 112 clinical strains of Serratia, isolated from patients hospitalized in Poland in 2010–2012. Identification of strains was confirmed using MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) system. Detection of class 1, 2 and 3 integrase DNA sequence was performed by multiplex-PCR. Amplicons obtained in the PCR reactions were purified and then sequenced bidirectionally. <h4>Results</h4> Among the analyzed strains, Serratia marcescens was a predominant species (103/112, 92.0%). All three classes of integrase DNA sequence were detected in the analyzed strains of Serratia spp. DNA sequence of class 3 integron, besides integrase gene, revealed three gene cassettes (dfrB3, blaGES-7,blaOXA/aac(6ʹ)-Ib-cr). BLAST analysis of DNA sequence revealed that class 3 integron was carried on 9448 bp plasmid which was named pPCMI3 – whole sequence of its DNA was submitted to GenBank NCBI (National Center for Biotechnology Information) – NCBI MH569711. <h4>Conclusion</h4> In this study, we identified a new plasmid pPCMI3 harboring class 3 integron. This is the first report of a gene oxa/aac(6ʹ)-Ib-cr coding for a novel fusion protein, which consists of OXA β-lactamase and acetyltransferase aac(6ʹ)-Ib-cr. In the analyzed strains, class 1 and 2 integrons were also detected. Among the strains with class 1 integron, nine contained cassette array 5ʹCS-aadA2-ORF-dfrA12-3ʹCS, and two – cassette array 5ʹCS-aacC1-ORF-ORF-aadA1-3ʹCS, which were not previously reported in Serratia spp.
Project description:AIMS:This study was carried to reveal the genetic mechanisms of trimethoprim/sulfamethoxazole (SXT) resistance. METHODS:Among 300 clinical Stenotrophomonas maltophilia isolates from China, resistance determinants such as sul and dfrA genes, integrons and transposase were examined using PCR, DNA sequencing and thermal asymmetric interlaced PCR (TAIL-PCR). Data were analyzed using SPSS 20.0. RESULTS:Of the 300 isolates, 116 (38.7%) were resistant to SXT. An alarming trend of increased resistance to SXT were found over the 10-year period. The positive rates of sul and class 1 integrase (intI1) increased gradually with the development of SXT resistance over the 10-year period. Multiple logistic regression analyses indicated that the genes of qacE?1-sul1 (81% vs 46.2%, p = 0.000), sul2 (50.9% vs 9.8%, p = 0.000), intI1 (83.6% vs 65.8%, p = 0.000), dfrA12 (25% vs 3.3%, p = 0.000), dfrA17 (15.5% vs 3.8%, p = 0.000) and dfrA27 (4.3% vs 1.6%, p = 0.01) were more prevalent in SXT-resistant isolates than SXT-susceptible isolates except dfrA1(p = 0.83) and dfrA5(p = 0.18). Sequencing data revealed 12 types of resistance gene cassettes (aar-3-dfrA27, dfrA12-aadA2, dfrA17-aadA5, cmlA1, aacA4, aadA5, arr-3-aacA4, aadA1, aadB-aadA4, aacA4-catB8-aadA1, aadB-aac(6')-II-blaCARB-8 and aac(6')-II-blaCARB-8) located in the class 1 integron in 163 isolates (87% SXT-resistant vs 33.7% SXT-susceptible isolates, p = 0.000). A novel finding was the aar-3-dfrA27 (KC748137) gene cassette. The gene of sul2 linked to transposase in 50 SXT- resistant and 7 SXT- susceptible isolates was detected by TAIL-PCR. CONCLUSIONS:The findings demonstrated a higher prevalence of sul, dfrA, intI1 and resistance gene cassettes in class 1 integron in SXT-resistant clinical S. maltophilia isolates in China. The sul1 and dfrA genes located in integrons and the sul2 linked to transposase may imply wide and rapid dissemination of resistance gene in bacteria.
Project description:BACKGROUND:Proteus mirabilis is an opportunistic pathogen, commonly associated with complicated urinary tract infections (UTIs). UTIs caused by multidrug-resistant Proteus mirabilis have increased worldwide. Multidrug-resistance of Gram-negative enteric bacteria is usually associated with class 1 integrons. PURPOSES:To investigate the prevalence and characterize gene cassettes of class 1 integrons in multidrug-resistant P. mirabilis Methods: From 2006 to 2008, 314 P. mirabilis isolates from urine were collected from a regional teaching hospital. Antimicrobial resistance of the isolates was determined by disk diffusion methods. The phenotypic confirmatory test of extended-spectrum ?-lactamase (ESBL) production was performed as described in the Clinical and Laboratory Standards Institute (CLSI) guideline. The genetic organization of the class 1 integron cassettes was investigated by PCR, cloning, and sequencing of the regions surrounding these genes. RESULTS:Seventy-nine (25%, 79/314) P. mirabilis isolates were ESBL-producing and most ESBL-producing P. mirabilis were positive for blaCTX-M. Class 1 integrons were presented in 76 isolates (24.2%, 76/314), and were more frequently found in ESBL-positive (55/79, 70%) than ESBL-negative (21/235, 8.9%) P. mirabilis isolates. The most prevalence of the cassettes encoded resistance genes were aminoglycoside (aac(6')-Ib, aacA7, aadAl, aadA2, and aadAla), trimethoprim (dfrAl and dfrA12) and chloramphenicol (catB3 and cmlA6). The most prevalent cassette of dfr12-orfF-aadA2 was found in 49 isolates. The cassette array aadB-catB3-oxa10-aadA1 was first found in P. mirabilis. The enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting patterns were detected in these 76 integron positive P. mirabilis isolates and belonged to 8 profiles. CONCLUSION:This study investigated the prevalence and characterized gene cassettes of class 1 integrons in MDR P. mirabilis isolates from urine samples. The frequency of gene cassettes in P. mirabilis were partially by clonal spread of the carriers and the results could provide information for effective antimicrobial therapy and infection control.
Project description:In the last few years, numerous cases of multidrug-resistant Achromobacter xylosoxidans infections have been documented in immunocompromised and cystic fibrosis patients. To gain insights into the molecular mechanisms and mobile elements related to multidrug resistance in this bacterium, we studied 24 non-epidemiological A. xylosoxidans clinical isolates from Argentina. Specific primers for plasmids, transposons, insertion sequences, bla(ampC), intI1, and intI2 genes were used in PCR reactions. The obtained results showed the presence of wide host range IncP plasmids in ten isolates and a high dispersion of class 1 integrons (n = 10) and class 2 integrons (n = 3). Four arrays in the variable region (vr) of class 1 integrons were identified carrying different gene cassettes as the aminoglycoside resistance aac(6')-Ib and aadA1, the trimethoprim resistance dfrA1 and dfrA16, and the ?-lactamase bla(OXA-2). In only one of the class 2 integrons, a vr was amplified that includes sat2-aadA1. The bla(ampC) gene was found in all isolates, confirming its ubiquitous nature. Our results show that A. xylosoxidans clinical isolates contain a rich variety of genetic elements commonly associated with resistance genes and their dissemination. This supports the hypothesis that A. xylosoxidans is becoming a reservoir of horizontal genetic transfer elements commonly involved in spreading antibiotic resistance.
Project description:In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated in Thailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB, aadA2, blaP1, dfrA1, and dfrA15 gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; beta-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2 resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among different V. cholerae O serotypes of mainly clinical origin in Thailand.
Project description:<h4>Background</h4>Antimicrobial resistance among enteric bacteria in Africa is increasingly mediated by integrons on horizontally acquired genetic elements. There have been recent reports of such elements in invasive pathogens across Africa, but very little is known about the faecal reservoir of integron-borne genes.<h4>Methods and findings</h4>We screened 1098 faecal Escherichia coli isolates from 134 mother-child pairs for integron cassettes by PCR using primers that anneal to the 5' and 3' conserved ends of the cassette regions and for plasmid replicons. Genetic relatedness of isolates was determined by flagellin and multi-locus sequence typing. Integron cassettes were amplified in 410 (37.5%) isolates and were significantly associated with resistance to trimethoprim and multiple resistance. Ten cassette combinations were found in class 1 and two in class 2 integrons. The most common class 1 cassette configurations were single aadA1 (23.4%), dfrA7 (18.3%) and dfrA5 (14.4%). Class 2 cassette configurations were all either dfrA1-satI-aadA1 (n = 31, 7.6%) or dfrA1-satI (n = 13, 3.2%). A dfr cassette was detected in 294 (31.1%) of trimethoprim resistant strains and an aadA cassette in 242 (23%) of streptomycin resistant strains. Strains bearing integrons carried a wide range of plasmid replicons of which FIB/Y (n = 169; 41.2%) was the most frequently detected. Nine isolates from five different individuals carried the dfrA17-aadA5-bearing ST69 clonal group A (CGA). The same integron cassette combination was identified from multiple distinct isolates within the same host and between four mother-child pairs.<h4>Conclusions</h4>Integrons are important determinants of resistance in faecal E. coli. Plasmids in integron-containing strains may contribute to dispersing resistance genes. There is a need for improved surveillance for resistance and its mechanisms of dissemination and persistence and mobility of resistance genes in the community and clinical settings.
Project description:Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n?=?202), animals (n?=?304) and the environment (n?=?6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3'-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer.
Project description:Aeromonas spp. is one of the major pathogens of freshwater animals. There has been little research on the genetics of antimicrobial resistance associated with it in Iranian aquaculture. To remedy this lack in research, 74 multi-drug-resistant Aeromonas spp. were isolated from farmed diseased carp, trout, sturgeon, ornamental fish, crayfish, and corresponding water samples and examined for genomic integron sequences. Class 1 integrons, containing seven types of integron cassette arrays (dfrA1-aadA1, dfrA1-orfC, dfrA12-aadA2, dfrA12-orfF-aadA2, dfrA15, dfrB4-catB3-aadA1, aac(6')-Ib-cr-arr3-dfrA27) were found in 15% of the resistant isolates; no class 2 integrons were detected in any of the resistant isolates. As some tested isolates were resistant to more than two groups of antibiotics, our results demonstrated that freshwater animals in Iran could be a source of multiply drug-resistant Aeromonas spp. This finding suggests that the origin of the antimicrobial resistance of these animals be placed under increased surveillance in the future and that the use of antimicrobials be limited in aquaculture.
Project description:<h4>Background</h4>In this study a large random collection (n=2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007-2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products.<h4>Methodology/principal findings</h4>Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (?(2)) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6'-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae.<h4>Conclusions</h4>Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.