Resistance Gene Cassettes Research Articles

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes.

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

Resistance-Gene Cassettes Associated With Salmonella enterica Genotypes.

The epidemiology of salmonellosis is complex because of the diversity and different serotypes of Salmonella enterica (S. enterica) that occur in different reservoirs and geographic incidences. To determine the genotype distribution and resistance-gene content of 2 classes of integron among S. enterica isolates. Thirty-six S. enterica species were isolated and tested for their serological distribution and the resistance-gene contents of 2 classes of integron, as well as for their genetic diversity, using the pulsed-field gel electrophoresis (PFGE) genotyping method. Serogroups E (36.1%) and D (30.5%) were dominant among the isolates. All of the isolates in serogroup D belonged to the serovar enteritidis. The aadA1 gene was found within all resistance-gene cassettes. We observed 4 common and 26 single pulsotypes among the isolates, which indicated a high degree of genetic diversity among the isolates. Using the PulseNet International standard protocol, it was found that these isolates were different from those reported previously in Iran. The presence of a few common and new pulsotypes among the isolates suggests the emergence and spread of new clones of S. enterica in Iran.

Protoplasting, regeneration and transformation of medicinal mushroom Ganoderma multipileum using succinate dehydrogenase mutation gene as a selection marker

Ganoderma multipileum is a medicinal mushroom possessing potent medicinal secondary metabolites. However, in order to improve metabolic activity via molecular breeding, optimized protoplasting, regeneration and genetic transformation systems are mandatory but remain unexplored in G. multipileum. Thus, we sought to fill this gap by testing different parameters for protoplasting, regeneration, and genetic transformation based on the carboxin resistance marker gene. According to our results, the best viable protoplasting conditions were pH 6.0, mycelia age 4 days, enzyme concentration 7.5 mg/mL, 0.6 M sucrose osmoticum and 3 h of enzyme digestion. In a transformation study, a carboxin resistance gene cassette was constructed using PCR mutagenesis and restriction digestion of the native succinate dehydrogenase subunit B gene in G. multipileum. A PCR-restriction fragment length polymorphism strategy was also applied by PCR point mutagenesis to create an AgeI restriction site. The transformation system using carboxin resistant cassette proved amenable. The Sdh gene structure showed an intron structure similar to that of other Basidiomycetes and Ascomycetes. Alignment and analysis of the Sdh gene sequences in numerous fungi revealed conservation at a carboxin resistant point mutation site that could be applied to a wide range of fungal species. Further Sdh gene phylogenetic analysis revealed congruence with species phylogeny but with low bootstrap supporting values.

Diversity of Class 1 Integron Gene Cassette Rearrangements Selected under Antibiotic Pressure.

Integrons are bacterial genetic elements able to capture and express genes contained within mobile gene cassettes. Gene cassettes are expressed via a Pc promoter and can be excised from or integrated into the integron by integrase IntI. Although the mechanisms of gene cassette integration and excision are well known, the kinetics and modes of gene cassette shuffling leading to new gene cassette arrays remain puzzling. It has been proposed that under antibiotic selective pressure, IntI-mediated rearrangements can generate integron variants in which a weakly expressed gene cassette moves closer to Pc, thus leading to higher-level resistance. To test this hypothesis, we used an integron with four gene cassettes, intI1-aac(6')-Ib-dfrA15-aadA1-catB9, and applied selective pressure with chloramphenicol, resistance to which is encoded by catB9. Experiments were performed with three different Pc variants corresponding to three IntI1 variants. All three integrases, even when not overexpressed, were able to bring catB9 closer to Pc via excision of the dfrA15 and aadA1 gene cassettes, allowing their host bacteria to adapt to antibiotic pressure and to grow at high chloramphenicol concentrations. Integrase IntI1(R32_H39), reported to have the highest recombination activity, was able, when overexpressed, to trigger multiple gene cassette rearrangements. Although we observed a wide variety of rearrangements with catB9 moving closer to Pc and leading to higher chloramphenicol resistance, "cut-and-paste" relocalization of catB9 to the first position was not detected. Our results suggest that gene cassette rearrangements via excision are probably less cost-effective than excision and integration of a distal gene cassette closer to Pc. Integrons are bacterial genetic elements able to capture and express gene cassettes. Gene cassettes are expressed via a Pc promoter; the closer they are to Pc, the more strongly they are expressed. Gene cassettes can be excised from or integrated into the integron by integrase IntI. The kinetics and modes of gene cassette shuffling, leading to new gene cassette arrays remain puzzling. We used an integron with 4 antibiotic resistance gene cassettes and applied selective pressure with the antibiotic for which resistance was encoded by cassette 4. All IntI variants were able to bring cassette 4 closer to Pc. Rearrangements occur via excision of the previous gene cassettes instead of cut-and-paste relocalization of the fourth gene cassette.

Molecular Detection of Genomic Islands Associated with Class 1 and 2 Integron in Haemophilus influenzae Isolated in Iran

Background:High levels of multidrug resistance are usually associated with mobile genetic elements that encode specific resistance genes. Integrons are important genetic elements involved in spreading antibiotic multi-resistance. In special cases, large exogenous segments in bacterial genomes form genomic islands, and one of the functions of these genomic islands is antibiotic resistance. Due to geographical heterogeneity in antibiotic resistance pattern, it is mandatory to determine resistance patterns that are region-specific rather than generalized.Objectives:The objective of this study was to detect class 1 and 2 integrons in clinical isolates of Haemophilus influenzae.Patients and Methods:Antibiogram tests were carried out for twenty clinical isolates collected from different patients admitted to the Milad hospital. The PCR reactions were performed using universal primers specified for Int1 and Int2 genes attributed to class 1 and 2 integrons. Also amplification of integrase genes related to genomic islands was investigated by designing specific primers.Results:Of the twenty isolates, all (100%) were resistant to clindamycin, chloramphenicol and tetracycline, 95% to amoxicillin, 50% to ceftriaxone, 45% to ciprofloxacin and 5% to azithromycin. Also, all isolates (100%) were sensitive to trimethoprim/sulfamethoxazole. Class 1 and 2 integrons were not detected in any of the isolates; however the integrase gene attributed to genomic islands was identified in twelve isolates.Conclusions:Antibiotic resistance gene cassettes may be carried on integron or other genetic elements. The purpose of this study was to detect integron or genomic islands involved in antibiotic resistance profile of the isolates of H. influenzae collected in this study.

Two novel CMY-2-type β-lactamases encountered in clinical Escherichia coli isolates.

BackgroundChromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal blaAmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene.FindingsBoth isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of blaCMY-46 and blaCMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-blaCMY-2-type-ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon.ConclusionsThis study describes two new blaCMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.

Detection of integron-associated gene cassettes and other antimicrobial resistance genes in enterotoxigenic Bacteroides fragilis

Twenty seven Enterotoxigenic Bacteroides fragilis (ETBF) strains isolated from children in Kolkata, India, were tested for their antimicrobial resistance, presence of integrons and resistance encoding genes. Almost all the strains (>90%) were resistant to two or more antimicrobials. About 59–92% of the strains were resistant to ampicillin, amoxicillin, streptomycin, tetracycline, ciprofloxacin and norfloxacin. Most of these antimicrobial agents have been used in the treatment of diarrhea and other infectious diseases. In addition, about half a number of strains (48–55%) were resistant to clindamycin, cefotaxime, ceftazidime, ampicillin/sulbactam and trimethoprim/sulfamethoxazole. Moxifloxacin and metronidazole resistance ranged from 30 to 40%. All strains however, were found to be susceptible to chloramphenicol and imipenem. Class 1 integrase (intI1) was detected in seven and class 2 integrase (intI2) in one of the twenty seven ETBF strains. Resistance gene cassettes carried by these integrons had different alleles of dfr or aad genes. Beside these integron-borne genes, other genes encoding different antimicrobial resistance were also detected. Resistance genes such as cep(A) and tet(Q) were detected in most of the ETBF strains. To the best of our knowledge, this work constituted the first extensive report from India on the detection of integrons and antimicrobial resistance genes in ETBF.

Identification and characterization of a serious multidrug resistant Stenotrophomonas maltophilia strain in China.

An S. maltophilia strain named WJ66 was isolated from a patient; WJ66 showed resistance to more antibiotics than the other S. maltophilia strains. This bacteraemia is resistant to sulphonamides, or fluoroquinolones, while the representative strain of S. maltophilia, K279a, is sensitive to both. To explore drug resistance determinants of this strain, the draft genome sequence of WJ66 was determined and compared to other S. maltophilia sequences. Genome sequencing and genome-wide evolutionary analysis revealed that WJ66 was highly homologous with the strain K279a, but strain WJ66 contained additional antibiotic resistance genes. Further analysis confirmed that strain WJ66 contained an amino acid substitution (Q83L) in fluoroquinolone target GyrA and carried a class 1 integron, with an aadA2 gene in the resistance gene cassette. Homology analysis from the pathogen-host interaction database showed that strain WJ66 lacks raxST and raxA, which is consistent with K279a. Comparative genomic analyses revealed that subtle nucleotide differences contribute to various significant phenotypes in close genetic relationship strains.

Construction of thyA deficient Lactococcus lactis using the Cre-loxP recombination system

Lactococcus lactis is widely used in the food industry as a food-grade non-pathogenic bacterium. In this study, we constructed the thymidylate synthase gene (thyA) deficient strain (NZ9000-3) derived from L. lactis NZ9000 using the Cre-loxP recombination system. Two loxP (lox66 and lox71) sites were integrated into the chromosome at target sites, and the thyA gene was replaced by a chloramphenicol resistance gene (cat) cassette. The recombination between the two loxP sites was very efficient when the Cre recombinase was expressed by a constructed pNZTS-Cre plasmid, and the thyA-null strain was isolated after removal of the cre gene. Then the relationship between the thyA-null strain and a thymidine requirement was analyzed. Results showed that the thyA gene was successfully knocked out, and the thyA-null strain could not grow well in minimal medium. Afterwards, pMG36e-thyA plasmid was constructed and transformed into NZ9000-3. The good screening efficiency revealed that as the selection marker thyA gene was as efficient as the erythromycin resistance gene, this suggested the thyA gene could be used as a food-grade selection marker for L. lactis to be used in food and industry applications.

Multidrug-Resistant IncA/C Plasmid inVibrio choleraefrom Haiti

Multidrug-Resistant IncA/C Plasmid in<i>Vibrio cholerae</i>from Haiti

Polymorphism of gene cassette promoter variants of class 1 integron harbored in S. Choleraesuis and Typhimurium isolated from Taiwan.

Integrons, mobile genetic units, capture and incorporate antibiotic resistance gene cassette by site-specific recombination. Class 1 integrons are widespread and associated with dispersion of antibiotic resistance among Gram-negative bacteria. The expression of gene cassette in Class 1 can vary, based on the Pc promoter but seldom from another promoter hiding downstream of Pc, called P2. To probe distribution and prevalence of gene cassette promoter variants, we analyzed 169 S. Choleraesuis and 191 S. Typhimurium isolates from humans and animals, finding 95.27% occurrence of integrin among S. Choleraesuis, 83.25% among S. Typhimurium. PCR-RFLP analysis identified four promoters (PcS+P2, PcWTGN-10+P2, PcH1+P2, and PcWTGN-10+P2-GGG) in said integron-positive isolates; major types in S. Choleraesuis and S. Typhimurium were PcS+P2 and PcWTGN-10+P2, respectively. Likewise, β-galactosidase assay rated promoter strength of variants by transcriptional fusion constructs to show extended -10 promoter (TGn/-10 promoter) in Pc and three-nucleotide insertion (GGG) between -35 and -10 region of P2 improving promoter strength of gene cassette.

Evaluation of Antibiotic Resistant Gene in Abattoir Environment

Pseudomonas aeruginosa is a ubiquitous gram-negative pathogen with susceptibility to cause opportunistic infections in humans. Among the total of 75 isolated presumptive Pseudomonas aeruginosa, by cultural and morphology characteristic, 55 were identified as Pseudomonas aeruginosa by the specie-specific primer employing PCR assay which was more sensitive for confirmation of the isolates. Fifty-five Pseudomonas aeruginosa isolates were screened using PCR for the presence of integrons and associated resistance gene cassette. Thirty-four isolates harbored class 1 integrons (61.8%), of which 27 isolates (79.4%) carried gene cassettes. PCR was performed targeting the presence of these genes:- blaOXA 11(32.4%); blaIMP 5(14.7%); blaAMPC 5(14.7%); blaTEM 4(11.8%); and TetC 2(5.8%). Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and environmental Pseudomonas aeruginosa stains. Multiresistance was observed in the isolates, revealing strong correlation between integron presence and multi-resistance. These results demonstrate that abattoir milieu is potential reservoirs of various antibiotics resistance genes, thus constituting a serious health risk to the communities dependent on the receiving water bodies.Keywords: Abattoir, Class 1 integron, Pseudomonas aeruginosa, Multi-resistance, Gene cassette

Detection and characterization of class 1 integron-associated gene cassettes from Pseudomonas aeruginosa isolates in southern Taiwan

In this study, detection and characterization of class 1 integron-associated gene cassettes from Pseudomonas aeruginosa isolates in southern Taiwan were investigated. The study focused on the association between integron-associated resistance gene cassettes and multidrug resistance in P. aeruginosa isolates. Using polymerase chain reaction amplification, DNA sequencing, and basic local alignment search tool analysis, a total of 22 different types of gene cassette arrays were detected in 162 class 1 integron-positive P. aeruginosa isolates. We first identified 11 different types of new gene cassette arrays within class 1 integron in P. aeruginosa isolates, including aac(6′)-II-catB2-aadA2, aac(6′)-II-aadA2, aac(6′)-II-catB2, aacA4-aadA15, aacC1-orfA-orfB-aadA1, cm1A-aadA1, catB3-blaOxA-10-aadA15, aacA4-catB8-aadA1, aadB-orfF1-aadA11, dfrB1, and dfrB4a-aacA4-aacA4-aadA1. Of these, aac(6′)-II-catB2-aadA2 was the most frequently found gene cassette. Twenty-one (21/162, 12.9%) strains carrying two different types of gene cassette arrays of catB3-blaOxA-10-aadA15 and aac(6′)-II-catB2-aadA2 were also simultaneously present in the P. aeruginosa isolates. A novel dfrB4a gene, different from the dfrB4 gene detected in the gene cassette array of dfrB4a-aacA4-aacA4-aadA, was characterized. A metallo-β-lactamaseo gene was also found to be carried on the gene cassette of blaVIM-3-orf2a-aacA4-aadB-aacA4 inserted in a class 1 integron obtained from meropenem-resistant P. aeruginosa isolates.

Phenotypic and genotypic characterization of antimicrobial resistance in Escherichia coli isolates

Background:Themajor influenceson the increase and spreadof antimicrobial resistant bacteria are the use of antimicrobial agents in human medicine and their use in livestock for therapy and growth promotion. To generate baseline data to use in future risk assessment of antimicrobial resistance, a number of surveillance program on the local, continental and global scale have been initiated. The prevalence of resistance in commensal E. coli is a good indicator for the selective pressure by antibiotics use and resistance problems to be expected in pathogenic bacteria. The aim of this studywas to determine and compare the occurrence of antimicrobial resistance phenotypes and genotypes among E. coli strains recovered from animal sources. Methods & Materials: Fifty-eight (58) E. coli strains were isolated from animal sources (Cattle, pig and chicken). MIC was determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Strains were screened and mapped by PCR methods for antimicrobial resistant genes, integrons and gene cassettes. Results: The resistance profile of E. coli for ampicillin (100%), cefotaxime (100%), ceftazidime (83%), ciprofloxacin (43%) (C), tetracycline (100%) (T), gentamicin (90%) (G), kanamycin (77%) (K), streptomycin (100%) (S), chloramphenicol (97%) (Ch) sulphamethoxazole (100%) (Su) and trimethoprime (97%) (T). Eight-Five per cent (85%) of E. coli harbored class 1 integron and variable gene cassettes were revealed. A prevalence of gene cassettes that present resistance to streptomycin and trimethoprime was observed. The resistance genes determinant present in gentamicin resistance were aac(3’)-IIc, aac(6’)-Ib and ant(2”)-1a. The occurrence of aph(3)-Ia in kanamycin resistant strainswas 68%. The prevalence of sul1 (80%) and sul2 (100%) was observed. The prevalence of tet(A) is higher than tet(B) in tetracycline-resistant strains. The gene determinants for chloramphenicol resistance (cat1 and catB) were detected. Differences in the rates and profile of resistance were observed, possibly reflecting differences in antibiotic use regimens among these strains. Conclusion: This study shows that multi-drug resistant E. coli isolates are prevalent in animal sources and a considerable proportion of E. coli strains are resistant to a variety of antimicrobial agents.Genotypic characterization revealedhighnumberofdiverse resistance determinants.

Virulence genes, antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain

The aim of this study was to determine the presence of virulence genes and antibiotic resistance profiles in 164 Escherichia coli strains isolated from birds (feral pigeons, hybrid ducks, house sparrows and spotless starlings) inhabiting urban and rural environments. A total of eight atypical enteropathogenic E. coli strains were identified: one in a house sparrow, four in feral pigeons and three in spotless starlings. Antibiotic resistance was present in 32.9% (54) of E. coli strains. The dominant type of resistance was to tetracycline (21.3%), ampicillin (19.5%) and sulfamethoxazole (18.9%). Five isolates had class 1 integrons containing gene cassettes encoding for dihydrofolate reductase A (dfrA) and aminoglycoside adenyltransferase A (aadA), one in a feral pigeon and four in spotless starlings. To our knowledge, the present study constitutes the first detection of virulence genes from E. coli in spotless starlings and house sparrows, and is also the first identification worldwide of integrons containing antibiotic resistance gene cassettes in E. coli strains from spotless starlings and pigeons.

The consequences of a sudden demographic change on the seroprevalence pattern, virulence genes, identification and characterisation of integron-mediated antibiotic resistance in the Salmonella enterica isolated from clinically diarrhoeic humans in Egypt

The present study was undertaken to identify and characterise integrons and integrated resistance gene cassettes among eight multidrug-resistant (MDR) Salmonella serovars isolated from humans in Egypt. Virulotyping by polymerase chain reaction (PCR) was used for the detection of the presence of virulence genes. Integron PCR was used to detect the presence of class 1 in the MDR strains. The associated individual resistance gene cassettes were identified using specific PCRs. The isolated serovars were Salmonella Grampian (C1; 2/5), Larose (C1; 1/5), Hato (B; 1/5) and Texas (B; 1/5). Among the Salmonella serovars, five Salmonella isolates showed the highest resistance to amoxicillin, ampicillin, chloramphenicol, lincomycin, gentamicin, nalidixic acid, streptomycin and trimethoprim (100%), followed by neomycin, norfloxacin and tetracycline (80%), while the lowest resistance was recorded to colistin sulphate and ciprofloxacin in percentages of 20 and 40%, respectively. The invA, avrA, ssaQ, mgtC, siiD and sopB genes were detected in all isolates (100%), while the spvC and gipA genes were totally (100%) absent from all isolates. The remaining three virulence genes were diversely distributed as follows: the bcfC gene was detected in all isolates except Salmonella Hato (80%); the sodC1 gene was detected only in Salmonella Grampian and Salmonella Texas (60%); and the sopE1 gene was detected only in Salmonella Grampian, Hato and Texas (60%). Class 1 integrons were detected in 90% of the MDR isolates, comprising serovars Muenster, Florian, Noya, Grampian, Larose, Hato and Texas. Of the class 1 integron-positive isolates, 45% harboured Salmonella genomic island 1 (SGI1) either right junction or right and left junction having an A-C-S-T phenotype. Of the class 1 integron-positive isolates, 44% harboured integron gene cassette aadA2, while 11% harboured the floR gene present in multidrug resistance flanked by two integrons of SGI1. The results of the present study indicate that class 1 integrons carrying gene cassettes conferring resistance mainly to aminoglycosides are widespread among the MDR Salmonella serovars isolated from humans in Egypt, indicating the important role of these genetic elements in the dissemination of multidrug resistance.

Carriage of Class 1 and 2 Integrons in Acinetobacter baumannii and Pseudomonas aeruginosa Isolated from Clinical Specimens and a Novel Gene Cassette Array: blaOXA-11-cmlA7

The dissemination of antibiotic resistance genes between bacteria leads to serious problems in the treatment of infectious diseases. It has been shown that resistance genes can also be carried by the integrons. There are limited studies regarding the carriage of class 1 and 2 integrons in Acinetobacter baumannii and Pseudomonas aeruginosa clinical strains in Turkey. The aims of this study were to investigate the carriage rates of class 1 and class 2 integrons in A.baumannii and P.aeruginosa strains isolated from clinical samples in Abant Izzet Baysal University Hospital, and to characterize the antibiotic resistance gene cassettes in these integrons by sequence analyses. A total of 137 strains (77 A.baumannii and 60 P.aeruginosa) isolated from various clinical specimens (56% were sputum, 19% wound, 11% urine, 11% blood, 3% catheter), between March 2010-December 2012, were included in the study. The identification and antibiotic susceptibility tests of the isolates were performed by Vitek 2 Compact (bioMérieux, France) and BD Phoenix 100 (Becton Dickinson, USA) systems. The presence of integrons were screened by PCR method using specific primer pairs targeting class 1 (intI1) and 2 (intI2) integrase regions. All the samples that revealed integron amplification were subjected to DNA sequence analysis, both in the forms of cloned products and PCR amplicons. In the study, the highest susceptibility rates were found against colistin (96%) and tigecycline (78%) in A.baumannii, and against piperacillin/tazobactam (97%) and piperacillin (93%) in P.aeruginosa isolates. The highest resistance rate was determined for piperacillin/tazobactam (95%) in A.baumannii strains. The presence of intI1 gene was detected in 33% (26/77) of A.baumannii and 10% (6/60) of P.aeruginosa isolates. When variable regions in intI1 positive strains were amplified by PCR, eight (8/77, 10%) A.baumannii and three (3/60, 5%) P.aeruginosa strains were found to harbor antibiotic resistance gene cassettes. IntI2 gene was not detected in any of the isolates. Resistance to piperacillin/tazobactam, ceftazidime, cefepime, ceftriaxone and ampicillin/sulbactam was detected as the common resistance pattern in all integron-positive A.baumannii strains, whereas resistance to ceftazidime, gentamicin and ciprofloxacin was the common pattern in all integron-positive P.aeruginosa strains. DNA sequence analysis of variable regions of integrons indicated that two separate gene cassette arrays (aacC1-aadA1 and aac(3)-1) were carried by A.baumannii strains, and two types of gene cassette arrays (blaOXA-30-aadA1 and blaOXA-11-cmlA7) were carried by P.aeruginosa strains. To our best knowledge, this is the first report of the gene sequence of blaOXA-11-cmlA7 defined in an integron gene cassette of P.aeruginosa.

Molecular Detection of Antimicrobial Resistance Gene Cassettes Associated with Class 2 Integron in Salmonella Serovars Isolated in Iran

Aim: Salmonella is an important food-borne pathogen in humans and a broad range of animals. Antimicrobial resistance in Salmonella spp. is a serious health problem in human and veterinary medicine worldwide. The aim of this study was to detect integrons, the natural recombination systems that can be transferred in companion with mobile genetic Research Article British Microbiology Research Journal, 4(1): 132-141, 2014 133 elements and play a major role in spreading antibiotic resistance genes in clinical isolates. Place and Duration of Study: Salmonella clinical isolates were provided by a number of institutes and hospitals over the country through the years 2008-2009. Methodology: Antimicrobial susceptibility testing and serotyping were carried out for eighty four epidemiologically unrelated clinical isolates of Salmonella serovars collected from different provinces of Iran through the years 2008-2009. PCR assays were carried out to detect intI2 gene (integrase I attributed to class 2 integron) and internal variable regions (IVRs) of class 2 integron. These sequences were deposited in EMBL/GenBank database (www.ncbi.nlm.nih.gov). Results: Eleven isolates (13.1%) which were resistant to at least 4 groups of antimicrobial agents were considered as MDR (multidrug resistant) Salmonella serovars. PCR assays detected intI2 gene (integrase I attributed to class 2 integron) and internal variable regions (IVRs) of class 2 integron in Fourteen (16.7%) and eleven (78.6%) of Salmonella clinical isolates respectively. Analysis of the sequence data revealed 3 gene cassette arrays deposited in Genbank databases including the dhfrA1 (0.75 kb), dfrA14lsp (1 kb), dhfrA1sat2-aadA1 (3 kb) with three IVR distribution patterns. An artifact PCR product of 2 kb was reported in this study to be amplified together with IVRs of class 2 integrons which was associated with the fhuEptsG genes. Conclusions: Presence of MDR Salmonella serovars demonstrates that antimicrobial selection pressure is widespread in our clinical settings. Detection of class 2 integron carrying gene cassettes which confer resistance to different classes of antibiotics such as aminoglycosides, and trimethoprim confirms that integron-mediated antimicrobial gene cassettes are prevalent in Salmonella serovars isolated in Iran.

Molecular characterization of enterohemorrhagic Escherichia coli isolates from cattle.

ABSTRACTA total of 21 (4.3%) enterohemorrhagic E. coli strains were isolated by biochemical tests and identification of the eae+stx1+stx2+ genotype from 490 stool samples obtained from calves with diarrhea during 1-year period from a major farm in Tehran, Iran. All of the strains showed resistance to ampicillin, ciprofloxacin, trimethoprim, streptomycin, chloramphenicol and tetracycline, while 19% showed resistance to gentamicin. Out of 21 EHEC strains, 11 (53%) harbored class 1 integron. Two different amplification products, which were approximately 750 and 1,700 bp in size, were obtained from amplified variable regions (in-F/in-R primers) in 3 (14.3%) and 4 (19%) of the EHEC isolates, which corresponded to dfrA7(dihydrofolate reductase type I) and dfrA1/aadA1(dihydrofolate reductase/aminoglycoside adenyltransferase) resistance gene cassettes, respectively, and this was confirmed by sequencing. Genotyping analysis revealed a total of 16 pulsotypes that corresponded to 16 isolates with the similarity indices of 62% and 30% for the most and least similar isolates, respectively, 9 of which harbored class 1 integron. Analysis of pulsotypes showed an extensive diversity among the isolates harboring integron, which is indicative of a lack of any significant genetic relatedness among the isolates. No obvious relation could be deduced between integron content and special pulsotypes. The little data available on the genotyping patterns of EHEC isolates from cattle and their resistance gene contents emphasize the need to establish genotyping databases in order to monitor and source track the source of emergence and spread of new resistant and integron-carrying genotypes.