Transposon Tn5281 is the main distributor of the aminoglycoside modifying enzyme gene among isolates of Enterococcus faecalis in Tehran hospitals

Infections with high levels of gentamicin-resistant (HLGR) isolates of Enterococcus faecalis are common in Tehran hospitals. Genes encoding such resistance are transmissible by conjugation at high frequency. The purpose of this study was to determine the existence of Tn5281 and its flanking aminoglycoside modifying enzyme gene aac(6')-aph(2") among 102 HLGR isolates of E. faecalis cultured from patients at three hospitals in Tehran, Iran. These isolates were detected by disks containing 120 microg of gentamicin and made 65% of all E. faecalis during the study period. DNA was extracted from HLGR isolates and subjected to PCR assays targeting aac(6')-aph(2") and conjugative transposon Tn5281. The amplified aac(6')-aph(2") gene was labeled with digoxigenin and probed with Tn5281 amplicons in dot blot hybridization assays. The aac(6')-aph(2") gene was detected in 91%-92% (n = 93) of the HLGR isolates. All isolates containing aac(6')-aph(2") were positive in long-PCR targeting Tn5281 and the probe hybridized with Tn5281 amplicons. The number of HLGR isolates of E. faecalis has increased considerably in Tehran hospitals. Tn5281 is the main cause of transmission of aac(6')-aph(2") to different isolates of E. faecalis in the hospitals studied.     

The role of bla(OXA-like carbapenemase) and their insertion sequences (ISS) in the induction of resistance against carbapenem antibiotics among Acinetobacter baumannii isolates in Tehran hospitals

This study aimed to evaluate the occurrence and dissemination of bla(OXA-like) carbapenemase genes and their insertion sequences among Acinetobacter baumannii isolates, taken from different hospitals in Tehran city and also their roles in the induction of resistance to carbapenem drugs. A total number of 100 non duplicate Acinetobacter baumannii with different origins, were isolated from patients with proved nosocomial infections at eight university hospital in Tehran city. Antimicrobial susceptibility of these strains was done by E-test against 7 antimicrobial agents according to CLSI guideline. PCR of bla(OXA-51-like), bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), IS(ABA-1), IS(1133) was carried out by specialized primers and then these strains were typed by REP-fingerprinting. Colistin, imipenem and meropenem were the most sensitive antibiotics against Acinetobacter baumannii isolates with 96%, 51% and 51% sensitivity respectively. All the isolates had a bla(OXA-51-like) intrinsic to these species. The rates of bla(OXA-23), 23 and 58-like were 38%, 32% and 1% respectively. Coexistence of bla(OXA-51/23/24-like) was observed among 16% of these isolates. All bla(OXA-23-like) carbapenemase genes had only one IS(ABA1). REP fingerprinting showed 5 genotypes among carbapenem resistant isolates, 16 of them being genotype A. This study emphasized on the major role of bla(OXA-like) carbapenemase, particularly bla(OXA-23-like) carbapenemase and their IS(ABA1), in the dissemination of carbapenem resistant Acinetobacter baumannii. This study confirmed a presumptive role of IS element neighboring the carbapenemase gene in the elevation of resistance to carbapenem drug among Acinetobacter baumannii isolates for the first time in Iran.     

Characterization of antibiotic resistance genes linked to class 1 and 2 integrons in strains of Salmonella spp. isolated from swine

The aim of this study was to characterize the antibiotic resistance profiles, the integron-associated resistance determinants, and the potential ability of transferring these determinants by conjugation in Salmonella enterica isolated from swine. Fifty-four strains of Salmonella spp. were isolated from healthy swine. The percentages of resistance, determined by the plate dilution method were as follows: oxytetracycline (41%), streptomycin (39%), sulphamethoxazol+trimethoprim (19%), enrofloxacin-ciprofloxacin (13%), and amoxicillin (0%). The most important resistance serovars were Salmonella Branderburg, Salmonella Derby, Salmonella Typhimurium, and Salmonella Heidelberg. The oxytetracycline-resistant strains amplified the genes tetA (36%), tetB (64%); and the strains resistant to streptomycin and trimethoprim amplified the genes aadA1 (100%) and dfrA1 (100%), respectively. None of the fluoroquinolone-resistant strains amplified the gene qnr. Ten strains amplified the class 1 integron harboring the cassette aadA1. Six strains amplified the class 2 integron harboring the cassettes dfrA1, sat1, and aadA1. The conjugation assays showed that 2 strains transferred the tetA and aadA1 genes and the class 1 integron to a recipient strain. Taken together, the results obtained in this study show a high percentage of resistance in and the presence of integrons in strains of S. enterica isolated from swine. This information should support the implementation of regulations for the prudent use of antimicrobial agents in food-producing animals.     

Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China

Aims: To characterize the molecular diversity of class 1 integrons and antibiotic resistance (AR) genes of Enterobacteriaceae strains recovered from aquatic habitats in Jinan, Shandong Province, China. Methods and Results: Six hundred and thirty‐eight antimicrobial‐resistant Enterobacteriaceae isolated from wastewater were examined for class 1 integron. Of these, 293 were positive for the class 1 integrase gene intI1; among these, 34 gene cassettes and 29 AR genes were detected. Twenty‐nine distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP). Seven strains harboring novel gene cassette arrays were subjected to further study, in which antimicrobial susceptibility profiles were determined, and the presence of other AR genes outside of the integrons was assayed. Several of the resistance determinants were found to be transferable by conjugation or transformation. Conclusions: This study established the assessment of class 1 integron and antimicrobial resistance gene patterns among environmental Enterobacteriaceae. Also, a restriction enzyme EcoRII was employed to develop a rapid and simple method for characterizing gene cassette arrays by RFLP analysis, which facilitated further study of novel gene cassette arrays. Significance and Impact of Study: These data not only illustrated the diversity of class 1 integron gene cassettes but also provided direct evidence that integrons mobilized gene cassettes, generating new linkages of resistance genes, and they could be integrated in gene transfer units such as conjugative plasmids to contribute to the dissemination of AR genes by horizontal gene transfer (HGT) in aquatic environments.     

Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals

Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.     

Class 1 and class 2 integrons in multidrug-resistant gram-negative bacteria isolated from the Salmon River, British Columbia

Using an enrichment protocol, we isolated 16 gram-negative, multidrug-resistant strains of known or opportunistic bacterial pathogens from the Salmon River in south-central British Columbia from 2005 to 2009, and investigated the genetic basis of their resistance to a variety of antibiotics. Of the 16 strains, 13 carried class 1 integrons and three carried class 2 integrons. Genes found in cassettes associated with the integrons included those for dihydrofolate reductases (dfrA1, dfrA12, dfrA17, and dfrB7), aminoglycoside adenyltransferases (aadA1, aadA2, aadA5, and aadB), streptothricin acetyltransferase (sat), and hypothetical proteins (orfF and orfC). A new gene cassette of unknown function, orf1, was discovered between dfrA1 and aadA5 in Escherichia sp. Other genes for resistance to tetracycline, chloramphenicol, streptomycin, and kanamycin (tetA, tetB, tetD; catA; strA-strB; and aphA1-Iab, respectively) were outside the integrons. Several of these resistance determinants were transferable by conjugation. The detection of organisms and resistance determinants normally associated with clinical settings attest to their widespread dispersal and suggest that regular monitoring of their presence in aquatic habitats should become a part of the overall effort to understand the epidemiology of antibiotic resistance genes in bacteria.     

Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey

We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, bla _oxA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens. This study was presented in part at the 2^nd World Conference on Magic Bullets, held October 3–5, 2008 in Nurnberg, Germany.     

Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii strains isolated from patients of intensive care units (ICU)

The aim of this study was to investigate the prevalence of ESBL and MBL encoding genes among A. baumannii isolates. In this cross sectional study, 100 A. baumannii strains were isolated from ICU wards of 3 educational hospitals of Hamadan City, Iran in 2011. Phenotypic identification of the production of ESBLs and MBLs has been carried out by using E-test and DDST methods, respectively. PCR technique was used for amplification of the ESBL and MBL encoding genes, namely: CTX-M, SHV, TEM, OXA-51, VIM-Family, IMP-Family, SPM-1, SIM-1, and GIM-1. Eighty seven (87%), 95 (95%), 98 (98%) and 95 (95%) out of 100 A. baumannii isolates were resistant to imipenem, meropenem, ceftazidime and cefotaxime, respectively. Also, 99% and 7% of the isolates were MBLs and ESBLs produced phenotypically. Thirty (30%), 20 (20%) and 58 (58%) out of 100 A. baumannii isolates have been confirmed to harbor the bla_VIM-family, TEM and SHV genes, respectively. Our results show no significant relationship between the detected gens with production of MBLs and ESBLs in spite of high prevalence of MBL encoding and drug resistant A. baumannii. Probably some other genes rather than what we studied are involved in phenotypic production of MBLs and ESBLs and subsequent drug resistance in Hamadan area, Iran.     

Interaction of dihydrofolate reductase and aminoglycoside adenyltransferase enzyme from Klebsiella pneumoniae multidrug resistant strain DF12SA with clindamycin: a molecular modelling and docking study

Klebsiella pneumoniae strain DF12SA (HQ114261) was isolated from diabetic foot wounds. The strain showed resistance against ampicillin, kanamycin, gentamicin, streptomycin, spectinomycin, trimethoprim, tetracycline, meropenem, amikacin, piperacillin/tazobactam, augmentin, co-trimoxazole, carbapenems, penicillins and cefoperazone, and was sensitive to clindamycin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing two genes, a dihydrofolate reductase (DHFR) (PF00186), which confers resistance to trimethoprim; and aminoglycoside adenyltransferase (AadA) (PF01909), which confers resistance to streptomycin and spectinomycin. A class 1 integron in K. pneumoniae containing these two genes was present in eight (18.18 %) out of 44 different diabetic foot ulcer (DFU) patients. Hence, there is a need to develop therapeutics that inhibit growth of multidrug resistant K. pneumoniae in DFU patients and still achieve amputation control. Am attempt was made to create a 3D model and find a suitable inhibitor using an in silico study. Rational drug design/testing requires crystal structures for DHFR and AadA. However, the structures of DHFR and AadA from K. pneumoniae are not available. Modelling was performed using Swiss Model Server and Discovery Studio 3.1. The PDBSum server was used to check stereo chemical properties using Ramachandran plot analysis of modeled structures. Clindamycin was found to be suitable inhibitor of DHFR and AadA. A DockingServer based on Autodock & Mopac was used for docking calculations. The amino acid residues Ser³², Ile⁴⁶, Glu⁵³, Gln⁵⁴, Phe⁵⁷, Thr⁷², Met⁷⁶, Val⁷⁸, Leu⁷⁹, Ser¹²², Tyr¹²⁸, Ile¹⁵¹ in case of DHFR and Phe³⁴, Asp⁶⁰, Arg⁶³, Gln⁶⁴, Leu⁶⁸, Glu⁸⁷, Thr⁸⁹, Val⁹⁰ for AadA were found to be responsible for positioning clindamycin into the active site. The study identifies amino acid residues crucial to ‘DHFR and AadA -drug’ and ‘DHFR and AadA -inhibitor’ interactions that might be useful in the ongoing search for a versatile DHFR and AadA -inhibitor.     

Emergence of dhfrXVb and blaCARB-4 gene cassettes in class 1 integrons from clinical Pseudomonas aeruginosa isolated in Amazon region

Integrons play a role in horizontal acquisition and expression of genes, as well as gene reservoir, contributing for the resistance phenotype, particularly relevant to bacteria of clinical importance. We aimed to determine the composition and the organization of the class 1 integron variable region present in Pseudomonas aeruginosa clinical isolates from Brazil. Strains carrying class 1 integrons were resistant to the majority of antibiotics tested, except to imipenem and ceftazidime. Sequence analysis of the integron variable region revealed the presence of the blaCARB-4 gene into two distinct cassette arrays: aacA4-dhfrXVb-blaCARB-4 and aadB-aacA4-blaCARB-4. dhfrXVb gene cassette, which is rare in Brazil and in P. aeruginosa species, was found in one isolate. PFGE analysis showed the spread of blaCARB-4 among P. aeruginosa clones. The occurrence of blaCARB-4 and dhfrXVb in Brazil may contribute for developing resistance to clinically important antibiotics, and shows a diversified scenarium of these elements occurring in Amazon clinical settings, where no study about integron dynamics was performed to date.     

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.     

Class 1 integrons and tetracycline resistance genes in alcaligenes, arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil

The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.     

The role of anaerobic digestion in controlling the release of tetracycline resistance genes and class 1 integrons from municipal wastewater treatment plants

In this study, the abilities of two anaerobic digestion processes used for sewage sludge stabilization were compared for their ability to reduce the quantities of three genes that encode resistance to tetracycline (tet(A), tet(O), and tet(X)) and one gene involved with integrons (intI1). A two-stage, thermophilic/mesophilic digestion process always resulted in significant decreases in the quantities of tet(X) and intI1, less frequently in decreases of tet(O), and no net decrease in tet(A). The thermophilic stage was primarily responsible for reducing the quantities of these genes, while the subsequent mesophilic stage sometimes caused a rebound in their quantities. In contrast, a conventional anaerobic digestion process rarely caused a significant decrease in the quantities of any of these genes, with significant increases occurring more frequently. Our results demonstrate that anaerobic thermophilic treatment was more efficient in reducing quantities of genes associated with the spread of antibiotic resistance compared to mesophilic digestion.     

Studies on New Delhi Metallo-Beta-Lactamse-1 producing Acinetobacter baumannii isolated from donor swab in a tertiary eye care centre, India and structural analysis of its antibiotic binding interactions

Gram-negative bacilli, Enterobacteriaceae and Non-fermentors with resistance to carbapenems and metallo beta-lactams are the major cause of concern in clinical problems in current human healthcare. The most highly emerging dreadful Metallo Beta-lactamses is New Delhi metallo-beta-lactamase (blaNDM-1) which confers resistance to carbapenems; susceptible only to colistin and, less consistently to tigecycline, leading to no therapeutic options. In the present study, we demonstrate the effects of cephalosporins and carbepenems on biofilm producing A. baumanii clinical isolate and also to infer the probable inhibitory binding mode through molecular docking studies. The result of MIC on Biofilm producing A. baumanii and the docking analysis results were found to be concordant. Moreover, we also found cephalosporins and carbepenem groups to interact with 162-166 region of blaNDM-1, which is unique for NDM-1 and also documented to be a potential drug targeting region.     

Evaluation of biofilm production and prevalence of the icaD gene in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains isolated from patients with nosocomial infections and carriers

Staphylococcus aureus is one of the most important etiological agents responsible for healthcare-associated infections and is capable of producing many virulence factors including biofilm. The aim of the present study was to analyze the correlation between the presence of the icaD and icaA genes and the ability to produce biofilm in vitro in 302 methicillin-resistant (MRSA) and 268 methicillin-sensitive S. aureus (MSSA) strains isolated in the Provincial Hospital in Gdansk. Presence of the icaD and icaA genes was detected by PCR and the ability to produce biofilm in vitro was measured both spectrophotometrically and via Congo Red Agar plate culture methods. We found that 91% of MRSA strains harbored the icaD gene. Moreover, all icaD-negative strains were icaA-positive. Of MRSA and MSSA strains, 47% and 69%, respectively, produced biofilm in vitro. The level of consistency between the two applied phenotypic methods was 96%. Additionally, we found that strains with the same biofilm status may be present in asymptomatic carriers and cause infections.     

Identification and analysis of genes from the mouse otic vesicle and their association with developmental subprocesses through in situ hybridization

The otic vesicle (otocyst) occupies a pivotal position in inner ear development, bridging the gap between otic placode determination, and morphogenesis of vestibular and auditory compartments. The molecular mechanisms underlying the progressive subdivision of the developing inner ear into different compartments, and the molecular control and execution of the different developmental processes involved, are largely unknown. Since relatively few genes have been implicated in these processes, we have undertaken this study to identify genes involved in these early embryonic stages. We have used cDNA subtractions of mouse otic vesicle against adult liver cDNA, and describe a set of 280 candidate genes. We have also performed otic vesicle RNA hybridizations against DNA chips to not only confirm the efficacy of the library approach, but also to investigate the utility of DNA array alternatives. To begin to dissect potential developmental roles, we investigated the spatial pattern of gene expression for a selected set of 80 genes in developing mouse embryos at mid-gestation by whole-mount in situ hybridization. These data illustrate the compartmentalisation of gene expression in the otic vesicle for the majority of genes tested, and furthermore, implicate many of the genes tested with distinct developmental subprocesses.     

Molecular survey of Tamyb10-1 genes and their association with grain colour and germinability in Chinese wheat and Aegilops tauschii

Journal of Genetics - To investigate allelic variation of Myb10-1 genes in Chinese wheat and to examine its association with germination level in wheat, a total of 582 Chinese bread wheat cultivars...