Aminoglycoside-modifying Enzyme Genes Research Articles

Detecting the frequency of aminoglycoside modifying enzyme encoding genes among clinical isolates of methicillin-resistant Staphylococcus aureus.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) plays an important role in causing many serious nosocomial infections. In this study, the antimicrobial susceptibility and the frequency of aminoglycoside modifying enzyme encoding genes among clinical isolates of methicillin-resistant Staphylococcus aureus was investigated from two university hospitals of Zanjan province of Iran. Methods: In this study, the antimicrobial susceptibility of MRSA isolates to various antibiotics was investigated by the disk diffusion method. Multiplex PCR assays were used for the determination of aminoglycoside modifying enzyme (AME) genes and staphylococcal cassette chromosome mec (SCCmec) types in MRSA strains. Results: All 58 MRSA isolates were sensitive to vancomycin. Resistance to penicillin G, oxacilin, gentamicin, erythromycin, clindamycin, kanamycin, and tobramycin was found in 96.4%, 98.3%, 51.7%, 53.4%, 55.2%, 62% and 58.6% of the isolates, respectively. The most prevalent AME genes were aac(6′)/aph(2′′) (48.3 %) followed by ant(4)-Ia (24%). The aph(3′)-Ia gene was the least frequent AME gene among MRSA isolates (19%). Of the 58 tested MRSA isolates, 5 (8.6%) were harboured SCCmec type I, 11 (19%) SCCmec type II, 20 (34.5%) SCCmec type III, 17 (29.3%) SCCmec type IVa, 1 (1.7%) SCCmec type IVb, 2 (3.4%) SCCmec type IVc, 11 (19%) SCCmec type IVd, and, 18 (31%) SCCmec type V. Nineteen isolates were not typeable. Conclusion: In conclusion, the aac (6′)/aph (2′′) was the most common aminoglycoside modifying enzyme gene and SCCmec type II and V were the most frequent types detected in hospital isolates, respectively.

Identification of aac(2')-I Type b aminoglycoside-modifying enzyme genes in resistant Acinetobacter baumannii

The aim of this study was to investigate the mechanism underlying the drug resistance of Acinetobacter baumannii toward aminoglycosides. A total of 32 A. baumannii strains were identified by molecular identification and subsequently isolated. The isolates were then amplified by polymerase chain reaction to analyze the 9 aminoglycoside-modifying enzyme genes and 7 16S rRNA methylase genes. Five types of aminoglycoside-modifying enzyme genes and 1 type of 16S rRNA methylase gene were detected in the 32 drug-resistant A. baumannii strains. Positive genes included 7 detection modes, of which the all-6-gene-positive mode aac(2')-Ib+aac(3)-I+aac(6')-Ib+ant(3'')-I+aph(3')-I+armA exhibited the largest number of strains (12, 37.5%). The resistance of A. baumannii against aminoglycosides resulted from the presence of 5 types of aminoglycoside-modifying enzyme genes and the 16S rRNA methylase gene armA. This study is the first to isolate the aac(2')-Ib aminoglycoside-modifying enzyme gene from A. baumannii in a domestic clinical setting.

Prevalence of aminoglycoside modifying enzyme and 16S ribosomal RNA methylase genes among aminoglycoside-resistant Escherichia coli isolates.

Aminoglycoside resistance determinants among 188 aminoglycoside-resistant blood culture Escherichia coli isolates from a tertiary hospital in Hong Kong, from 2004 to 2010 were investigated. Overall, 91% had aac(3)-II, 12.2% had aac(6')-Ib/Ib-cr, and 5.4% had the methylase genes (rmtB, armA). Aminoglycoside-resistant isolates with aac(')-Ib/Ib-cr, rmtB, and armA often had coresistance to multiple other antibiotics.

High rate of aminoglycoside resistance in CTX-M-15 producing Klebsiella pneumoniae isolates in Tehran, Iran.

To determine the prevalence of extended-spectrum beta lactamases (ESBLs), the bla(CTX-M) genes, and aminoglycoside modifying enzymes genes in clinical isolates of Klebsiella pneumoniae (K. pneumoniae). We collected 200 nonduplicate clinical isolates of K. pneumoniae in hospitals in Tehran, Iran. We determined antibacterial susceptibility and confirmed ESBL production via the disk diffusion and minimum inhibitory concentration (MIC) methods. We identified bla(CTX-M) and aminoglycoside modifying enzymes genes via polymerase chain reaction (PCR). We detected 72 (36.0%) ESBL-positive K. pneumoniae, in which the bla(CTX-M-15) was dominant (62.5%). A total of 54.0% of isolates were resistant to at least 1 tested aminoglycoside; also, we detected aac(6')-Ib in 42.5% of isolates and aac(3)-IIa in 35.1% of them. We observed a high rate of aminoglycoside-resistant genes (71.0%) among bla(CTX-M-15)-carrying isolates. We report that CTX-M-15 is the dominant type of CTX-M, which associates with entities that have high aminoglycoside resistance. Continuous surveillance and monitoring of this entity are needed because the codissemination of multiple drug-resistant genes with K. pneumoniae may become a serious therapeutic problem.

Amikacin resistance in Staphylococcus pseudintermedius isolated from dogs.

Staphylococcus pseudintermedius is the most common microorganism isolated from canine pyoderma and postoperative wound infections. The prevalence of methicillin-resistant S. pseudintermedius (MRSP) has increased, and recently, isolates that are resistant not only to methicillin but also to other classes of antibiotic drugs, including aminoglycosides, have become common. A total of 422 S. pseudintermedius isolates collected from 413 dogs were analyzed for amikacin and methicillin resistance using broth microdilution and disk diffusion testing. Methicillin-resistant isolates were significantly (P < 0.0001) more likely to be resistant to amikacin (37%, 31/84) than were methicillin-susceptible isolates (7%, 22/338). Additionally, resistance to non-β-lactam antibiotics was significantly associated with resistance to amikacin irrespective of methicillin resistance. Among the 422 isolates, 32 that tested positive for amikacin resistance by broth microdilution or disk diffusion testing were investigated further for the presence of aminoglycoside-modifying enzyme genes using multiplex PCR. Of these isolates, 66% (21/32) were methicillin resistant. In contrast to previous studies of Staphylococcus aureus, the most prevalent gene detected was aph(3')-IIIa found in 75% (24/32) of isolates followed by aac(6')/aph(2") and ant(4')-Ia in 12% (4/32) and 3% (1/32), respectively. Understanding the differences in antimicrobial resistance gene carriage between different species of Staphylococcus may improve antimicrobial drug selection for clinical therapy and provide insights into how resistance develops in S. pseudintermedius.

Genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii from Beijing, China

The objective of this study was to investigate the genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii clinical isolates from Beijing, China. 173 A. baumannii clinical isolates from hospitals in Beijing from 2006 to 2009 were first subjected to high level aminoglycoside resistance (HLAR, MIC to gentamicin and amikacin>512µg/mL) phenotype selection by broth microdilution method. The strains were then subjected to genetic basis analysis by PCR detection of the aminoglycoside modifying enzyme genes (aac(3)-I, aac(3)-IIc, aac(6′)-Ib, aac(6′)-II, aph(4)-Ia, aph(3′)-I, aph(3′)-IIb, aph(3′)-IIIa, aph(3′)-VIa, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, ant(2″)-Ia, ant(3″)-I and ant(4′)-Ia) and the 16S rRNA methylase genes (armA, rmtB and rmtC). Correlation analysis between the presence of aminoglycoside resistance gene and HLAR phenotype were performed by SPSS. Totally 102 (58.96%) HLAR isolates were selected. The HLAR rates for year 2006, 2007, 2008 and 2009 were 52.63%, 65.22%, 51.11% and 70.83%, respectively. Five modifying enzyme genes (aac(3)-I, detection rate of 65.69%; aac(6′)-Ib, detection rate of 45.10%; aph(3′)-I, detection rate of 47.06%; aph(3′)-IIb, detection rate of 0.98%; ant(3″)-I, detection rate of 95.10%) and one methylase gene (armA, detection rate of 98.04%) were detected in the 102 A. baumannii with aac(3)-I+aac(6′)-Ib+ant(3″)-I+armA (detection rate of 25.49%), aac(3)-I+aph(3′)-I+ant(3″)-I+armA (detection rate of 21.57%) and ant(3″)-I+armA (detection rate of 12.75%) being the most prevalent gene profiles. The values of chi-square tests showed correlation of armA, ant(3″)-I, aac(3)-I, aph(3′)-I and aac(6′)-Ib with HLAR. armA had significant correlation (contingency coefficient 0.685) and good contingency with HLAR (kappa 0.940). The high rates of HLAR may cause a serious problem for combination therapy of aminoglycoside with β-lactams against A. baumannii infections. As armA was reported to be able to cause high level aminoglycoside resistance to most of the clinical important aminoglycosides (gentamicin, amikacin, tobramycin, etc), the function of aminoglycoside modifying enzyme gene(s) in A. baumannii carrying armA deserves further investigation.

The prevalence of aminoglycoside-modifying enzymes among coagulase negative staphylococci in Iranian pediatric patients.

In spite of widespread emergence of aminoglycoside resistance, these drugs are still used in the treatment of staphylococcal infections. This study aimed to investigate the distribution of aminoglycoside resistance and genes encoding aminoglycoside - modifying enzymes (AMEs) as well as Staphylococcal Cassette Chromosome mec (SCCmec) type in coagulase negative staphylococci (CoNS) in pediatric patients. Totally, 93 CoNS isolates were examined for susceptibility to aminoglycosides using disk diffusion and/or E-test methods. AMEs genes and SCCmec types were detected using multiplex PCR. Strain typing was performed using repetitive extragenic palindromic (REP) - PCR assay. The non-susceptibility rates to kanamycin, tobramycin, gentamicin, amikacin and netilmicin were 73%, 59%, 49.5%, 16% and 7.5%, respectively. aac(6')-Ie-aph(2″)-Ia, ant(4')-Ia and aph(3')-IIIa were encountered in 56 (60.2%), 38 (40.8%) and 18 (19.3%) isolates, respectively. In aac(6')-Ie-aph(2″)-Ia- positive isolates, the non- susceptibility rates to kanamycin, gentamicin, tobramycin, amikacin and netilmicin were 83%, 74%, 73%, 49% and 43%, respectively. SCCmec types included type IV (n = 31), I (n = 17), II (n = 5), III (n = 4), and V (n = 2). Three isolates had two types; I + III (n = 2) and III + IV (n = 1) whereas 11 isolates were non-typeable. AMEs genes carriers were distributed frequently into type IV. We found diverse fingerprint patterns among our isolates. In conclusion, there was a strong correlation between alarming rate of aminoglycoside resistance and methicillin resistance. Discordances between phenotypic and genotypic detection of aminoglycoside resistance were discernible. AMEs genes might be related to SCCmec types.

Emergence of SCCmec type III with variable antimicrobial resistance profiles and spa types among methicillin-resistant Staphylococcus aureus isolated from healthcare- and community-acquired infections in the west of Iran

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen of public health importance. The prevalence of MRSA and its antimicrobial resistance pattern, as well as SCCmec and spa types, remain unclear both in the community and in the hospitals of the western region of Iran. One hundred MRSA isolates were collected from different hospitals in the west of Iran during 2010-2011. Antimicrobial susceptibility testing to 15 antimicrobial agents was carried out by disk agar diffusion (DAD) method in accordance with the Clinical and Laboratory Standards Institute guidelines. Vancomycin minimum inhibitory concentrations (MICs) were evaluated by a broth microdilution method. The Etest was used for the detection of highly gentamicin-resistant MRSA. A combination of single and multiplex PCR was used for the detection of different resistance genes, including beta-lactamase, aminoglycoside modifying enzymes (AMEs), and macrolide-lincosamine, and for SCCmec typing of MRSA isolates. Genotyping of MRSA isolates was performed via spa typing. All tested isolates were susceptible to quinupristin-dalfopristin, linezolid, and vancomycin, but were resistant to penicillin (100%), erythromycin (50%), clindamycin (27%), and gentamicin (18%). MIC50 and MIC90 was 256 μg/ml among gentamicin-resistant MRSA. The most prevalent AME genes among aminoglycoside-resistant isolates were aac(6')-1e-aph(2")-1a (77.8%), aph(3')-IIIa (38.9%), and ant(4')-1a (27.8%). Nearly all tetracycline- and erythromycin-resistant MRSA had ermA and/or ermC but not ermB. Five SCCmec types and subtypes, 13 spa types, and four BURP groups (A-D) were identified. SCCmec types III (45%) and IVc (24%), spa type t701 (30%), and new spa type t12311 (15%) were the most prevalent among MRSA isolates. This study showed the emergence of MRSA with SCCmec type III and with spa types t12311, t10740, t1234, t1991, and t2651 with different phenotypic and genotypic antimicrobial resistance in the west of Iran. We found different SCCmec and spa types distributed among nosocomial and non-nosocomial MRSA in the west of Iran.

OXA- and GES-type β-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital

We determined the antibiotic susceptibility and genetic mechanisms of resistance in clinical strains of Acinetobacter baumannii from Istanbul, Turkey. A total of 101 clinical strains were collected between November 2011 and July 2012. Antimicrobial susceptibility was performed using the Vitek 2 Compact system and E-test. Multiplex PCR was used for detecting blaOXA-51-like, blaOXA.-23-like, blaOXA-40-like and blaOXA-58-like genes. ISAba1, blaIMP-like, blaVIM-like, blaGES, blaVEB, blaPER-2, aac-3-Ia and aac-6'-Ib and NDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains were positive for blaOXA-51, 79 strains carried blaOXA-23 and one strain carried blaOXA-40. blaOXA-51 and blaOXA-23 were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of blaOXA-51. GES-type carbapenemases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains) while blaPER-2, blaVEB-1, blaNDM-1, blaIMP- and blaVIM-type carbapenemases were not observed. Aminoglycoside modifying enzyme (aac-3-Ia and aac-6'Ib) genes were detected in 13 and 15 strains, respectively. Ninety-seven (96%) A. baumannii strains were defined as MDR and of these, 98% were extensively drug resistant (sensitive only to colistin). Colistin remains the only active compound against all clinical strains. As seen in other regions, OXA-type carbapenemases, with or without an upstream ISAba1, predominate but GES-type carbapenemases also appear to have a significant presence. REP-PCR analysis was performed for molecular typing and all strains were collected into 12 different groups. To our knowledge, this is the first report of GES-11 and OXA-40 in A. baumannii from Turkey.

Multidrug-resistant genes of aminoglycoside-modifying enzymes and 16S rRNA methylases in Acinetobacter baumannii strains.

We examined the distribution of genes of aminoglycoside-modifying enzymes and 16S rRNA methylases in multidrug-resistant Acinetobacter baumannii to explore the association of these genes with drug resistance. Strains isolated from clinical specimens were screened using an automatic microbial identification system, and 9 aminoglycoside-modifying enzyme and 6 16S rRNA methylase genes were analyzed using polymerase chain reaction and verified by DNA sequencing. Next, sequence alignment was carried out using the Chromas software and a susceptibility test was performed using the Kirby-Bauer disk diffusion method. Genes encoding aminoglycoside-modifying enzymes were detected in all 20 strains of multidrug-resistant A. baumannii. The positive rates of aac(3')-I, aac(6')-Ib, ant(3'')-I, and aph(3')-I were 90.0, 90.0, 85.0, and 35.0%, respectively. However, genes encoding 16S rRNA methylases were not positively detected in the 20 strains of multidrug-resistant A. baumannii. The resistance of multidrug-resistant A. baumannii may be associated with aminoglycoside-modifying enzyme genes but not with 16S rRNA methylase genes.

Short communication: Genetic characterization of antimicrobial resistance in Acinetobacter isolates recovered from bulk tank milk

A total of 176 Acinetobacter isolates, including 57 Acinetobacter baumannii originally obtained from 2,287 bulk tank milk (BTM) samples in Korea was investigated for the genetic basis of antimicrobial resistance using molecular methods. In addition, the occurrence and cassette content of integrons were examined and the genetic diversity of A. baumannii strains identified was evaluated. Aminoglycoside-modifying enzyme genes were detected in 15 (88.2%) of the 17 aminoglycoside-resistant Acinetobacter isolates tested. The most common aminoglycoside-modifying enzyme gene identified was adenylyltransferase gene aadB (n=9), followed by phosphotransferase genes aphA6 (n=7) and aphA1 (n=5). Of the 31 isolates resistant to tetracycline, tet(39) was detected in 20 of them. The genetic basis of resistance to sulfonamide was identified in 15 (53.6%) of 28 trimethoprim-sulfamethoxazole-resistant isolates and 9 (32.1%) of them carried both sul1 and sul2 genes. A blaADC-7-like gene was detected in 1 β-lactam-resistant A. baumannii. Furthermore, class 1 integron was identified in 11 Acinetobacter isolates. Two gene cassettes dfrA15, conferring resistance to trimethoprim, and aadA2, conferring resistance to aminoglycosides, were identified in 8 Acinetobacter isolates. None of the isolates was positive for class 2 or class 3 integrons. Pulsed-field gel electrophoresis revealed that most of the A. baumannii strains from BTM samples were genetically diverse, indicating that the occurrence of A. baumannii strains in BTM was not the result of dissemination of a single clone. Elucidation of resistance mechanisms associated with the resistance phenotype and a better understanding of resistance genes may help in the development of strategies to control infections, such as mastitis, and to prevent further dissemination of antibiotic resistance genes. To the best of our knowledge, this is the first report of molecular characterization of antimicrobial-resistant Acinetobacter spp. from milk.

ARG-ANNOT, a New Bioinformatic Tool To Discover Antibiotic Resistance Genes in Bacterial Genomes

ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) is a new bioinformatic tool that was created to detect existing and putative new antibiotic resistance (AR) genes in bacterial genomes. ARG-ANNOT uses a local BLAST program in Bio-Edit software that allows the user to analyze sequences without a Web interface. All AR genetic determinants were collected from published works and online resources; nucleotide and protein sequences were retrieved from the NCBI GenBank database. After building a database that includes 1,689 antibiotic resistance genes, the software was tested in a blind manner using 100 random sequences selected from the database to verify that the sensitivity and specificity were at 100% even when partial sequences were queried. Notably, BLAST analysis results obtained using the rmtF gene sequence (a new aminoglycoside-modifying enzyme gene sequence that is not included in the database) as a query revealed that the tool was able to link this sequence to short sequences (17 to 40 bp) found in other genes of the rmt family with significant E values. Finally, the analysis of 178 Acinetobacter baumannii and 20 Staphylococcus aureus genomes allowed the detection of a significantly higher number of AR genes than the Resfinder gene analyzer and 11 point mutations in target genes known to be associated with AR. The average time for the analysis of a genome was 3.35 ± 0.13 min. We have created a concise database for BLAST using a Bio-Edit interface that can detect AR genetic determinants in bacterial genomes and can rapidly and easily discover putative new AR genetic determinants.

Dissemination of Aminoglycoside-Modifying Enzymes and 16S rRNA Methylases AmongAcinetobacter baumanniiandPseudomonas aeruginosaIsolates

The purpose of the present study was to investigate the diversity of the genes encoding aminoglycoside-modifying enzymes (AMEs) and their associations with resistance phenotypes and clonality in Acinetobacter baumannii and Pseudomonas aeruginosa isolates. Seventy six P. aeruginosa and 75 A. baumannii isolates were collected from three University affiliated hospitals in Tehran. MIC determination of amikacin and gentamicin as well as the disk diffusion method for tobramycin, netilmicin, and kanamycin were carried out. Nine AMEs genes and three RNA methylases were investigated in all isolates using the PCR method. Clonality for A. baumannii and P. aeruginosa was investigated using repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus PCR, respectively. aph(3')-VIa (90.6%) and aph(3')-IIb (61.8%) were the most prevalent AME genes in A. baumannii and P. aeruginosa, respectively. Eight (26%) amikacin highly resistant A. baumannii isolates were positive for armA methylase. Phenotypes and clonality did not link to the genetic determinants of resistance to aminoglycosides in our isolates. AMEs genes are disseminated in different clones of A. baumannii and P. aeruginosa isolates in Iran. Other than AMEs, there are more complex and multifactorial mechanisms that result in aminoglycoside-resistant phenotypes.

Emergence of a novel GyrA variant from a strain of multidrug-resistant Klebsiella pneumoniae isolated from in China

A strain of Klebsiella pneumoniae (NB029) was positive for blaTEM (beta-lactamase gene), aph(3’)Ⅰ(aminoglycoside modifying enzyme gene), gyrA (quinolone resistance gene), trbC (genetic marker of plasmids), tnp513, IS26, IS903 (genetic markers of transposons and insertion sequences), intⅠ1 (genetic marker of integrons), qacE△1 (drug efflux gene) by PCR assays. So these positive genes confer multidrug-resistant phenotype. And the presence of gyrA, confirmed as a novel variant (Genbank accession number: JN232083), was determined by sequencing and Blastsguin algorithm. The double mutations at codon 83 and codon 87 in the quinolone resistance-determining region of the gyrA gene lead to high level quinolone resistance.

Emergence of five kinds of aminoglycoside-modifying enzyme genes simultaneously in a strain of multidrugresistant Escherichia coli in China

A strain of Escherichia coli was positive for 5 aminoglycoside modifying enzyme genes (aac(3)-I, aac(6′)-Ib-cr, ant(3′)-1, aadA5, and aph(3′)-I) in PCR assays. And these positive genes confer resistance to aminoglycosides (gentamicin and tobramycin). This is the first report of emergence of five kinds of aminoglycoside-modifying enzymes genes simultaneously in E. coli worldwide.

Increasing prevalence of high-level gentamicin resistant enterococci: An emerging clinical problem

Enterococci have recently been recognized as a causative organism of intractable infections. This study investigated the species prevalence, antimicrobial resistance and characterization of high-level gentamicin resistant (HLGR) enterococci. A total of 163 enterococci were isolated from various clinical samples collected from hospitalized patients in Riyadh, Saudi Arabia. The Enterococcus faecalis was the most frequent species (58.9%), followed by Enterococcus faecium (26.9%), Enterococcus avium(6.1%), Enterococcus gallinarum (2.5%), Enterococcus durans (1.8%), Enterococcus hirae (1.2%), Enterococcus casseliflavus (1.2%) and Enterococcus cecorum (0.6%). High-level of resistances to gentamicin and streptomycin were detected by the disc diffusion method, in 20.2% of isolates. The prevalence of resistances among β-lactam antibiotics and glycopeptides were very low. All Enterococcus isolates were identified phenotypically by conventional methods and genotypically by polymerase chain reaction (PCR) targeting aminoglycoside modifying enzyme (AMEs) genes including aac(6`)-Ie-aph(2``)-Ia, aph(2``)-Ib, aph(2``)-Ic, aph(2``)-Id, aph(3`)-IIIa and ant(4`)-Ia. The genetic diversity of E. faecalis presenting HLGR was assessed by pulsed-field gel electrophoresis (PFGE) of chromosomal deoxyribonucleic acid (DNA) after SmaI digestion. Most of the high level aminoglycoside resistant isolates contained genes coding for the bifunctional AMEs AAC(6`)-APH(2``), ANT(6`) and APH(3`) but not the ANT(4`). The results demonstrated that the spread of the aac(6`)-Ie-aph(2``)-Ia gene was responsible for HLGR among enterococci isolated in Riyadh hospital. Key words: Enterococci, resistance, Enterococcus faecalis, high-level gentamicin resistant (HLGR).

Diversity of aminoglycoside modifying enzyme genes among multidrug resistant Acinetobacter baumannii genotypes isolated from nosocomial infections in Tehran hospitals and their association with class 1 integrons

The aim of the present study was to investigate, for the first time, the diversity of the genes encoding aminoglycoside-modifying enzymes (AME) and their association with class 1 integrons in Iranian Acinetobacter baumannii strains. A total of 100 multidrug resistant A. baumannii, isolated from eight distinct hospitals in Tehran, were enrolled in this study. Susceptibility of these isolates to antimicrobial agents including gentamicin and amikacin was determined by E-test. Aminoglycoside resistant isolates were then tested by PCR for AME genes, including aphA6, aacC1, aacC2, aacA4, aadB, aadA1, classes 1 integron, 5'-CS-3' and typed by RAPD PCR. The rate of resistance to imipenem, meropenem, gentamicin and amikacin were 39%, 39%, 38% and 32%, respectively. Intermediate resistance phenotype to gentamicin and amikacin was observed in 2% and 5% of all the isolates, respectively. After aph6 with 90% (n = 36/40), aadA1, aacC1 and aadB with 82.5% (n = 33/40), 65% (n = 26/40) and 20% (n = 8/40) were the most prevalent AME genes among aminoglycosides resistant A. baumannii isolates. A combination of two to four different resistance genes was observed in 39 of 40 strains (97.5%), with a total of 7 different combinations. PCR of integrase genes revealed that AME gene was associated with 67% of class 1 integrons. RAPD analysis showed three predominant genotypes A (n = 20), B (n = 10) and 10 unrelated genotypes. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.

The Major Aminoglycoside-Modifying Enzyme AAC(3)-II Found inEscherichia coliDetermines a Significant Disparity in Its Resistance to Gentamicin and Amikacin in China

The aim of this study was to investigate the prevalence of aminoglycoside-modifying enzymes in Escherichia coli in different areas of China and to explore the relationship between pandemic enzyme type and bacterial resistance to antimicrobial agents in China. Gentamicin- or etimicin-resistant clinical isolates of E. coli were collected from different areas of China, and the in vitro antibacterial activity of 11 aminoglycoside agents was determined using standard (Clinical and Laboratory Standards Institute) agar dilution methods. Twelve aminoglycoside-modifying enzyme genes were detected by PCR and confirmed by DNA sequencing. A total of 205 E. coli strains were collected from nine hospitals in seven cities. All strains were highly resistant to gentamicin or etimicin, whereas resistance to tobramycin, netilmicin, and kanamycin was slightly lower. However, less than 15% of isolates were resistant to amikacin and isepamicin. Of the gentamicin-resistant strains, 88.2% and 86.7% were sensitive to isepamicin and amikacin, respectively. Five aminoglycoside-modifying enzyme genes were detected in 191 strains, whereas the remaining 14 strains were negative. The most common gene type was aac(3)-II (162 strains), followed by aac(6')-I (50 strains), ant(3″)-I (28 strains), aph(3')-II (20 strains), and ant(2″)-I (20 strains). Ninety-five strains yielded aac(3)-II only, whereas the others contained two or three genes. The three main gene combinations were aac(6')-I/aac(3)-II (28 strains), aac(3)-II/ant(3″)-I (11 strains), and aac(3)-II/aac(6')-I (10 strains). Regional bacterial resistance and enzyme distribution were roughly similar, although minor differences were found in Guangzhou, Jinan, and Dalian, which were the sources of most of the amikacin- or isepamicin-resistant strains. Chinese clinical isolates of E. coli remain highly resistant to gentamicin and etimicin, but are susceptible to amikacin and isepamicin. The dominant type of aminoglycoside-modifying enzyme, AAC(3)-II, might be the main source of the disparity in E. coli resistance to different aminoglycoside agents.

Study on the resistant genes of metal β-lactamase(MBLs) of the multi-drug resistant mechanism of Pseudomonas aeruginosa

Objective To survey the distribution of aminoglycoside-modifying enzyme genes in multi-drug-resistant Pseudomonas aeruginosa(MRPA) isolated in our hospital and provide epidemiologic data for clinic. Methods Eighteen strains of Pseudomonas aeruginosa were collected randomly from 2007 to 2009 in our hospital. Isolates were further evaluated for resistance to 14 kinds of anti-bateria mainly by VITEK-32 instrument and some by K-B method as a supplement. Three-dimensional extract test were used to screen the phenotype of MBL production. Five genes， including blaIMP， blaVIM， blaSPM， blaGIM and blaSIM， were amplified by polymerase chain reaction (PCR). Results Of the eighteen PA， nine was resistant to imipenem. Three-dimensional extract test showed that five were positive for MBLs. The PCR results showed that four were positive for blaVIM， one was positive for blaGIM， and one was positive both for blaVIM and blaGIM. Conclusions MBLs production was the majior mechanism for MRPA resistant to imipenen in our hospial and the genes of MBLs is of diversity.

Aminoglycoside Resistance and Susceptibility Testing Errors in Acinetobacter baumannii-calcoaceticus Complex

Antimicrobial resistance is depleting the pharmacopeia of agents clinically useful against Gram-negative bacilli. As the number of active agents diminishes, accurate susceptibility testing becomes critical. We studied the susceptibilities of 107 isolates of the Acinetobacter baumannii-calcoaceticus complex to amikacin, gentamicin, and tobramycin using disk diffusion, Etest, as well as the Phoenix, Vitek 2, and MicroScan automated systems, and compared the results to those obtained by broth microdilution. Genes encoding aminoglycoside-modifying enzymes (AMEs) were detected by multiplex PCR, and clonal relationships were determined by pulsed-field gel electrophoresis. Tobramycin was the most active aminoglycoside (27.1% of isolates were susceptible). Disk diffusion and Etest tended to be more accurate than the Vitek 2, Phoenix, and MicroScan automated systems; but errors were noted with all methods. The Vitek 2 instrument incorrectly reported that more than one-third of the isolates were susceptible to amikacin (a very major error). Isolates were polyclonal, with 26 distinct strains, and carried multiple AME genes unrelated to the strain type. The presence of the ant(2")-Ia gene was statistically associated with resistance to each aminoglycoside. The AME genotype accounted for the resistance profile observed in a minority of isolates, suggesting the involvement of multiple resistance mechanisms. Hospital pharmacy records indicated the preferential use of amikacin over other aminoglycosides in the burn intensive care unit, where aminoglycoside resistance is prevalent. The resistance in that unit did not correlate with a predominant strain, AME genotype, or total annual aminoglycoside consumption. Susceptibility to tobramycin increased, even though susceptible isolates carried AME genotypes predicting the inactivation of tobramycin. Determination of the relative contribution of multiple concurrent resistance mechanisms may improve our understanding of aminoglycoside resistance in the Acinetobacter baumannii-calcoaceticus complex.