The prevalence of aminoglycoside-modifying enzyme and virulence genes among enterococci with high-level aminoglycoside resistance in Inner Mongolia, China

ABSTRACT This study highlights the prevalence of aminoglycoside-modifying enzyme genes and virulence determinants among clinical enterococci with high-level aminoglycoside resistance in Inner Mongolia, China. Screening for high-level aminoglycoside resistance against 117 enterococcal clinical isolates was performed using the agar-screening method. Out of the 117 enterococcal isolates, 46 were selected for further detection and determination of the distribution of aminoglycoside-modifying enzyme-encoding genes and virulence determinants using polymerase chain reaction -based methods. Enterococcus faecium and Enterococcus faecalis were identified as the species of greatest clinical importance. The aac(6')-Ie-aph(2")-Ia and ant(6')-Ia genes were found to be the most common aminoglycoside-modifying enzyme genes among high-level gentamicin resistance and high-level streptomycin resistance isolates, respectively. Moreover, gelE was the most common virulence gene among high-level aminoglycoside resistance isolates. Compared to Enterococcus faecium, Enterococcus faecalis harbored multiple virulence determinants. The results further indicated no correlation between aminoglycoside-modifying enzyme gene profiles and the distribution of virulence genes among the enterococcal isolates with high-level gentamicin resistance or high-level streptomycin resistance evaluated in our study.

Distribution of genes encoding aminoglycoside‑modifying enzymes among clinical isolates of methicillin‑resistant staphylococci.

The objective of this study was to determine the distribution of genes encoding aminoglycoside‑modifying enzymes (AMEs) and staphylococcal cassette chromosome mec (SCCmec) elements among clinical isolates of methicillin‑resistant staphylococci (MRS). Antibiotic susceptibility test was done using Kirby–Bauer disk diffusion method. The presence of SCCmec types and AME genes, namely, aac (6’)‑Ie‑aph (2’’), aph (3’)‑IIIa and ant (4’)‑Ia was determined using two different multiplex polymerase chain reaction. The most encountered AME genes were aac (6′)‑Ie‑aph (2’’) (55.4%) followed by aph (3’)‑IIIa (32.3%) and ant (4’)‑Ia gene (9%). SCCmec type I (34%) was predominant in this study. In conclusion, the aac (6’)‑Ie‑aph (2’’) was the most common AME gene and SCCmec type I was most predominant among the MRS isolates.

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>512 µg/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.

aac(6')-Ⅰ b gene in drug-resistant strains of Pseudomonas aeruginosa / 中华临床感染病杂志

Objective To investigate the distribution of aminoglycoside modifying enzyme genes (AMEs) and 16S rRNA methylase genes in drug-resistant strains of Pseudomonas aeruginosa. Methods Twenty strains of drug-resistant Pseudomonas aeruginosa were isolated from sputum and wound secretion samples collected from the First People's Hospital of Huai' an in Jiangsu province. Eight AMEs [aac(3)-Ⅰ , aac(3)-Ⅱ, aac(6')-Ⅰb,aac(6')-Ⅱ, ant{2)-Ⅰ ,ant(3)-Ⅰ , ant(4')-Ⅰ , aph(3')-Ⅱb] and 6 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD, npmA) were analyzed by PCR and verified by DNA sequencing. Results Out of 20 strains of Pseudomonas aeruginosa, aac(6')-Ⅱ was positive in 8 strains (40.0% ) , ant2- Ⅰ in 8 strains (40.0% ) , aac(3)-Ⅱ in 5 strains (25.0% ) , aac(6')- Ⅰ b in 2 strains (10.0% ) and rmtB in 1 strains (5.0% ) , respectively. The rest 9 genes were not detected. Among 2 strains harboring aac(6')- Ⅰ b, DNA sequencing confirmed that 1 was aac(6')- Ⅰ b (the clssical type) and another was aac(6')- Ⅰ b-cr. Conclusion Gene aac(6')- Ⅰ b-cr exists in drug-resistant Pseudomonas aeruginosa, and it has modifying effect on both aminoglycosides and quinolones.

16S rRNA Methylase Gene and Aminoglycoside-modifying Enzyme Genes in Pseudomonas aeruginosa Isolated from Burned Patients / 中华医院感染学杂志

OBJECTIVE To investigate the 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes in Pseudomonas aeruginosa isolated from burned patients. METHODS GNS-448 and K-B tests were performed to detect the susceptibility to 19 kinds of antimicrobial agents against these strains. 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencing. RESULTS The 32 isolated strains were all resistant to ampicillin,cefuroxime,cefoxitin,SMZ-TMP,The sensitive rates to amikacin and gentamicin were 68% and 46.9%,respectively. The resistant rates to imipenem and meropenem were 68.8% and 59.4%,respectively. The 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes including aac(6')-Ⅰb,aac(6')-Ⅱ,ant(3″)-Ⅰ,ant(2″)-Ⅰ and rmtB were found and positive rates were 9.4%,3.1%,28.1%,25.0% and 3.1%,respectively. A novel subtype of aac(6')-Ⅰb was reported firstly. CONCLUSIONS There are high positive percentage of 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes in P. aeruginosa isolated from burned patients. P. aeruginosa resistance to aminoglycoside relates to the existence of 16S rRNA methylase gene and aminoglycoside-modifying enzyme genes.

Aminoglycoside-modifying Enzyme and 16S rRNA Methylase Gene Expressions in Acinetobacter baumannii / 中华医院感染学杂志

OBJECTIVE To understand aminoglycoside-modifying enzyme and 16S rRNA methylase gene expressions in Acinetobacter baumannii in Xinjiang region.METHODS 20 A.baumannii strains were isolated and test the anti-bacterial drug sensitivity.PCR methods were used to test aminoglycoside-modifying enzyme and 16S rRNA methylase gene.RESULTS From thirteen A.baumannii strains detected the aminoglycoside-modifying enzyme genes,the detection rate was 65%;in which aac(3)-Ⅰ gene was positive in 4 strains(20%),aac(3)-Ⅱ gene in 8 strains(40%),aac(6′)-Ⅰad gene in 4 strain(20%),ant(3″)-Ⅰ gene in 4 strain(20%),and ant(2″)-Ⅰ gene was positive in 1 strain(5%).The aac(6′)-Ⅰ b and aac(6′)-Ⅱ gens and were not detected;the 16S rRNA gene methylation was negative.CONCLUSIONS There are aminoglycoside-modifying enzyme genes existing in A.baumannii,no 16S rRNA methylase gene was detected in Xinjiang region.

Drug-resistance and Aminoglycoside Modifying Enzymes Genes in Acinetobacter baumannii Isolated from ICU / 中华医院感染学杂志

OBJECTIVE To investigate the drug-resistance and aminoglycoside modifying enzymes (AME) in Acinetobacter baumannii (ABA) isolated from ICU. METHODS K-B method was conducted to detect the sensitivity to 14 common antibiotics of 291 strains of ABA isolated from 2005 to 2007;PCR was used to detect AME genes of partial bacteria isolated in 2005. RESULTS The drug-resistance rates of 13 kinds of antibiotics were 33.0-91.8%,but cefoperazone/sulbactam was low (12.7%). The rate of amikacin,gentamicin and tobramycin was 41.2%,61.2% and 61.5%. Twenty-three ABA strains were AME gene positive from total 27 ABA strains and the total positive rate was 74.4%.We found 21 strains with aac(3)-Ⅰ (77.8%),23 strains with aac(6′)-Ⅰ (85.2%) and 23 strains with ant(3″)-Ⅰ (85.2%). Two strains were found to have 2 kinds of AME genes,21 strains had 3 kinds of AME genes. The rest of ABA strains had not AME genes. CONCLUSIONS ABA isolated from ICU has strong drug-resistance rate and a high carrier rate,it is a critical area of preventing and controlling hospital infection.

Distribution of Genes Encoding Aminoglycoside Modifying Enzymes and Type Staphylococcal Chromosomal Cassette mec in Methicillin-resistant Staphylococcus aureus from Non-tertiary Hospitals / 감염과화학요법

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.

Distribution of Genes Encoding Aminoglycoside Modifying Enzymes and Type Staphylococcal Chromosomal Cassette mec in Methicillin-resistant Staphylococcus aureus from Non-tertiary Hospitals / 감염과화학요법

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.

Genotyping of ?-Lactamases,Aminoglycoside Modifying Enzymes and Chlorhexidine-sulfanilamide from Acinetobacter baumannii / 中华医院感染学杂志

OBJECTIVE To investigate the coding genes of ?-lactamases,aminoglycoside modifying enzymes and the drug-resistant to chlorhexidine-sulfanilamide genes on 20 Acinetobacter baumannii isolates in Xinjiang.METHODS Twenty strains of A.baumannii were isolated from hospitalized patients,and 9 kinds of ?-lactamases genes,3 kinds of aminoglycoside modifying enzymes genes and drug-resistant to chlorhexidine-sulfanilamide genes were detected.The drug-resistant to chlorhexidine-sulfanilamide genes were labeled and cluster analysis was performed to analyze the affinity of strain.RESULTS The detection rates of ?-lactamases coding genes of TEM,ADC and SHV groups were 65%,60% and 5%,respectively.The others were not found in all 20 isolates tested.The detection rates of aminoglycoside modifying enzymes coding genes of aac(3)-Ⅰ,aac(6′)-Ⅰ and aac(3″)-Ⅰwere 60%,65%and 70%,respectively.And the detection rates of qacE△1-stull genes were 70%.There were 9 strains showed clone transmission according to cluster analysis.CONCLUSIONS Drug-resistance of the 20 strains to ?-lactam and aminoglycosides is connected with ?-lactamases and aminoglycoside modifying enzymes,and there exists clone transmission.

Multiplex PCR for the Detection of Genes Encoding Aminoglycoside Modifying Enzymes and Methicillin Resistance among Staphylococcus Species

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method.

Multiplex PCR for the Detection of Genes Encoding Aminoglycoside Modifying Enzymes and Methicillin Resistance among Staphylococcus Species

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method.