Aminoglycoside-modifying Enzyme Genes Research Articles

Prevalence of Genes Encoding Aminoglycoside-Modifying Enzymes in Clinical Isolates of Gram-Positive Cocci in Iran: A Systematic Review and Meta-Analysis.

Introduction: Several studies have investigated the genes encoding aminoglycoside-modifying enzymes (AMEs) among gram-positive cocci (GPC) such as Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), coagulase-negative staphylococci (CoNS), and Enterococcus spp. in Iran; however, a comprehensive analysis has not yet been performed. Thus, the present systematic review and meta-analysis was conducted to determine the prevalence of genes encoding AMEs among GPC in Iran. Methods: A systematic review of the data published in the English and Persian languages from January 2000 to October 2018 was performed by searching different electronic databases (Medline, Embase, Web of Science, and the Iranian Database). Meta-analysis was performed by using the Comprehensive Meta-Analysis (Biostat V2.2) software. Cochran's Q and I2 statistics were used to test heterogeneity, and publication bias was assessed by using funnel plot and Begg's and Egger's tests. Results: Out of 117 studies, 28 were considered eligible for inclusion in the current meta-analysis. The most prevalent AMEs gene among GPC was aac(6')-Ie-aph(2'')-Ia, with a prevalence of 97.7% (95% CI; 94.4-99) in high-level gentamicin-resistant enterococci and 67.7% (95% CI; 59.2-75.2) in MRSA. The second most common gene was ant(4')Ia, with a prevalence of 45.3% (95% CI; 23.9-68.6) in MRSA. Conclusions: It was ultimately determined that the prevalence of AMEs genes among GPC had reached alarming levels in Iran; therefore, aminoglycosides should be prescribed with caution by clinicians. The implementation of a regional and nationwide surveillance system to monitor antimicrobial resistance, especially aminoglycosides, and increasing the awareness of AMEs genes among clinicians are essential to guiding empirical and pathogen-specific therapy.

The association of acetylacetate (Acr AB-Tol C) and QepA genes with multiple antibiotic resistance among Escherichia coli clinical isolates

The role of efflux pumps in antibiotic resistance development among Escherichia coli may have been underappreciated. The objective of this study was assessment the association of AcrAB-TolC efflux pumps and qepA genes with resistance to common antibiotics among E. coli isolates. A total of 200 E. coli isolates were obtained from diverse samples of inpatients. Minimum inhibitory concentrations and Kirby–Bauer disk diffusion tests were determined for ceftazidime, cefotaxime, imipenem, gentamicin, and tetracycline. The Acr-AB-TolC and qepA genes were amplified using PCR technique and their association with antibiotics was also evaluated using Chi-square test. A majority of isolates (64%) were retrieved from gastrointestinal samples, followed by urinary tract infections (33%), and bloodstream (3%). All the isolates were resistant to ampicillin (100%), followed by cefazolin (59%), and cefoxitin (58%). However, 100% of the isolates showed susceptibility to fosfomycin. The prevalence of acrA, acrB, and qepA genes was 94% (n = 188), 86% (n = 172), and 8% (n = 16), respectively. The acrA and acrB were significantly associated with resistance to cefoxitin and cefazolin (P < 0.01), ceftazidime (P < 0.01), carbapenems (P = 0.022), and tetracycline (P = 0.0112). In addition, qepA gene was significantly associated with tetracycline resistance (P = 0.032). None of the patients had death outcome. A majority of E. coli isolates harbored the AcrAB genes, but qepA was observed among lower number of the isolates. It is notable that three strains lacked the extended spectrum beta-lactamase and carbapenemases and none of multidrug resistant strains carried tet and aminoglycoside modifying enzymes genes. Over-expression of efflux pumps has been increasingly is associated with clinically relevant antibiotic resistance. For this reason, the expression and functionality of efflux pumps should be more investigated profoundly and be compared between drug-resistant and drug-susceptible isolates.

Detection and molecular characterisation of amikacin-resistant Mycobacterium abscessus isolated from patients with pulmonary disease.

The aim of this study was to investigate the molecular mechanisms conferring amikacin (AMK) resistance in Mycobacterium abscessus clinical isolates. A total of 194M. abscessus clinical isolates were collected from patients with pulmonary disease during the period 2012-2017. AMK susceptibility was determined by the broth microdilution method. Whole-genome data were used for identification of mutations in resistance-associated genes. Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the gene transcriptional level. AMK showed high in vitro killing activity against M. abscessus, with an MIC50 of 8mg/L and an MIC90 of 16mg/L. Five isolates (2.6%) were resistant to AMK (MIC>1024mg/L), of which four (80.0%) harboured a resistance-associated rrs mutation A1408G. qRT-PCR analysis showed that most of the AMK-resistant isolates (4/5; 80.0%) overexpressed the transcriptional regulator gene whiB7 and the multidrug-efflux transporter gene tap. However, overexpression of the aminoglycoside-modifying enzyme gene eis2 was only observed in one (20.0%) AMK-resistant isolate. The AMK resistance rate in M. abscessus clinical isolates in this study was low (2.6%). The A1408G mutation in rrs and overexpression of WhiB7 and Tap were the predominant mechanisms of AMK resistance in M. abscessus.

Mechanisms of Fluoroquinolone and Aminoglycoside Resistance in Keratitis-Associated Pseudomonas aeruginosa.

Global emergence of multidrug resistant (MDR) strains limits therapeutic efficacy in Pseudomonas aeruginosa corneal ulcers. Identifying the primary causal factors of resistance shall improve clinical management. In this study, we sought to identify the underlying mechanisms of fluoroquinolone and aminoglycoside resistance in MDR, non-MDR, and drug susceptible P. aeruginosa (n = 19) strains obtained from keratitis patients. Phenotypic assays were performed to study the bacterial growth kinetics, efflux, permeability, and biofilm formation. Mutational alteration of target genes (DNA sequencing), relative expression of efflux system genes (real time PCR), and detection of aminoglycoside modifying enzyme (AME) genes (PCR) were done by molecular methods. We repeatedly found the mutations in quinolone resistance determining region of fluoroquinolone target genes, gyrA and parC, and the presence of AME genes, aph(3″)-I and aph(6)-I, in all MDR isolates. Furthermore, the MDR isolates were largely characterized by slower growth, cytotoxic type III secretion system genotype, better biofilm-forming ability, and the presence of additional AME genes. The non-MDR isolates were resensitized upon inhibition of active efflux or enhancement of membrane permeability. Altogether this study highlights target gene alteration and enzymatic drug modification as the major mechanisms of quinolone and aminoglycoside resistance in P. aeruginosa keratitis isolates.

Aminoglycoside resistance determinants in multiresistant Escherichia coli and Klebsiella pneumoniae clinical isolates from Turkish and Syrian patients.

Escherichia coli and Klebsiella pneumoniae are frequently found resistance to aminoglycosides in Turkey. The aim of this study was to investigate aminoglycoside resistance in clinical isolates of E. coli and K. pneumoniae from Turkey using both phenotypic and genotypic methods and screening for the prevalence of gene coding for common aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase genes. A total of 88 consecutive, non-duplicated E. coli (n = 65) and K. pneumoniae (n = 23) isolates showing resistance or intermediate resistance to amikacin and/or gentamicin were collected between October 2013 and May 2015 from clinical samples received at Gaziantep Dr. Ersin Arslan Training and Research Hospital. Seventeen isolates were obtained from Syrian patients. Isolates resistant to any of the two aminoglycosides were tested by PCR for seven AME genes, and 22 isolates with amikacin MIC ≥16mg/L were also tested for 16S rRNA methylase genes. In E. coli isolates, the most frequent genes were aac(6')-Ib (50 strains; 76.9%) and aac(3)-IIa (40 strains; 70.7%), followed by aph(3')-Ia (5 strains; 7.6%) and ant(2″)-Ia (2 strains; 3.1%). Among the 23 resistant K. pneumoniae isolates, the most prevalent gene was aac(3')-IIa (87.0%) followed by aac(6')-Ib (73.9%) and aph(3')-Ia (8.6%). The rmtC gene was detected in one K. pneumoniae isolate. Resistance to aminoglycosides in clinical isolates of E. coli and K. pneumoniae from our center is predominantly caused by AAC(6')-Ib and AAC(3)-II enzymes, while the occurrence of 16S rRNA methylases is so far limited.

Molecular drivers of emerging multidrug resistance in Proteus mirabilis clinical isolates from Algeria

The aim of this study was to characterise the molecular drivers of multidrug resistance in Proteus mirabilis isolated from Algerian community and hospital patients. A total of 166 P. mirabilis isolates were collected from two hospitals and eight private laboratories from four cities (Khemis Miliana, Aïn Defla, Oran and Chlef) located in northwestern Algeria. All isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antimicrobial susceptibility testing was performed by the disk diffusion and Etest methods. Genes encoding AmpC β-lactamases, extended-spectrum β-lactamases (ESBLs), quinolone resistance and aminoglycoside-modifying enzymes (AMEs) as well as plasmid replicon typing were characterised by PCR. Clonal relationships were also determined by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) typing and were compared with MALDI-TOF/MS proteomic typing. Of the 166 P. mirabilis isolates, 14 (8.4%) exhibited resistance to important antibiotics, including amoxicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin and ciprofloxacin, of which 4/14 (28.6%) had an ESBL genotype (blaCTX-M-2) and 10 (71.4%) had an AmpC/ESBL genotype (blaCMY-2/blaTEM-1). AME genes were detected in all isolates, including ant(2'')-I, aac(3)-I, aac(6')-Ib-cr and aac(3)-IV. The qnrA gene was identified in 13 isolates (7.8%). ERIC-PCR showed one predominant clone, with eight blaCMY-2-producing isolates from UHC Oran belonging to profile A clustering together in the MALDI-TOF/MS dendrogram. Here we report the first description of AME and plasmid-mediated quinolone resistance genes among ESBL- and/or AmpC β-lactamase-producing P. mirabilis isolates from community- and hospital-acquired infections in northwestern Algeria.

Development of a Luminex xTAG Assay for the Rapid Detection of Five Aminoglycoside Resistance Genes Both in Staphylococci and Enterococci.

Resistance to aminoglycoside antibiotics is now common in pathogenic bacteria, making treatment of infections difficult. The rapid spread of resistance is mainly related to the dissemination of genes encoding aminoglycoside-modifying enzymes (AMEs). Staphylococci and enterococci are opportunistic human pathogens capable of causing a wide range of infections. Isolates from clinical cases are often found to be resistant to aminoglycosides. The aim of the present study was to develop a bead-based xTAG assay for the simultaneous detection of five prevalent aminoglycoside resistance genes in staphylococci and enterococci, including aac(6')-Ie-aph(2″)-Ia, aph(3')-IIIa, ant(4')-Ia, ant(9)-Ia, and ant(6)-Ia. The limit of detection ranged from 10 to 1000 copies/μL of input purified plasmid DNA. Twenty-two bacterial isolates from clinical samples were examined using the newly developed xTAG assay and also by conventional PCR to determine the relative performance of each. The results obtained by xTAG assay showed higher detection rates and accuracy for AME genes than conventional PCR. It indicated that the xTAG-multiplex PCR method is a high-throughput tool for rapid identification of AME genes.

Comparison of antibiotic resistance and virulence in vancomycin-susceptible and vancomycin-resistant Enterococcus faecium strains

Aim. Today, infections caused by vancomycin-resistant Enterococcus faecium (VRE) are a major problem in the healthcare system. The aim of this study was to compare the antibiotic resistance and virulence traits between vancomycin-susceptible E. faecium (VSE) and VRE clinical isolates.Material and Methods. Studies were performed on 66 E. faecium (32 VRE and 34 VSE) strains. Susceptibility testing and identification were performed, and strains were examined for ß-lactamase, hemolysin and biofilm production. Isolates were tested for the presence of 5 van genes, 8 virulence genes and 6 aminoglycoside-modifying enzyme (AME) genes. Obtained amplicons were subjected to electrophoretical separation and DNA sequencing.Results. Among 32 VRE isolates, 28 were found to have the VanA phenotype, and 4 the VanB. The most frequent resistance and virulence profile among VRE strains was resistance to ampicillin, imipenem, gentamicin, streptomycin, teicoplanin, and vancomycin with enterococcal surface protein (esp), endocarditis antigen (efaA), collagen adhesin (acm), and hialuronidase (hyl) genes; among VSE: resistance to ampicillin, imipenem, gentamicin, streptomycin with esp, efaA, acm, and hyl genes. Conclusions. Our findings prove that both VRE and VSE strains were well equipped with virulence and resistance genes, although VRE strains were characterized by a greater variety and a higher number of these genes. However, statistical analysis revealed no significant differences between VSE and VRE strains (p &gt; 0.05). Nevertheless, our results suggest that VRE strains may slowly acquire and incorporate resistance and virulence genes, due to their ability to survive in a hospital environment for a long time.

Emergence of aph(3')-VI and accumulation of aminoglycoside modifying enzyme genes in KPC-2-possessing Enterobacter aerogenes isolates from infections and colonization in patients from Recife-PE, Brazil.

The objective of this study was to characterize genes of aminoglycoside modifying enzymes (AMEs) in colonizing and infecting isolates of E. aerogenes harboring bla KPC from patients at a public hospital in Recife-PE, Brazil. We analyzed 29 E. aerogenes clinical isolates resistant to aminoglycosides. AMEs genes were investigated by PCR and sequencing. Colonizing and infecting isolates mainly presented the genetic profiles aac(3)-IIa/aph(3')-VI or ant(2")-IIa/aph(3')-VI. This is the first report of aph(3')-VI in E. aerogenes harboring bla KPC in Brazil. The results highlight the importance in establishing rigorous methods for the surveillance of resistance genes, especially in colonized patients.

In vitro activity of Plazomicin against Enterobacteriaceae isolates carrying genes encoding aminoglycoside-modifying enzymes most common in US Census divisions

Aminoglycoside-nonsusceptible isolates of Escherichia coli, Klebsiella, Proteus, and Enterobacter species (480/3675) from US hospitals collected during 2014–2015 were screened for 16S rRNA methyltransferase and aminoglycoside-modifying enzyme (AME) genes. Only 5 isolates had high aminoglycoside MICs and carried 16S rRNA methyltransferases. AME genes were observed among 89.7% (426/475) of isolates and the most common genes were aac(3)-IIa (n = 270) and aac(6′)-Ib (n = 269). Among other genes, ant(2″)-Ia, aac(3)-Iva, and aph(3′)-VIa were observed among 36, 23, and 3 isolates, respectively. Forty-nine (10.3%) isolates yielded negative results for the investigated AME genes. Plazomicin (MIC50/90, 0.5/1 μg/ml) inhibited 99.3% of the AME-carrying isolates at its susceptible breakpoint while amikacin, gentamicin, and tobramycin inhibited 90.1%, 20.9%, and 18.3%, respectively. Plazomicin was approved by the US Food and Drug Administration in June 2018 for the treatment of complicated urinary tract infections when limited treatment options are available. This agent displayed activity against isolates carrying AMEs that were resistance to other aminoglycosides and comparator agents.

High level aminoglycoside resistant enterococci in hospital-acquired urinary tract infections in Mansoura, Egypt.

Resistance to different antimicrobial agents is increasing in enterococci and effective treatment represents a major health concern. The aim of this study was to determine the antimicrobial resistance patterns and the frequency of high level aminoglycoside resistance (HLAR) among enterococci. A total of 80 enterococcal isolates, (73 Enterococcus faecalis, 7 Enterococcus faecium) were collected from patients with hospital acquired urinary tract infections (UTI) at Mansoura University hospitals in Egypt. Antimicrobial susceptibility testing was performed via the disc diffusion method. PCR was used for identification of species and detection of aminoglycoside-modifying enzymes genes (AME). All enterococcal isolates were sensitive to vancomycin and linezolid. Fifty-three isolates exhibited HLAR. Our results show that HLAR was mediated by the presence of multiple AMEs genes. The aac(6')-Ie-aph(2')-Ia gene was associated with aph(3')-IIIa and ant(6)-Ia gene in 69% of HLAR isolates. This study showed that enterococci isolated from hospital acquired UTI were resistant to multiple antibiotics. Furthermore, the frequency of high level gentamicin resistance (HLGR) was higher than high level of streptomycin resistance (HLSR). The most common AME genes were aph(3')-IIIa and ant(6)-Ia followed by aac(6')-Ie-aph(2')-Ia.

1964. Microbiological Outcomes With Plazomicin (PLZ) vs. Colistin (CST) in Patients With Bloodstream Infections (BSI) Caused by Carbapenem-Resistant Enterobacteriaceae (CRE) in the CARE Study

BackgroundPLZ is a next-generation aminoglycoside with structural modifications that protect it from aminoglycoside-modifying enzymes (AMEs) and in vitro activity against multidrug-resistant (MDR) Enterobacteriaceae, including aminoglycoside- and carbapenem-resistant strains. In the CARE study, PLZ was associated with improvement in 28-day all-cause mortality vs. CST in patients with CRE BSI. We report the microbiological outcomes in the CARE study by pathogen and key resistance mechanism.MethodsCARE was a multinational, open-label trial that enrolled BSI patients with documented or presumed CRE into two cohorts. Patients in the randomized cohort received PLZ (15 mg/kg q24h IV) or CST (300-mg load [CST base activity] then 5 mg/kg/day IV) plus adjunctive tigecycline or meropenem. Patients in the observational cohort received PLZ plus investigator’s choice of adjunctive agent. Treatment duration was 7–14 days. Isolate identification and susceptibility testing were conducted by a central laboratory. Whole-genome sequencing was used to identify AME and carbapenemase genes. Microbiological outcomes were assessed in patients with confirmed CRE who received ≥1 dose of study drug (mMITT population).ResultsOf 45 BSI patients enrolled, 43 had confirmed CRE (mMITT), including Klebsiella pneumoniae (n = 42) and Enterobacter aerogenes (n = 1). Against CRE, PLZ MICs ranged from 0.12 to >128 µg/mL; 25/28 (89.3%) isolates from PLZ-treated patients had a PLZ MIC ≤4 µg/mL, while 3 had a PLZ MIC ≥128 µg/mL and a confirmed 16S ribosomal methyltransferase gene. CST MICs ranged from 0.25 to >128 µg/mL; 6/16 (37.5%) isolates from CST-treated patients had an MIC >2 µg/mL. There were 47 distinct Enterobacteriaceae pathogens isolated from 43 patients, and of these, AME genes were detected in 43/47 (91.5%), most commonly aac(6’)-Ib (n = 29). Carbapenemase genes were detected in 45/47 (95.7%) isolates, most commonly blaKPC (n = 33). PLZ demonstrated higher microbiological eradication rates than CST against CRE, including AME- and carbapenemase-producing isolates (table).ConclusionThe results provide evidence of the efficacy of PLZ-based therapy for patients with BSI due to MDR Enterobacteriaceae, including AME- and carbapenemase-producing organisms. Disclosures A. W. Serio, Achaogen, Inc.: Employee and Shareholder, Salary. A. Smith, Achaogen, Inc.: Employee and Shareholder, Salary. K. M. Krause, Achaogen, Inc.: Employee, Salary. I. Galani, Achaogen, Inc.: Scientific Advisor, Research funding and honoraria. MSD: Scientific Advisor, Honoraria. A. C. Gales, MSD: Consultant and Speaker, Consulting fee. Pfizer: Consultant and Speaker, Consulting fee. BD: Consultant, Consulting fee. Bayer: Consultant, Consulting fee. A. Jubb, Achaogen, Inc.: Employee and Shareholder, Salary. L. E. Connolly, Achaogen, Inc.: Consultant, Consulting fee.

Aminoglycoside-modifying enzyme and 16S ribosomal RNA methyltransferase genes among a global collection of Gram-negative isolates

The prevalence of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases among 200 Gram-negative clinical isolates resistant to different aminoglycosides and collected worldwide during 2013 was evaluated. Selected AMEs and 16S rRNA methyltransferase genes were screened by PCR/sequencing among 49 Acinetobacter spp., 52 Pseudomonas aeruginosa and 99 Enterobacterales. In total 72 isolates carried aac(6')-lb variants (36.0% overall; 55.6% Enterobacterales): 30 aac(6')-Ib-cr, 21 aac(6')-Ib and 21 aac(6')-Ib-like displaying substitutions L119S (alone or in combination with V71A or R173K) or S100G. Ten aph(3')-VI variants were detected among 35 isolates (46.9% of Acinetobacter spp.). Nineteen isolates carried variants of aac(3)-I, with aac(3)-Ia (n=13, mostly Acinetobacter spp.) being the most prevalent. Other AME genes detected were ant(3″)-Ia (n=41), ant(2″)-Ia (n=24), aac(3)-IIe (n=23), aac(3)-IId (n=21), aac(6')-Im (n=13, mostly P. aeruginosa), aacA8 (n=3), aac(3)-IIf (n=1) and aac(3)-IVa (n=1). Among 42 isolates resistant to amikacin, gentamicin and tobramycin tested for 16S rRNA methyltransferase genes, 21 (50.0%) tested positive; armA was most common (n=14), but 4 isolates carried rmtB1, 2 rmtF1 and 1 new variant rmtB4. Over 60 gene combinations, consisting of one to four AMEs and 16S rRNA methyltransferases, were observed. Cloning genes not previously characterised revealed diverse aminoglycoside resistance patterns for some AMEs, but expected results for rmtB4. Studies broadly evaluating these aminoglycoside resistance genes are needed. Using agents stable in the presence of these resistance genes might help overcome resistance.

Structure analysis of transposons carrying the aac(6′)-aph(2″) gene in Enterococcus faecalis isolated in Beijing, China, and comparison of their transfer efficiency

Transfer of aac(6')-aph(2″) transposons mediating high-level gentamicin resistance (HLGR) in Enterococcus faecalis is a serious problem in the clinic. However, factors affecting the transfer of aac(6')-aph(2″) have not yet been elucidated. The current study aimed to examine the genetic and molecular basis of HLGR in E. faecalis strains isolated in Beijing (China) and to clarify the relationship between transfer efficiency of aac(6')-aph(2″) transposons and the transposon structure/location. A total of five transposon structures were identified by PCR mapping of the corresponding transposon regions, including a Tn5281-like non-truncated transposon and four truncated transposons. A plasmid location study of aac(6')-aph(2″) by Southern blot following S1-PFGE and filter mating conjugation experiments demonstrated that plasmid location rates correlated with conjugation-positive rates. Chromosome walking to identify the sequence upstream of a representative type III truncated transposon found a truncated aph(2″)-Ia region, and further PCR analysis of this region among strains from different groups revealed similar a positive rate trend as the transposon plasmid location rate and conjugation-positive rate. In conclusion, aac(6')-aph(2″) transposons were of different structures in E. faecalis strains from Beijing, with two new transposon structures that have not been reported elsewhere. Presence of the truncated aph(2″)-Ia region upstream of some truncated transposons suggests recombination between aminoglycoside-modifying enzyme genes. Possible links exist among plasmid location, conjugation and the presence of truncated aph(2″)-Ia upstream of the transposon.

Molecular epidemiology of aminoglycoside resistance in clinical isolates of Klebsiella pneumoniae collected from Qazvin and Tehran provinces, Iran

Production of aminoglycoside modifying enzymes (AMEs) and 16S rRNA methylases are two main resistance mechanisms against these antibiotics. This study determined the frequency of AMEs and 16 s rRNA methylase genes among aminoglycoside non-susceptible K. pneumoniae isolates and evaluated their clonal relationship by enterobacterial repetitive intergenic consensus (ERIC)-PCR. A total of 177 K. pneumoniae isolates were collected from hospitals of Qazvin and Tehran, Iran. The identification of isolates was done by standard laboratory methods and API 20E strips. Aminoglycosides susceptibility was determined by Kirby-Bauer method and AMEs and 16S rRNA methylase encoding genes were studied by PCR and sequencing methods. Clonal relatedness of isolates was assessed by ERIC-PCR method. In total, 74% of isolates were non-susceptible to the aminoglycosides used in the study among those kanamycin 110 (62.1%), tobramycin 91 (51.4%), and gentamycin 87 (49.2%) showed the highest rates of resistance whereas netilmicin and amikacin revealed high susceptibility rates of 67.8% and 61.0%, respectively. Of 130 aminoglycoside non-susceptible isolates, 91.5% were positive for the presence of aac(6′)-Ib as the most dominant gene followed by aac(3)-II (78.5%), aph(3′)-IIIa (14.6%), ant(4′)-Ia (3.1%), and armA (7.7%) either alone or in combination. ERIC-PCR results showed 67.7% of non-susceptible isolates had different banding patterns followed by three distinct clones including A (16.2%), B (10.8%), and C (5.4%). Among those isolates carrying AMEs genes, 85 (68%) isolates belonged to independent groups and 21 (16.8%), 12 (9.6%), and 7 (5.6%) isolates belonged to groups A, B, and C, respectively, whereas 7 (70%) of 16S rRNA methylase-producing isolates belonged to independent groups. Our results revealed high prevalence of AMEs with the emergence of armA genes among the genetically unrelated resistant isolates of K. pneumonia in Iran, suggesting the need for more effective therapeutic strategies to reduce the selection pressure and better management of the patients infected with these resistant isolates.

Distribution of Aminoglycoside-Modifying Enzymes and Molecular Analysis of the Coagulase Gene in Clinical Isolates of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus.

Enzymatic alteration of aminoglycosides by aminoglycoside-modifying enzymes (AMEs) is the major mechanism of resistance to aminoglycosides. The purpose of this study was to determine the frequency of AME genes, staphylococcal chromosomal cassette mec (SCCmec) types, and molecular analysis of the coagulase (coa) gene in Staphylococcus aureus strains isolated from clinical specimens. Totally, 102 S. aureus were tested by disk diffusion and microbroth dilution methods for susceptibility to aminoglycosides. AMEs genes and SCCmec types were determined by multiplex polymerase chain reaction (PCR). For polymorphism analysis, the 3' end region of the coa gene was amplified by PCR and the products were then subjected to restriction digestion with HaeIII enzyme. Of the 102 S. aureus, 42 (41.2%) were methicillin-resistant S. aureus (MRSA). Thirty-five (83%) of MRSA strains were resistant to kanamycin, 32 (76.2%) to tobramycin, 30 (71.4%) to gentamicin, 25 (59.5%) to amikacin, and 10 (23.8%) to netilmicin. The aac(6')-Ie-aph(2″) was the most frequent gene among MRSA isolates 19 (45.2%), followed by aph(3')-IIIa 8 (19%), ant(4')-Ia 6 (14.3%), and aph(2″)-Id 2 (4.8%). SCCmec types included type I 10 (23.8%), II 1 (2.4%), III 21 (50%), and IV 7 (16.7%). Three (7.2%) isolates were nontypeable. Digestion of the PCR products of the coa gene yielded 19 distinct restriction fragment length polymorphism patterns. In conclusion, given the alarming rate of resistance to aminoglycosides among MRSA, the monitoring of aminoglycoside resistance and AME genes should be performed to limit the spread of aminoglycoside resistance among MRSA isolates. Several variants of the coa gene were found in the studied isolates, although the majority of the MRSA isolates belonged to a limited number of coagulase types.

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates from a Korean hospital that carry blaOXA-23

To elucidate the epidemicity of carbapenem-resistant Acinetobacter baumannii (CRAB), we investigated the antimicrobial susceptibility, the genetic basis of antimicrobial resistance, and the ability to form biofilms of 147 CRAB isolates collected between 2013 and 2015 from a Korean hospital based on sequence types (STs). Six different STs were identified: ST191 (n=47) and ST208 (n=36) were clones that had already been identified in the study hospital, whereas ST229 (n=28), ST369 (n=18), ST357 (n=17), and ST552 (n=1) were previously unknown. All the CRAB isolates exhibited an extensively drug-resistance. Twelve isolates, including ST191 and ST208, were resistant to tigecycline, and two were resistant to colistin. All the isolates carried ISAbaI-blaOXA-23 structures. The presence of the armA gene and/or a combination of aminoglycoside-modifying enzyme genes (aacC1, aadA1, aacA4, aphA1, and aphA6) was responsible for high-level resistance to aminoglycosides (minimal inhibitory concentrations≥256mg/L). All the ST229 isolates carried the blaPER-1 gene, whereas all the ST357 and ST369 isolates carried both aacA4 and aadA1. The ST229 isolates exhibited the greatest tendency to form biofilms, whereas the ST369 isolates exhibited significantly less tendency to form biofilms than other isolates. In conclusion, we discovered clonal diversity in the CRAB isolates from the study hospital. The resistant gene profiles and biofilm formation capabilities of the emerging CRAB STs differed from those of the circulating STs. The potential relationship between these genotypic and phenotypic traits and the epidemic capacity of CRAB STs requires further investigation.

Application of protein typing in molecular epidemiological investigation of nosocomial infection outbreak of aminoglycoside-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosan has emerged as an important pathogen elated to serious infections and nosocomial outbreaks worldwide. This study was conducted to understand the prevalence of aminoglycoside (AMG)-resistant P. aeruginosa in our hospital and to provide a scientific basis for control measures against nosocomial infections. Eighty-two strains of P. aeruginosa were isolated from clinical departments and divided into AMG-resistant strains and AMG-sensitive strains based on susceptibility test results. AMG-resistant strains were typed by drug resistance gene typing (DRGT) and protein typing. Five kinds of aminoglycoside-modifying enzyme (AME) genes were detected in the AMG-resistant group. AMG-resistant P. aeruginosa strains were classified into three types and six subtypes by DRGT. Four protein peaks, namely, 9900.02, 7600.04, 9101.25 and 10,372.87Da, were significantly and differentially expressed between the two groups. AMG-resistant P. aeruginosa strains were also categorised into three types and six subtypes at the distance level of 10 by protein typing. AMG-resistant P. aeruginosa was cloned spread in our hospital; the timely implementation of nosocomial infection prevention and control strategies were needed in preventing outbreaks and epidemic of AMG-resistant P. aeruginosa. SELDI-TOF MS technology can be used for bacterial typing, which provides a new method of clinical epidemiological survey and nosocomial infection control.

Insertional inactivation of oprD in carbapenem-resistant Pseudomonas aeruginosa strains isolated from burn patients in Tehran, Iran.

In this study, we report the insertion sequence ISPpu21 in the oprD porin gene of carbapenem-resistant Pseudomonas aeruginosa isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for P. aeruginosa isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of oprD and mex efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS–PAGE. The genetic relationship between the P. aeruginosa isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of P. aeruginosa harboured the ISPpu21 insertion element in the oprD gene. The extended-spectrum β-lactamase-encoding gene in ISPpu21-carrying isolates was blaTEM. PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The rmtA, aadA, aadB and armA genes were positive in all ISPpu21 harbouring isolates. The relative expression levels of the mexX, mexB, mexT and mexD genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the oprD in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain P. aeruginosa PAO1. Evaluation of the outer membrane protein by SDS–PAGE suggested that oprD was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing ISPpu21 were shown to be clonally related. The present study describes a novel molecular mechanism, ISPpu21 insertion of the oprD gene, associated with carbapenem resistance in clinical P. aeruginosa isolates.

Frequency of 16S rRNA Methylase and Aminoglycoside-Modifying Enzyme Genes among Clinical Isolates of Acinetobacter baumannii in Iran

Multidrug-resistantAcinetobacter baumannii(MDR-AB) is an important nosocomial pathogen which is associated with significant morbidity and mortality, particularly in high-risk populations. Aminoglycoside-modifying enzymes (AMEs) and 16S ribosomalRNA (16S rRNA) methylation are two important mechanisms of resistance to aminoglycosides. The aim of this study was to determine the prevalence of 16S rRNA methylase (armA, rmtA, rmtB, rmtC, and rmtD), and the AME genes [aac(6')-Ib, aac(3)-I, ant(3'')-I, aph(3')-I and aac(6')-Id], among clinical isolates of A. baumannii in Tehran, Iran. Between November 2015 to July 2016, a total of 110 clinical strains of A. baumannii were isolated from patients in two teaching hospitals in Tehran, Iran. Antimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute guidelines. The presence of genes encoding the AMEs and 16S rRNA methylases responsible for resistance was investigated by multiplex polymerase chain reaction. The results showed that colistin was an effective antibiotic and could be used as a last-resort treatment of infections caused by MDR-AB. The resistance rate to aminoglycosides were 100%, 96.36% and 90.9% for tobramycin, gentamicin and amikacin, respectively. In this study, AME genes of aac(6')-Ib, aac(3)-I and ant(3'')-I were most prevalent among the isolated strains. Markedly high resistance to tobramycin, gentamicin and amikacin was noted in current study. Our results suggested that modifying enzyme genes in conjunction with methylation of 16S rRNA might contribute to aminoglycoside resistance induced in vivo in A. baumannii. Further studies are required to determine the prevalence of the aminoglycoside resistance genes in other hospitals of Iran.