High-level Gentamicin-resistant Isolates Research Articles

High-level gentamicin resistance mediated by Aac(6')-Ie-aph(2")-Ia gene in Enterococcus species isolated from clinical samples in Northern India.

CONTEXT:Enterococci are known to cause life-threatening infections which are difficult to treat as the organism harbors innate resistance to many antibiotics and can amass resistance toward many others through plasmid-mediated genetic exchange.AIMS:The study evaluates the drug susceptibility profile of various Enterococcus species isolated from various patient specimens submitted for bacteriological analysis and check the incidence of aac(6′) Ie-aph(2”) Ia gene.SETTING AND DESIGN:This in vitro cross-sectional study was executed at bacteriology laboratory of a 470 bedded hospital in New Delhi.MATERIALS AND METHODS:Drug susceptibility testing was carried out on enterococcal isolates. High-level gentamicin-resistant (HLGR) isolates detected by micro broth dilution assay were then subjected to molecular detection of aac(6′) Ie-aph(2”) Ia gene.STATISTICAL ANALYSIS USED:The level of significance was established by Chi-square test.RESULTS:Among the 182 enterococcal stains detected, 76.9% were Enterococcus faecalis and 20.3% were Enterococcus faecium. 12.08% strains were vancomycin resistant. 39% expressed resistance toward high-level gentamicin (HLG) and this finding was significantly higher in E. faecium than E. faecalis. HLGR strains expressed a higher degree of resistance to other drugs in contrast to non-HLGR isolates. In 67 out of 71 HLGR isolates the bifunctional gene was detected.CONCLUSION:Considerable presence of HLG and vancomycin resistance in the clinical isolates is alarming and should be taken seriously. The study shows high dissemination of aac(6′)-Ie-aph(2”)-Ia gene among Enterococci isolated from the region.

High-Level Resistance to Aminoglycosides Among Multidrug Resistant Non-faecalis and Non-faecium Enterococci.

Enterococci are considered as important causative pathogens of a variety of community and hospital-acquired infections. Due to the development of multidrug resistant (MDR) enterococci and the emergence of strains possessing high-level resistance to antimicrobial agents, treatment of their infections has been more complicated. In addition to more prevalent species of the Enterococcus genus, non-faecalis/non-faecium species are also responsible for severe healthcare-associated infections. Therefore, this study was designed to investigate high-level gentamicin resistance among the clinical isolates of non-faecalis and non-faecium enterococci in Shiraz, in the southwest of Iran. A total of 28 non-faecalis/non-faecium spp. were isolated from various infections. They were identified by the conventional methods. Antimicrobial resistance patterns, multidrug resistance, and high-level gentamicin resistance were determined, according to CLSI guidelines and related definitions. Detection of aminoglycoside resistance genes was also performed using standard procedures. All of the isolates were MDR (100%), and 75% of them were high-level gentamicin resistant (HLGR) (MIC ≥ 500 μg/mL). The distributions of aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa resistance genes were 82.1% and 75%, respectively. The aph(2")-Ib, aph(2")-Ic, aph(2")-Id, and ant(4')-Ia genes were not found in any isolate. Although vancomycin resistance was observed in 19 (67.8%) isolates, all of the isolates were susceptible to linezolid and fosfomycin. Our data indicate a high rate of MDR non-faecalis/non-faecium isolates. Furthermore, high-level gentamicin resistance was notable and all of the HLGR isolates harbored at least one of aac(6')-Ie-aph(2'')-Ia or aph(3')-IIIa resistance gene.

Antimicrobial resistance monitoring of commensal Enterococcus faecalis in broiler breeders

Enterococcus faecalis (E. faecalis) has rapidly acquired resistance to multiple antimicrobials, and the antimicrobial resistance of E. faecalis from broiler breeders has been implicated in its vertical transmission to their offspring. The objective of this study was to investigate the antimicrobial resistance and genetic diversity of commensal E. faecalis isolated from the broiler breeder farms. Among a total of 229 E. faecalis isolates from 9 broiler breeder farms, the highest resistance rate was observed in tetracycline (78.2%), followed by doxycycline (58.1%) and erythromycin (43.7%), and the prevalence of antimicrobial resistance showed significant differences among the 9 broiler breeder farms (P < 0.05). The tetM gene (77.1%) and ermB gene (85.0%) were detected at the highest levels in 179 TE-and 100 E-resistant isolates, respectively. Twenty-four high-level gentamicin-resistant isolates carried aac(6″)Ie-aph(2″)-la gene, and 9 high-level ciprofloxacin-resistant isolates showed point mutations in both gyrA and parC genes. All high-level gentamicin-resistant or high-level ciprofloxacin-resistant isolates showed one of the two different virulence gene patterns, ace-asa1-efaA-gelE complex or ace-efaA-gelE complex. These results indicate that constant epidemiological monitoring at the breeder level is required to prevent the pyramidal transmission of antimicrobial-resistant E. faecalis.

Clonal dissemination of high-level gentamicin-resistant isolates of Enterococcus faecalis within auniversity hospital in southeastern Iran.

Combination of acell wall-active antibiotic with an aminoglycoside confers a synergistic effect in the treatment of some severe enterococcal infections. Unfortunately, with the emergence of enterococci with high-level resistance to aminoglycosides, particularly to gentamicin, the efficacy of the synergistic combinations has decreased. In this study, high-level gentamicin-resistant (HLGR) isolates of enterococci and the diversity of the genes encoding aminoglycoside-modifying enzymes (AMEs) as well as putative clonal dissemination of HLGR isolates were investigated in auniversity hospital in southeastern Iran. The minimum inhibitory concentration of gentamicin was determined and HLGR isolates were investigated for AME genes. Genetic similarity between isolates was analyzed using repetitive extragenic palindromic (rep)-Polymerase Chain Reaction (PCR) assay. Of 150 Enterococcus isolates, 62isolates including Enterococcus faecalis (n = 46) and E.faecium (n = 16) were identified as HLGR. The most prevalent AME genes in both species were as follows: aph(3')-IIIa (n = 44), aac(6')-Ie-aph(2')-Ia (n = 36), and ant(4')-Ia (n = 15). The rep-PCR analysis showed clonality among E.faecalis isolates, so that 27isolates were grouped in seven clusters representing similarity greater than 95%. No link between AME determinants and clusters was found. Clonal spread of HLGR isolates of E.faecalis was found within our hospital. More rigorous recommendations are required to avoid dissemination of such resistant microorganisms in the hospital setting.

High-level aminoglycoside resistance and distribution of aminoglycoside resistance genes among Enterococcus spp. clinical isolates in Tehran, Iran.

Enterococci have gained attention during the past decade as important nosocomial pathogens. Their increasing prevalence has been paralleled by the occurrence of multidrug-resistant and high-level aminoglycoside-resistant strains. This study isolated Enterococcus spp. from hospital samples and determined their antibiotic resistance profile, focusing on aminoglycosides, and associated resistance mechanisms. A total of 195 enterococci from hospital samples in Tehran were studied. Isolates were identified by biochemical reactions. Antimicrobial resistance was determined by disk diffusion. The vancomycin MIC for vancomycin-resistant isolates was determined by agar dilution. Detection of aminoglycoside resistance genes and intI1 and intI2 gene was performed by PCR. The majority of isolates were Enterococcus faecalis (65.1%), followed by Enterococcus faecium (31.8%), Enterococcus gallinarum (2.6%) and Enterococcus solitarius (0.5%). According to antibiogram results, 42.1% of isolates were high-level gentamicin-resistant (HLGR) and 40.5% were high-level streptomycin-resistant (HLSR). There was a high prevalence of aac(6')-Ie-aph(2")-Ia (96.3%) among HLGR isolates. ant(6)-Ia and aadA were identified in 93.7% and 64.6% of HLSR isolates, respectively. aph(2'')-Ic was detected in 7 isolates (3.6%) and aph(2'')-Ib in only 4 isolates (2.1%); no isolates harboured aph(2'')-Id, intI1 or intI2. Multidrug resistance was higher among HLGR and HLSR isolates compared with non-HLGR and non-HLSR isolates, which may result in limited treatment options. More than 50% of isolates were susceptible to aminoglycosides, thus correct identification in clinical laboratories and administration of these antibiotics can result in decreased used of antibiotics such as vancomycin and linezolid and help to reduce the emergence of resistance to these drugs.

Faecal carriage of high-level aminoglycoside-resistant and ampicillin-resistant Enterococcus species in healthy Iranian children.

High-level aminoglycoside, ampicillin and vancomycin resistance and virulence genes among enterococcal isolates collected from healthy middle-school children in Ardabil, Iran, during 2016 were investigated. Totally, 305 faecal specimens were collected. Isolates underwent antimicrobial susceptibility testing, virulence gene detection and molecular typing. Totally, 409 enterococcal isolates were collected, comprising Enterococcus faecium (235; 57.5%), Enterococcus faecalis (56; 13.7%) and other Enterococcus spp. (118; 28.9%). Overall, 71 (17.4%), 11 (2.7%) and 10 (2.4%) isolates were identified as high-level streptomycin-resistant (HLSR), high-level gentamicin-resistant (HLGR) and ampicillin-resistant (AR), respectively. Among HLSR isolates, 40 (56.3%), 5 (7.0%) and 26 (36.6%) were E. faecium, E. faecalis and other Enterococcus spp., respectively. Among HLGR isolates 4 (36.4%) and 7 (63.6%) and among AR isolates 7 (70.0%) and 3 (30.0%) were E. faecium and other Enterococcus spp., respectively. Accordingly, 21.6%, 3.6% and 3.3% of subjects were colonised with HLSR, HLGR and AR Enterococcus spp. Carriage of HLGR, HLSR and AR isolates was associated with prior antibiotic consumption (P≤0.05). Additionally, male sex and antacid consumption were associated with AR enterococcal carriage. Moreover, 69 (97.2%), 10 (90.9%) and 9 (90.0%) of HLSR, HLGR and AR isolates were multidrug-resistant, respectively. No vancomycin-resistant enterococci were detected. ERIC-PCR revealed high genetic diversity among isolates. gelE and asa1 were major virulence genes both in E. faecalis and E. faecium. Presence of gelE was associated with HLSR and HLGR phenotypes (P≤0.05). Community intestinal carriage of HLSR enterococci was high; however, carriage of HLGR and AR enterococci was low.

High prevalence of vancomycin and high-level gentamicin resistance in Enterococcus faecalis isolates.

Multiple drug-resistant enterococci are major cause of healthcare-associated infections due to their antibiotic resistance traits. Among them, Enterococcus faecalis is an important opportunistic pathogen causing various hospital-acquired infections. A total of 53 E. faecalis isolates were obtained from various infections. They were identified by phenotypic and genotypic methods. Determination of antimicrobial resistance patterns was done according to CLSI guidelines. The isolates that were non-susceptible to at least one agent in ≥3 antimicrobial categories were defined as multidrug-resistant (MDR). Detection of antimicrobial resistance genes was performed using standard procedures. According to MDR definition, all of the isolates were MDR (100%). High-level gentamicin resistance was observed among 50.9% of them (MIC ≥ 500 μg/ml). The distributions of aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa genes were 47.2% and 69.8%, respectively. The aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id, and ant(4')-Ia genes were not detected. Vancomycin resistance was found in 45.3% of strains. The vanA gene was detected in 37.7% of isolates, whereas vanB and vanC1 genes were not observed in any strain. Erythromycin resistance rate was 79.2% and the frequencies of ermB and ermC genes were 88.6% and 69.8%, respectively. The ermA and msrA genes were not present in any of the isolates. Our data indicate a high rate of MDR E. faecalis strains. All of high-level gentamicin-resistant isolates carried at least one of aac(6')-Ie-aph(2'')-Ia or aph(3')-IIIa genes. Distribution of vanA was notable among the isolates. In addition, ermB and ermC were accountable for resistance to erythromycin.

Species distribution, antibiotic resistance and virulence traits in canine and feline enterococci in Tunisia.

In order to investigate the possible role of dogs and cats in the carriage and potential dissemination of resistant enterococci, seventy faecal samples from dogs and cats were tested for enterococci. Fifty-eight enterococci were recovered. Isolates were identified as Enterococcus faecium (n = 31) and E. faecalis (n = 14) E. durans (n = 6), E. casseliflavus (n = 2), E. hirae and E. gallinarum (2 isolates each). Enterococcal isolates showed resistance to ciprofloxacin (n = 35), erythromycin (n = 31), tetracycline (n = 25), kanamycin (n = 15), streptomycin (n = 13), pristinamycin (n = 11), gentamicin (n = 10), chloramphenicol (n = 8), and linezolid (n = 6). The gene erm(B) was detected in 22 out of 31 erythromycin-resistant enterococci. All tetracycline-resistant enterococci carried tet(M) and/or tet(L) genes. The gene aac(6')-Ie-aph(2″)-Ia was identified in five of high-level gentamicin-resistant isolates, the genes aph(3')-IIIa and/or aac(6')-Ie-aph(2″)-Ia in eleven high-level kanamycin-resistant isolates and the gene ant(6)-Ia in eleven high-level streptomycin-resistant isolates. Only one strain harboured cat(A) gene, and five strains contained vat(E) or vat(D) genes. Virulence genes gel(E) (21 strains), esp (11 strains) and cylA/cylB (5 strains) were detected. High genetic diversity was demonstrated among E. faecium isolates by pulsed-field gel electrophoresis (PFGE). Dogs and cats can be carriers of antibiotic-resistant enterococci in their faeces that could shed into the household environment.

Analysis of Aminoglycoside Modifying Enzyme Genes Responsible for High-Level Aminoglycoside Resistance among Enterococcal Isolates.

Enzymatic modification results in high-level resistance to aminoglycoside (HLAR), which eliminates the synergistic bactericidal effect of combined exposure to a cell wall-active agent and an aminoglycoside. So aim of the study was to determine prevalence of HLAR enterococcal isolate and to study distribution of aminoglycoside modifying enzyme genes in them. A total of 100 nonrepeat isolates of enterococci from various clinical samples were analyzed. As per Clinical and Laboratory Standards Institute guidelines enterococci were screened for HLAR by Kirby-Bauer disc diffusion method. Minimum inhibitory concentration of all isolates for gentamicin and streptomycin was determined by E-test. Multiplex polymerase chain reaction (PCR) was carried out for HLAR enterococcal isolates to identify aminoglycoside modifying enzymes genes responsible for resistance. 60% isolates were found to be high-level gentamicin resistant (HLGR) whereas 45% isolates were found to be high-level streptomycin resistant (HLSR). By multiplex PCR 80% HLGR isolates carried bifunctional aminoglycoside modifying enzyme gene aac(6′)-Ie-aph(2′′)-Ia whereas 18 out of 45 high-level streptomycin resistant, that is, 40%, isolates carried aph(3′)-IIIa. However, aph(2′′)-Ib, aph(2′′)-Ic, aph(2′′)-Id, and ant(4′)-Ia genes which encode other aminoglycosides modifying enzymes were not detected. Bifunctional aminoglycoside modifying enzyme gene aac(6′)-Ie-aph(2′′)-Ia is the predominant gene responsible for HLAR.

Increasing Prevalence of Aminoglycoside-Resistant Enterococcus faecalis Isolates Due to the aac(6')-aph(2") Gene: A Therapeutic Problem in Kermanshah, Iran.

Background:Enterococci are important pathogens in nosocomial infections. Various types of antibiotics, such as aminoglycosides, are used for treatment of these infections. Enterococci can acquire resistant traits, which can lead to therapeutic problems with aminoglycosides.Objectives:This study was designed to identify the prevalence of, and to compare, the aac(6’)-aph(2”) and aph(3)-IIIa genes and their antimicrobial resistance patterns among Enterococcus faecalis and E. faecium isolates from patients at Imam Reza hospital in Kermanshah in 2011 - 2012.Patients and Methods:One hundred thirty-eight clinical specimens collected from different wards of Imam Reza hospital were identified to the species level by biochemical tests. Antimicrobial susceptibility tests against kanamycin, teicoplanin, streptomycin, imipenem, ciprofloxacin, and ampicillin were performed by the disk diffusion method. The minimum inhibitory concentrations of gentamicin, streptomycin, kanamycin, and amikacin were evaluated with the microbroth dilution method. The aminoglycoside resistance genes aac(6’)-aph(2”) and aph(3”)-IIIa were analyzed with multiplex PCR.Results:The prevalence of isolates was 33 (24.1%) for E. faecium and 63 (46%) for E. faecalis. Eighty-nine percent of the isolates were high-level gentamicin resistant (HLGR), and 32.8% of E. faecium isolates and 67.2% of E. faecalis isolates carried aac(6’)-aph(2”). The prevalence of aph(3”)-IIIa among the E. faecalis and E. faecium isolates was 22.7% and 77.3%, respectively.Conclusions:Remarkably increased incidence of aac(6’)-aph(2”) among HLGR isolates explains the relationship between this gene and the high level of resistance to aminoglycosides. As the resistant gene among enterococci can be transferred, the use of new-generation antibiotics is necessary.

Emergence of antimicrobial resistance and virulence factors among the unusual species of enterococci, from North India.

Several enterococcal species are increasingly being reported from clinical infections, besides the major species. This study was undertaken to determine the prevalence of unusual enterococcal species and their antimicrobial susceptibility patterns, virulence factors, and molecular characterization. The study was conducted in Department of Microbiology and associated Tertiary Care University Hospital in North India. Enterococcal isolates were collected for a period of 2 years from clinical specimens. Identification and elaborate phenotypic characterization was done biochemically. All the isolates were tested by Kirby-Bauer disc diffusion method and breakpoint minimum inhibitory concentration for susceptibility against standard antibiotics. Screening for vancomycin-resistant enterococci (VRE), high-level aminoglycoside resistance was done on brain heart infusion agar incorporated with 6 μg/ml vancomycin, 500 μg/ml gentamicin, and 2000 μg/ml streptomycin, respectively. VRE isolates were tested for the presence of vanA, vanB, and vanC genes and high-level gentamicin resistant (HLGR) isolates for aac-6'- aph-2' gene by polymerase chain reaction (PCR). Hemolysin and gelatinase production, hemagglutination and biofilm formation were detected along with asa1, gelE, esp, hyl, and cylA genes by multiplex PCR. Of 403 enterococci, 93 (23.07%) isolates were identified as unusual species and atypical variants. Resistance of 52.68%, 46.23%, 44.08%, and 6.45% for ampicillin, ciprofloxacin, high strength gentamicin, and vancomycin, respectively were noted. Presence of vanC gene in Enterococcus gallinarum and Enterococcus casseliflavus isolates and vanA gene in Enterococcus durans and Enterococcus hirae and aac-6'- aph-2'' gene was found in 33.14% (14/41) of the HLGR isolates. The most frequent virulence factor was biofilm production. Only a few isolates harbored asa1 (2), gelE (9), and hyl (3) genes. Considerable prevalence of pathogenic unusual species of enterococci was seen along with their emerging drug resistance and virulence. Complete identification and routine speciation is essential to limit their emergence as major species in near future.

Genotypes of aminoglycoside-modifying enzyme and clinical study of high-level gentamycin resistant enterococcus

To determine the antibiotics resistance, aminoglycoside-modifying enzymes and homology of high-level gentamycin resistant enterococcus in clinical specimens. The high-level gentamicin resistant (HLGR) isolates were screened by the agar method and the resistance of 14 antimicrobial agents was determined by K-B method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates. The ratio of HLGR was 64.2% (68/106). Among the HLGR,there were no isolates resistant to linezolid, vancomycin and tecoplanin, and Enterococcus faecium was more resistant to beta-lactam antibiotics and quinolone than Enterococcus faecalis. The positive rate of aac(6')-Ie-aph(2')-Ia was 92.6% and 3 isolates had the resistance gene mostly similar to aph(2')-Id. And among 51 HLGR isolates from the hospitalized patients, PFGE grouped 17 E. faecalis isolates into 4 clusters (A-D), and 33 E. faecium isolates into 8 clusters (A-H) with A cluster as predominant. HLGR has become the important antibiotic resistance bacteria which results in nosocomial infection; and aac(6')-Ie-aph(2')-Ia is the main aminoglycoside-modifying enzyme gene which causes HLGR.

Genotypic diversity and epidemiology of high-level gentamicin resistant Enterococcus in a Chinese hospital

To investigate the antibiotics resistance of Enterococcus, the aminoglycoside-modifying enzymes (AME) and homology of high-level gentamicin resistant (HLGR) Enterococcus in clinical specimens for the implementation of effective infection control measures. The resistance of 13 antimicrobial agents was determined by Kirby-Bauer (K-B) or agar dilution method. And the HLGR and high-level streptomycin resistant (HLSR) isolates were screened by agar screen. Production of beta-lactamases was tested by the nitrocefin disc method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates from in-patients. No isolates resistant to linezolid, vancomycin and teicoplanin were found. Ampicillin-resistant isolates did not produce beta-lactamases and 68 HLGR isolates were screened at the rate of 64.2%. The positive rate of aac(6')-Ie-aph(2'')-Ia was 86.8% and 3 isolates had the new AME gene designated aph(2'')-Ie mostly similar to aph(2'')-Id. Among 51 HLGR isolates from in-patients, PFGE grouped 17 Enterococcus faecalis (E. faecalis) isolates into 4 clusters (A-D), and 33 Enterococcus faecium (E. faecium) isolates into 8 clusters (A-H), of which the A cluster is the main. HLGR has become the important antibiotic resistance pathogen causing nocosomial infection. And the aac(6')-Ie-aph(2'')-Ia gene was the main aminoglycoside-modifying enzyme gene leading to HLGR.

High-level gentamicin-resistant isolates of oral streptococci and Aerococcus from blood specimens.

Seventy-nine isolates of oral streptococci and Aerococcus obtained from blood specimens between December 1997 and January 2000 were tested for high-level gentamicin resistance. Three isolates with minimum inhibitory concentrations (MICs) of > or =1024 microg/ml were selected as potential high-level gentamicin-resistant isolates. The DNA sequence of the structural gene encoding 6'-acetyltransferase-2"-phosphotransferase in the three high-level gentamicin-resistant isolates was investigated according to the method used for Enterococcus faecalis. The results of this gene analysis showed that all the DNA sequence of Aerococcus viridans no. 70, which had the highest MIC (2048 microg/ml), matched that of E. faecalis, whereas no polymerase chain reaction (PCR) products were obtained in the other two isolates that were tested. The mechanism of gentamicin resistance in these two isolates is unknown.