rRNA Methylase Genes Research Articles

Prevalence and molecular characteristics of 16s rRNA methylase gene rmtB in amikacin resistant Escherichia coli isolated from South Korea

Prevalence and molecular characteristics of 16s rRNA methylase gene rmtB in amikacin resistant Escherichia coli isolated from South Korea

Association of blaNDM-1 with blaKPC-2 and aminoglycoside-modifying enzyme genes among Klebsiella pneumoniae, Proteus mirabilis and Serratia marcescens clinical isolates in Brazil

ObjectivesCarbapenemase-producing Enterobacterales are frequently involved in healthcare-associated infections worldwide. The objectives of this study were to investigate (i) the frequency of the main genes encoding carbapenemases, 16S rRNA methylases and aminoglycoside-modifying enzymes (AMEs) as well as the mcr gene and (ii) the clonal relationship of enterobacteria isolates resistant to carbapenems and aminoglycosides from colonisation and infection in patients from hospitals in northeastern Brazil. MethodsAntimicrobial susceptibility was determined using an automated VITEK®2 system. Presence of carbapenemase, AME and 16S rRNA methylase genes as well as the mcr gene was determined by PCR and amplicon sequencing. Genetic variability was determined by ERIC-PCR. ResultsA total of 35 isolates resistant to carbapenems and aminoglycosides were selected for this study. Klebsiella pneumoniae was most common (45.7%), followed by Proteus mirabilis (28.6%) and Serratia marcescens (25.7%). AME genes were found in 97.1% of isolates, most commonly aph(3ʹ)-VI and aac(6')-Ib. The blaNDM-1 and blaKPC-2 genes were detected in 25.7% and 88.6% of isolates, respectively; five isolates harboured these genes concomitantly. According to the literature, this is the first report of the association of blaNDM-1 and blaKPC-2 in P. mirabilis and S. marcescens in Brazil. The isolates showed a multiclonal profile by ERIC-PCR. ConclusionThe emergence of blaNDM-1 associated with blaKPC-2 and AME genes in K. pneumoniae, P. mirabilis and S. marcescens isolates with a multiclonal profile is of concern as this limits therapeutic options. These results should alert medical authorities to establish rigorous detection methods to reduce the spread of these antimicrobial resistance genes.

Dissemination of International Clone II Acinetobacter baumannii Strains Coproducing OXA-23 Carbapenemase and 16S rRNA Methylase ArmA in Athens, Greece.

The aim of this study was to study the molecular epidemiology of 16S rRNA-methylase (RMT)-producing clinical Acinetobacter baumannii isolates from hospitals in Athens, Greece. Single-patient A. baumannii clinical isolates, coresistant to amikacin and gentamicin (n = 347), from five tertiary care hospitals, were submitted to minimum inhibitory concentration determination and molecular testing for carbapenemase and RMT genes. A. baumannii, resistant to amikacin and gentamicin, was isolated at participating institutions at a mean rate of 67.8%. Among them 93.7% harbored the armA. The vast majority (98.5%) of armA positive isolates were OXA-23 producers, assigned mainly (99.4%) to sequence group G1, corresponding to international clone (IC) II. Four isolates (all from the same hospital) were OXA-24 producers (1.2%), assigned to G6 corresponding to CC78 and only one isolate was OXA-58-producer, assigned to G2 (IC I). Apramycin was the most active agent inhibiting 99.7% of the isolates at ≤64 mg/L, whereas colistin, trimethoprim/sulfamethoxazole, minocycline, and tigecycline exhibited only sparse activity (S, <18%). RMT production is an emerging mechanism of resistance, capable of compromising the clinical efficacy of aminoglycosides. High prevalence of armA was observed among A. baumannii strains isolated in participating hospitals in Athens, which were mainly OXA-23 producers and belonged to IC II. Apramycin is a structurally unique aminoglycoside, currently used as a veterinary agent. Although it has not been evaluated for clinical use, apramycin appears worthy of further investigation for repurposing as a human therapeutic against difficult-to-treat pathogens.

High prevalence of carbapenemase, AmpC β-lactamase and aminoglycoside resistance genes in extended-spectrum β-lactamase-positive uropathogens from Northern India.

This study investigated the occurrence of extended-spectrum β-lactamase (ESBL) genes coexisting with carbapenemase, AmpC and aminoglycoside resistance gene in uropathogens in India. Antimicrobial susceptibility testing was performed by disk diffusion. Antimicrobial resistance genes were detected by multiplex PCR. Of 1516 consecutive urine samples, 454 (29.9%) showed significant bacteriuria with a single micro-organism, predominantly Escherichia coli (n=343), followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the uropathogens, 61 ESBL-producers were identified containing blaCTX-M-15 (n=32), blaCTX-M-15+blaOXA-2 (n=15), blaCTX-M-15+blaOXA-2+blaTEM-1 (n=6), blaOXA-2 (n=5), blaOXA-2+blaSHV-76 (n=1), blaTEM-1+blaSHV-76 (n=1) and blaTEM-1 (n=1). All ESBL genes were located on horizontally transferable plasmids of incompatibility types HI1, I1, FIA+FIB, FIA and Y. Among the 61 ESBL-producers, 59 harboured carbapenemase genes, including blaNDM-5 (n=48), blaNDM-5+blaOXA-48 (n=5), blaNDM-5+blaIMP (n=5) and blaNDM-5+blaIMP+blaVIM (n=1). ESBL-producing uropathogens also harboured 16S rRNA methylase genes, including rmtB (n=9), rmtA (n=4), rmtC (n=1) and armA (n=1). ESBL-positive isolates also contained AmpC genes, including blaCIT (n=8) and blaDHA-1 (n=1). Imipenem and gentamicin had the lowest resistance rates against the uropathogens. This is the first report showing the high prevalence of carbapenemases in ESBL-positive isolates in this area. Regular surveillance for such resistance mechanisms will be useful for health personnel to treat infections by these multidrug-resistant pathogens.

BlaIMP-45 and armA co-carrying multidrug resistance Pseudomonas isolates and characterization of their genetic environment

Objective The primary goal of this work is to discuss the molecular mechanism of multi-drug resistant Pseudomonas spp. resistance to carbapenam and aminoglycoside antibiotics. Methods From June 2012 to March 2013, six strains of P. aeruginosa and P. putida producing carbapenemasefrom 4 different district were collected. Species identification was performed using VITEK-2 compact system and by sequencing of 16S rDNA amplicons. Minimum inhibitory concentrations were determined by E-test method. Production of carbapenemase were detected by Carba NP method. Carbapenemase genes and aminoglycoside 16s rRNAmethylase genes were screened by PCR, and their subtypes combined with their immediate genetic context were worked out by assemble the sequence of overlapped PCR amplicons. SpeI-PFGE (Pulse field gel electrophoresis after SpeI enzyme digestion) were conducted to evaluate their clonal relatedness. S1-PFGE (Pulse field gel electrophoresis after S1 enzyme digestion) were conducted to conform the relatedness of plasmids they carried. Results Three multidrug resistant P. aeruginosa and three P. putida, were all positive for Carba NP test and conformed producing class B carbapenemase. PCR screening followed by sequencing confirmed carriage of blaIMP-45 and armA, which confer resistance to β-lactams (except aztreonam) and aminoglycosides. These two genes co-located in a Tn1548-associatedregion. SpeI-PFGE disclosed that these isolates were not clonal closely related to each other, except two P. aeruginosa isolates were clonal related. S1-PFGE results showed that these isolates all carried plasmids of large size (300-600 kb). Conclusions This study showed that Pseudomonas spp. isolates co-carried blaIMP-45 and armA were disseminated in clinical settings. Spread of these genes may attribute to horizontal gene transfer of related entities. Key words: Pseudomonas; Drug resistance, multiple, bacterial; Methyltransferases; Escherichia coli proteins; beta-Lactams

High-Level Resistance to Aminoglycosides due to 16S rRNA Methylation in Enterobacteriaceae Isolates.

Introduction: High-level aminoglycoside resistance due to methylase genes has been reported in several countries. The purpose of this study was to investigate the diversity of the genes encoding 16S rRNA methylase and their association with resistance phenotype in Enterobacteriacae isolates. Materials and Methods: Based on sampling size formula, from February to August 2014, a total of 307 clinical Enterobacteriaceae isolates were collected from five hospitals in northwest Iran. The disk diffusion method for amikacin, gentamicin, tobramycin, kanamycin, and streptomycin, as well as the minimum inhibitory concentration (MIC) for aminoglycosides (except streptomycin), was used. Six 16S rRNA methylase genes (armA, npmA, and rmtA-D) were screened by PCR and sequencing assays. Results: In this study, 220 (71.7%) of 307 isolates were aminoglycoside resistant and 40 isolates (18.2%, 40/220) were positive for methylase genes. The frequency of armA, rmtC, npmA, rmtB, and rmtA genes was 9.5%, 4.5%, 3.6%, 2.3%, and 1%, respectively. The rmtD gene was not detected in the tested bacteria. Sixty percent of positive methylase gene isolates displayed high-level resistance (MIC ≥512 μg/mL to amikacin and kanamycin; and MIC ≥128 μg/mL to gentamicin and tobramycin). Conclusions: The prevalence of resistance to aminoglycoside in Iran is high. Furthermore, there is a statistically significant association between amikacin and kanamycin resistance with the presence of rmtC and rmtB genes.

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.

High distribution of 16S rRNA methylase genes rmtB and armA among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran.

The emergence of 16S rRNA methylase genes encoded on plasmids confers high-level aminoglycoside resistance (HLAR). This study aimed to investigate the prevalence of 16S rRNA methylases among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran. A total of 68 E. cloacae clinical strains were collected between November 2017 and September 2018. The MICs of aminoglycosides were assessed using the agar dilution method. The presence of 16S rRNA methylase genes, including armA, rmtA to rmtH, and nmpA was evaluated by PCR. The transferability of 16S rRNA methylase-harboring plasmids was evaluated by conjugation assay. The genetic diversity of all isolates was evaluated by ERIC-PCR. The armA and rmtB genes were the only 16S rRNA methylase genes detected in this study (29 out of 68 isolates; 42.64%). The transferability by conjugation was observed in 23 rmtB or/and armA positive donors. HLAR phenotype was in 33 of 68 strains. Ten clonal types were obtained by ERIC-PCR and significant associations (p < 0.05) were between the clone types and aminoglycoside susceptibility, as well as with profile of the 16S rRNA methylase genes. In conclusion, both horizontal transfer and clonal spread are responsible for dissemination of the rmtB and armA genes among E. cloacae strains.

First report of New Delhi metallo-β-lactamase-6 (NDM-6) among Klebsiella pneumoniae ST147 strains isolated from dialysis patients in Iran

There has been an alarming health-related concern about the growth of New Delhi metallo-β-lactamase. The aims of this study include the phenotypic detection of β-lactamases and molecular characterization of NDM in Klebsiella pneumoniae isolates in Tehran, Iran. A total of 120 K. pneumoniae isolates were collected from hospitalized haemodialysis patients, Tehran, Iran from March 2014 to February 2017. Antibiotic susceptibility tests were conducted using Kirby-Bauer disc diffusion and Broth Microdilution methods according to Clinical and Laboratory Standards Institute guidelines. Metallo-β-lactamase was detected using the Combined Disc Diffusion Test (CDDT), and production of carbapenemase was screened using the Modified Hodge Test. NDM-producing K. pneumoniae strains were screened for the presence of mcr-1 gene, β-lactamase genes, and 16S rRNA methylase genes by Polymerase Chain Reaction and sequencing. Molecular typing of the strains was determined using Repetitive Sequence Based-PCR and Multilocus Sequence Typing. The blaNDM-6 gene was detected in 3 (2.5%) out of 120 isolates from dialysis patients. Also, the three isolates were positive for blaCTX-M-15,blaTEM extended-spectrum β-lactamase genes, armA type plasmid-mediated 16S rRNA methylase and CMY-type plasmid-mediated AmpC β-lactamase.The isolates were identified as MLST sequence type 147 (ST147). This is the first report of blaNDM-6 in K. pneumoniae strains, isolated in Iran.

Endogenous endophthalmitis caused by a multidrug-resistant hypervirulent Klebsiella pneumoniae strain belonging to a novel single locus variant of ST23: first case report in China

BackgroundEndogenous endophthalmitis caused by hypervirulent Klebsiella pneumoniae (HVKP) is an emerging infectious disease commonly with a devastating visual outcome. Most HVKP strains display a wild-type susceptibility profile to antibiotics. However, reports of antimicrobial-resistant HVKP have increased over time, which poses a serious therapeutic dilemma.Case presentationA 25-year-old man with a liver abscess and poorly controlled diabetes mellitus was admitted for endophthalmitis due to K. pneumoniae. The isolate displayed hypermucoviscosity as determined by a positive string test and exhibited resistance to ceftazidime, piperacillin-tazobactam and amikacin. Whole genome sequencing (WGS) analysis demonstrated the isolate to be a K1-serotype strain and belong to a novel single locus variant of ST23, ST2922. In addition to the virulence genes linked to HVKP, rmpA, magA, iucABCDiutA (aerobactin), ybtAPSTUX (yersiniabactin) and iroBDN (salmochelin), it was found to harbor extended-spectrum β-lactamase (ESBL) gene (blaCTX-M-14), AmpC β-lactamase gene (blaDHA), and 16S rRNA methylase gene (armA).ConclusionsThis is the first known case of endogenous endophthalmitis caused by a multidrug-resistant HVKP strain ever reported in China. Early diagnosis and treatment with intravenous and intravitreal injection of carbapenem were essential for a favorable visual outcome.

Clonal expansion and horizontal transmission of epidemic F2:A1:B1 plasmids involved in co-spread of rmtB with qepA and blaCTX-M-27 in extensively drug-resistant Salmonella enterica serovar Indiana isolates.

To investigate the prevalence and transmission of 16S rRNA methylase genes among Salmonella isolates from food animals in China. A total of 310 Salmonella isolates collected from food animals in seven provinces of China during 2016-17 were screened for 16S RMTase genes. The clonal relationship of the 16S RMTase-producing isolates and their plasmid contents were also characterized. rmtB and armA were respectively identified in 12 and 1 Salmonella enterica serovar Indiana (Salmonella Indiana) isolates from farmed ducks. These 13 isolates concurrently expressed high-level resistance to amikacin, cefotaxime and ciprofloxacin. They were assigned to seven distinct PFGE patterns and the high similarity among 10 of the 12 rmtB-carrying isolates suggests clonal expansion. The rmtB gene was co-transferred with blaCTX-M-27-qepA and qepA in eight and two of the isolates, respectively, and was located on F2:A1:B1 plasmids with sizes of 135 and 100 kb, respectively. These 10 rmtB-bearing plasmids showed four restriction patterns with a high similarity. Four representative rmtB-bearing plasmids were fully sequenced and they exhibited remarkable similarity and possessed typical FII backbones. The primary differences were located in the region between blaTEM-1 and ycgA. Furthermore, a novel MDR region (13.5 kb) was identified that contained qepA, rmtB and blaCTX-M-27. This is the first report, to our knowledge, of the prevalence and complete sequences of plasmids simultaneously containing rmtB, qepA and blaCTX-M-27. These findings underscore a major public health threat posed by epidemic F2:A1:B1 plasmids bearing qepA-rmtB-blaCTX-M-27 that are circulating in XDR Salmonella Indiana clonal isolates from waterfowl husbandry.

First report of colistin resistance among carbapenem-resistant Acinetobacter baumannii isolates recovered from hospitalized patients in Egypt.

Acinetobacter baumannii is an opportunistic pathogen that poses an increasing threat in the health-care community. Colistin is one of the promising options for treatment of multidrug-resistant A. baumannii. The current study investigated the emergence of colistin resistance among carbapenem-resistant strains of A. baumannii in Egypt. It involved identification of clinically recovered A. baumannii isolates using the VITEK-2 system, and screening of their antimicrobial susceptibilities using broth microdilution techniques. Characterizations of carbapenemase and 16S rRNA methyltransferase genes were performed using PCR. Colistin-resistance determinants were characterized by sequencing. Carbapenem-resistant A. baumannii isolates (n = 40) showed resistance to amoxicillin-clavulanic acid, cefotaxime, gentamicin and amikacin. Most isolates revealed resistance to ciprofloxacin (95%; n = 38) and co-trimoxazole (92.5%; n = 37). Resistance to tobramycin and doxycycline was 80% (n = 32) and 62.5% (n = 25), respectively. Only two A. baumannii isolates demonstrated colistin resistance. Carbapenemase activity was tested by modified Hodge test and 78% of isolates were positive. All isolates carried blaOXA-51-like genes whereas bla-OXA-23 was detected in 80% (n = 32) of isolates. Among 16S rRNA methylase genes, armA was detected in 22.5% (n = 9) of the isolates. Analyses of lpxA, lpxC, lpxD and pmrCAB genetic sequences suggest that colistin resistance could be attributed to mutations in pmrCAB genes. Alarmingly, colistin resistance was associated with high levels of resistance to other antimicrobials. The current findings represent a serious health-care problem capable of restraining future therapeutic options.

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.

A Gene Homologous to rRNA Methylase Genes Confers Erythromycin and Clindamycin Resistance in Bifidobacterium breve.

Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island.IMPORTANCEBifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains.

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.

Emergence of Various NDM-Type-Metallo-β-Lactamase-Producing Escherichia coli Clinical Isolates in Nepal.

Of 250 clinical isolates of Escherichia coli obtained in Nepal, 38 were carbapenem resistant, with MICs of imipenem or meropenem of ≥4 μg/ml. All 38 isolates harbored the following blaNDMs: blaNDM-1, blaNDM-3, blaNDM-4, blaNDM-5, blaNDM-7, blaNDM-12, and blaNDM-13 Most of these isolates also harbored the 16S rRNA methylase gene(s) armA, rmtB, and/or rmtC.

Emergence of Salmonella enterica serovar Indiana and California isolates with concurrent resistance to cefotaxime, amikacin and ciprofloxacin from chickens in China

The aim of this study was to investigate the prevalence and characterization of Salmonella concerning the poultry industry in China. A total of 170 non-duplicate Salmonella isolates were recovered from the 1540 chicken samples. Among the Salmonella isolates from chickens, the predominant serovars were S. enterica serovar Enteritidis (S. Enteritidis) (49/170, 28.8%), S. enterica serovar Indiana (S. Indiana) (37/170, 21.8%) and S. enterica serovar California (S. California) (34/170, 20.0%). High antimicrobial resistance was observed for ciprofloxacin (68.2%), amikacin (48.2%) and cefotaxime (44.7%). Of particular concerns were the 18 S. Indiana and 17 S. California isolates, which were concurrently resistant to cefotaxime, amikacin and ciprofloxacin. The blaCTX-M genes, 16S rRNA methylase genes (armA, rmtD or rmtC) and five plasmid-mediated quinolone resistance (PMQR) determinants (aac(6′)-Ib-cr, oqxAB, qnrB, qepA and qnrD) were identified in 18 S. Indiana and 17 S. California isolates. To clarify their genetic correlation, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were further conducted. PFGE profiles showed that the majority of S. Indiana and S. California isolates were clonally unrelated with a standard cut-off of 85%. The results of MLST demonstrated that ST17 and ST40 were the most common ST types in S. Indiana and S. California isolates, respectively. Our findings indicated that the multiple antibiotic resistant S. Indiana and S. California isolates were widespread in chicken in China and might pose a potential threat to public health.

Discrepant susceptibility to gentamicin despite amikacin resistance in Klebsiella pneumoniae by VITEK 2 represents false susceptibility associated with the armA 16S rRNA methylase gene.

Because we experienced gentamicin failure in Klebsiella pneumoniae bacteraemia that was susceptible to gentamicin despite amikacin resistance, as determined by VITEK 2, we evaluated the true susceptibility and mechanism of resistance. We screened 2818 K. pneumoniae isolates during a 1-year period at a university hospital and reviewed anti-microbial susceptibility reports using the VITEK 2 system. The minimum inhibitory concentration was substantiated by broth microdilution (BMD), and the presence of 16S rRNA methylase genes and aminoglycoside-modifying enzymes was also investigated. A total of 131 amikacin-resistant isolates from 19 patients were gentamicin non-resistant according to the VITEK 2 system. Among these, we were able to collect isolates from 12 patients (63.2 %), and a single isolate from each patient was tested. Eleven of the gentamicin non-resistant isolates (91.7 %) showed high-level resistance to both amikacin and gentamicin by BMD in association with the armA gene. Gentamicin is not an adequate treatment option for amikacin-resistant K. pneumoniae, even if VITEK 2 reports susceptibility.

Environmental Spread of New Delhi Metallo-β-Lactamase-1-Producing Multidrug-Resistant Bacteria in Dhaka, Bangladesh.

Resistance to carbapenem antibiotics through the production of New Delhi metallo-β-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor the possible source of the spread of these organisms in Dhaka, Bangladesh, we conducted a comparative analysis of wastewater samples from hospital-adjacent areas (HAR) and from community areas (COM), as well as public tap water samples, for the occurrence and characteristics of NDM-1-producing bacteria. Of 72 HAR samples tested, 51 (71%) samples were positive for NDM-1-producing bacteria, as evidenced by phenotypic tests and the presence of the blaNDM-1 gene, compared to 5 of 41 (12.1%) samples from COM samples (P < 0.001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among blaNDM-1-positive isolates, followed by Escherichia coli (29%), Acinetobacter spp. (15%), and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including blaCTX-M-1 (80%), blaCTX-M-15 (63%), blaTEM (76%), blaSHV (33%), blaCMY-2 (16%), blaOXA-48-like (2%), blaOXA-1 (53%), and blaOXA-47-like (60%) genes. Around 40% of the isolates contained a qnr gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying blaNDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multidrug-resistant bacteria as a potential public health threat. In view of the existing practice of discharging untreated liquid waste into the environment, hospitals in Dhaka city contribute to the potential dissemination of NDM-1-producing bacteria into the community.IMPORTANCE Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently described carbapenemase, and the blaNDM-1 gene, which encodes NDM-1, is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows a high prevalence of NDM-1-producing organisms in the wastewater samples from hospital-adjacent areas as a potential source for the spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines the characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple-antibiotic-resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

Wastewater is a reservoir for clinically relevant carbapenemase- and 16s rRNA methylase-producing Enterobacteriaceae

The aim of this study was to evaluate wastewater for carbapenemase-producing Enterobacteriaceae (CPE) and 16S rRNA methylase-producing Gram-negative bacteria (MPB) and to assess their occurrence following wastewater treatment. Wastewater samples were collected between June 2015 and March 2016 in the sewage network of the city of Basel (Switzerland) from sites located before and after influx of wastewater from the hospital into the sewage network. Samples were also obtained from the influent and effluent of the receiving wastewater treatment plant. Samples were screened for CPE and MPB using selective media. Escherichia coli and Klebsiella pneumoniae were typed by multilocus sequence typing (MLST). Carbapenemase and 16S rRNA methylase genes were identified by PCR and sequencing. Resistance profiles were determined by the disk diffusion test and Etest. The occurrence of CPE and MPB was increased downstream of hospital wastewater influx. Of 49 CPE isolates, 9 belonged to OXA-48-producing E. coli clone D:ST38, 7 were OXA-48-producing Citrobacter freundii, and 6 were KPC-2- or OXA-48-producing K. pneumoniae belonging to clonal complex 258. NDM (NDM-1, -5 and -9) and VIM (VIM-1) producers were detected sporadically. MPB included ArmA- and RmtB-producing E. coli and Citrobacter spp. Isolates corresponding to strains from wastewater were detected in the effluent of the treatment plant. Conclusively, CPE and MPB, predominantly OXA-48-producing Enterobacteriaceae, are readily detected in wastewater, survive wastewater treatment and are released into the aquatic environment. OXA-48-producers may represent an emerging threat to public health and environmental integrity.