rRNA Methylase Research Articles

Cooccurrence of NDM-1, ESBL, RmtC, AAC(6')-Ib, and QnrB in Clonally Related Klebsiella pneumoniae Isolates Together with Coexistence of CMY-4 and AAC(6')-Ib in Enterobacter cloacae Isolates from Saudi Arabia.

The aim of this study was to investigate the mechanisms responsible for resistance to antimicrobials in a collection of enterobacterial isolates recovered from two hospitals in Saudi Arabia. A total of six strains isolated from different patients showing high resistance to carbapenems was recovered in 2015 from two different hospitals, with four being Klebsiella pneumoniae and two Enterobacter cloacae. All isolates except one K. pneumoniae were resistant to tigecycline, but only one K. pneumoniae was resistant to colistin. All produced a carbapenemase according to the Carba NP test, and all were positive for the EDTA-disk synergy test for detection of MBL. Using PCR followed by sequencing, the four K. pneumoniae isolates produced the carbapenemase NDM-1, while the two E. cloacae isolates produced the carbapenemase VIM-1. Genotyping analysis by Multilocus Sequence Typing (MLST) showed that three out of the four K. pneumoniae isolates were clonally related. They had been recovered from the same hospital and belonged to Sequence Type (ST) ST152. In contrast, the fourth K. pneumoniae isolate belonged to ST572. Noticeably, the NDM-1-producing K. pneumoniae additionally produced an extended-spectrum ß-lactamase (ESBL) of the CTX-M type, together with OXA-1 and TEM-1. Surprisingly, the three clonally related isolates produced different CTX-M variants, namely, CTX-M-3, CTX-M-57, and CTX-M-82, and coproduced QnrB, which confers quinolone resistance, and the 16S rRNA methylase RmtC, which confers high resistance to all aminoglycosides. The AAC(6′)-Ib acetyltransferase was detected in both K. pneumoniae and E. cloacae. Mating-out assays using Escherichia coli as recipient were successful for all isolates. The blaNDM-1 gene was always identified on a 70-kb plasmid, whereas the blaVIM-1 gene was located on either a 60-kb or a 150-kb plasmid the two E. cloacae isolates, respectively. To the best of our knowledge, this is the first report of the coexistence of an MBL (NDM-1), an ESBL (CTX-M), a 16S rRNA methylase (RmtC), an acetyltransferase (AAC[6′]-Ib), and a quinolone resistance enzyme (QnrB) in K. pneumoniae isolates recovered from different patients during an outbreak in a Saudi Arabian hospital.

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.

NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (Solanum betaceum Cav.).

Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation and for embryogenesis research. Moreover, genetic transformation and cryopreservation procedures in many species rely on efficient SE protocols. We have been studying different aspects related to SE induction and somatic embryo development in tamarillo (Solanum betaceum Cav.), a small tree from the Solanaceae family. Previous proteomic analyses identified a protein (NEP-TC, 26.5 kDa) consistently present in non-embryogenic calluses of tamarillo, but absent in the embryogenic ones. In this work, the role of NEP-TC during SE was assessed by gene expression analysis and immunolocalization. The results obtained demonstrated that NEP-TC is a putative member of the SpoU rRNA methylase family. This protein, present in the cytoplasm and nucleus, is expressed in non-embryogenic cells and not expressed in embryogenic cells. Slightly enhanced SE induction levels in tamarillo plants with NEP-TC down-regulated levels also supports the role of this protein on SE induction. Heterologous expression was used to confirm NEP-TC rRNA methyltransferase activity, with enhanced activity levels when rRNA was used as a substrate. These data relate a putative member of the SpoU methylase family with plant morphogenesis, in particular with SE induction.

Emergence of Enterobacteriaceae co-producing CTX-M-15, ArmA and PMQR in Poland.

Plasmid-mediated extended-spectrum β-lactamases (ESBLs), 16S rRNA methylases and quinolone resistance mechanisms (PMQRs) are well-known agents conferring resistance to more than 1 antimicrobial in its group. The accumulation of these agents poses, therefore, a serious risk to public health. The objective of this study was to investigate the presence of common ß-lactamases and 16S rRNA methylases in Qnr-producing Enterobacteriaceae and their genetic relatedness. We examined 18 Qnr-producing isolates (Klebsiella pneumoniae n = 8, Enterobacter cloacae n = 6 and Escherichia coli n = 4) selected from a collection of 215 ciprofloxacin-resistant strains obtained from patients in a 1030-bed tertiary hospital from 1 March to 31 August 2010. The antibiotics minimum inhibitory concentration (MIC) was determined by E-test. The detection of common ß-lactamases, 16S rRNA methyltransferases and PMQR genes was performed by polymerase chain reaction (PCR) and sequencing. Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All the isolates tested were susceptible to carbapenems and colistin, while 16 were multidrug-resistant. Thirteen, 2 and 2 isolates carried qnrB1, qnrA1 and qnrS1, respectively. Ten of 13 qnrB1-positive Enterobacteriaceae also carried genes encoding for aac(6')-Ib-cr and at least 1 ESBL. The blaCTX-M-15 gene was the most common ESBL. The most prevalent combination of genes was qnrB1+aac(6')-Ib-cr+blaTEM-1+blaCTX-M-15. Two isolates of K. pneumoniae and E. cloacae were found to bear multiple extended range resistance traits: ArmA, CTX-M-15, QnrB1, and AAC (6')-Ib-cr. The PFGE showed that most of the isolates exhibited individual DNA patterns, whilst MLST assigned K. pneumoniae (n = 8) to 5 sequence types (STs) (ST15, ST323, ST336, ST147, and ST525), E. coli (n = 4) to 2 (ST131 and ST1431) and E. cloacae (n = 5) to 4 (ST90, ST89, ST133, and the novel ST407). Our findings reveal the accumulation of resistance traits and their important role in spreading of multiresistant bacteria among hospitalized patients.

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.

Nationwide epidemiology of carbapenem resistant Klebsiella pneumoniae isolates from Greek hospitals, with regards to plazomicin and aminoglycoside resistance

BackgroundTo evaluate the in vitro activities of plazomicin and comparator aminoglycosides and elucidate the underlying aminoglycoside resistance mechanisms among carbapenemase-producing K. pneumoniae isolates collected during a nationwide surveillance study in Greek hospitals.MethodsThree hundred single-patient carbapenemase-producing K. pneumoniae isolates were studied, including 200 KPC-, 50 NDM-, 21 VIM-, 14 KPC & VIM-, 12 OXA-48-, two NDM & OXA- and one KPC & OXA-producing isolates. Susceptibility testing was performed by broth microdilution, and minimum inhibitory concentrations (MICs) interpreted per EUCAST breakpoints. Carbapenemase-, aminoglycoside modifying enzyme- and 16S rRNA methylase- encoding genes were detected by PCR.ResultsOf 300 isolates tested, 5.7% were pandrug resistant and 29.3% extensively drug resistant. Plazomicin inhibited 87.0% of the isolates at ≤2 mg/L, with MIC50/MIC90 of 0.5/4 mg/L. Apramycin (a veterinary aminoglycoside) inhibited 86.7% of the isolates at ≤8 mg/L and was the second most active drug after plazomicin, followed by gentamicin (S, 43%; MIC50/MIC90, 4/> 256) and amikacin (S, 18.0%; MIC50/MIC90, 32/128). Twenty-three (7.7%) isolates (16 KPC-, 6 VIM- and one KPC & OXA-48-producers) exhibited MICs ≥64 mg/L for plazomicin, and harbored rmtB (n = 22) or armA (n = 1). AAC(6′)-Іb was the most common aminoglycoside modifying enzyme (84.7%), followed by AAC(3΄)-IIa (25.3%), while those two enzymes were co-produced by 21.4% of the isolates.ConclusionsPlazomicin retains activity against most carbapenemase-producing K. pneumoniae isolated from Greek hospitals, with MICs consistently lower than those of the other aminoglycosides, even in the presence of aminoglycoside modifying enzymes. Dissemination of 16S- rRNA methylases in 8% of the isolates is an unwelcome event that needs strict infection control measures and rigorous stewardship interventions.

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.

Prevalence of 16S rRNA methylases in Gram-negative bacteria derived from companion animals and livestock in Japan.

The emergence and spread of aminoglycoside-resistant bacteria are a public health concern. The acquisition of the genes encoding 16S rRNA methylases, such as armA, rmtA, and rmtB, confers high-level resistance to aminoglycosides. However, the prevalence has not been well investigated in Japanese veterinary fields. To determine the prevalence of 16S rRNA methylases in animals, we detected 16S rRNA methylases genes in Gram-negative bacteria from animals. Here, we report the isolation of rmtB and armA from two of the 446 Escherichia coli (0.5%) and one of the 103 Klebsiella spp. isolates (1.0%) from companion animals, respectively. However, none of the isolations were observed from 2445 E. coli isolates derived from livestock in Japan. The prevalence of 16S rRNA methylases in animals, especially in companion animals, should be carefully monitored in Japanese veterinary fields to avoid the spreading of the genes.

Endemic dissemination of different sequence types of carbapenem-resistant Klebsiella pneumoniae strains harboring bla NDM and 16S rRNA methylase genes in Kerman hospitals, Iran, from 2015 to 2017.

IntroductionThe emergence and spread of Klebsiella pneumoniae strains resistant to multiple antimicrobial agents are considered as a serious challenge for nosocomial infections.Materials and methodsIn this study, 175 nonrepetitive clinical isolates of K. pneumoniae were collected from hospitalized patients in Kerman, Iran. Extended-spectrum β-lactamases (ESBLs), AmpC, and carbapenemase-producing isolates were recognized by phenotypic methods. The resistance genes including efflux pumps oqxA/oqxB, 16S rRNA methylase, ESBL, AmpC, and carbapenemase were detected by PCR-sequencing method. Molecular typing was performed by enterobacterial repetitive intergenic consensus-PCR and multilocus sequence typing methods among blaNDM-positive isolates.ResultsThirty-seven (21.14%) isolates along with sequence types (STs): ST43, ST268, ST340, ST392, ST147, and ST16 were harbored blaNDM. ST43 in 2015 and ST268 during 2016–2017 were the most frequent STs among New Delhi metallo-beta-lactamase (NDM)-positive isolates. We found the distribution of some isolates with blaNDM, blaCTX-M, blaSHV, blaOXA, blaTEM, blaCMY, rmtC, and oqxA/oqxB. Enterobacterial repetitive intergenic consensus-PCR represented seven clusters (A–G) plus four singletons among NDM-positive isolates. This study provides the first report of blaNDM-1-positve K. pneumoniae along with ST268 as well as the spread of nosocomial infections with six different STs harboring blaNDM-1 and other resistance genes in hospital settings especially neonatal intensive care unit.ConclusionThe dissemination of various clones of NDM-producing K. pneumoniae can contribute to increase the rate of their spread in health care settings. Therefore, molecular typing and detection of resistance genes have an important role in preventing and controlling infection by limiting the dissemination of multidrug-resistant isolates.

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.

Diversity of aminoglycoside modifying enzymes and 16S rRNA methylases in Acinetobacter baumannii and Acinetobacter nosocomialis species in Iran; wide distribution of aadA1 and armA

PurposeAcinetobacter baumannii-calcoaceticus complex (ABC) make a great burden on health-care systems due to hospital-acquired infections and antibacterial resistance. Aminoglycoside in combination with other antibacterials used as treatment options. However, ABC species overcome this class of antibacterials in different ways. This study provides a comprehensive report on the distribution of aminoglycoside modifying enzymes (AMEs) and 16S rRNA methylase in Acinetobacter baumannii and Acinetobacter nosocomialis isolated from various provinces in Iran. MethodsDuring six month of study, from eight referral centers in seven provinces across the country, Iran, 178 A. baumannii and 43 A. nosocomialis isolates were collected. The minimum inhibitory concentration of amikacin, gentamicin, netilmicin, kanamycin and tobramycin were measured by microbroth dilution method. AMEs and 16S rRNA methylase variants were sought by PCR. ResultsHigh rates of resistance were seen in all centers. MIC50 and MIC90 for all A. baumannii and A. nosocomialis isolates from different centers were > 512 mg/L. The most frequent AME was ant(3″)-Ia (aadA1) in both of A. baumannii (74.1%) and A. nosocomialis (86%). armA was detected in A. baumannii and A. nosocomialis at the frequency of 41.6% and 67.4%, respectively. rmtA, B, C, D, aac(3)-Ia (aacC1) and aac(6′)-Im were not detected, neither in A. baumannii nor A. nosocomialis. Moreover, aac(6′)-Ih was only found in A. baumannii isolates. The distribution of some of the ARGs was limited to a definite center. ConclusionThe overall high-level carriage of ARGs in Acinetobacter species may limited usage of this class of antibacterials as a treatment option.

First report of blaOXA-24 carbapenemase gene, armA methyltransferase and aac(6′)-Ib-cr among multidrug-resistant clinical isolates of Proteus mirabilis in Algeria

Carbapenemase-producing, or carbapenem-resistant, Enterobacteriaceae are an emerging threat to human and animal health because they are resistant to many of the last-line antimicrobials available for treatment of infection. The aim of this study was to analyse the antimicrobial resistance patterns and their encoding genes of Proteus mirabilis isolated in Constantine, Algeria. A total of 108 Proteus, Morganella and Providencia (PMP) strains were isolated from a large variety of clinical specimens at University Hospital of Constantine in Algeria. Isolates were identified using the API 20E system and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Diagnostic accuracy was determined by independent comparison of each method to phylogenetic analysis based on 16S rRNA gene sequencing. Antimicrobial susceptibility was determined by the standard disk diffusion and Etest methods. The presence of antimicrobial resistance genes was screened for by PCR amplification and sequencing. A total of 72 PMP strains were multidrug-resistant (MDR). Among them, one P. mirabilis isolate was resistant to imipenem with a minimum inhibitory concentration (MIC) of ≥12μg/mL. PCR and sequencing showed the presence of various antimicrobial resistance genes, including blaCTX-M-15, blaTEM-1, blaTEM-2, blaPER-1, blaSHV-11, aadA1, aadA2, armA, aac(6')-Ib, aac(6')-Ib-cr, aac(3)-Ia and ant(2″)-I, forming different resistance profiles. Moreover, the blaOXA-24 gene was detected in the imipenem-resistant P. mirabilis strain. In this study, a MDR P. mirabilis isolate harbouring the blaOXA-24, armA 16S rRNA methylase and aac(6)-Ib-cr genes was found for the first time in Algeria.

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.

Antimicrobial Resistance in Escherichia coli.

Multidrug resistance in Escherichia coli has become a worrying issue that is increasingly observed in human but also in veterinary medicine worldwide. E. coli is intrinsically susceptible to almost all clinically relevant antimicrobial agents, but this bacterial species has a great capacity to accumulate resistance genes, mostly through horizontal gene transfer. The most problematic mechanisms in E. coli correspond to the acquisition of genes coding for extended-spectrum β-lactamases (conferring resistance to broad-spectrum cephalosporins), carbapenemases (conferring resistance to carbapenems), 16S rRNA methylases (conferring pan-resistance to aminoglycosides), plasmid-mediated quinolone resistance (PMQR) genes (conferring resistance to [fluoro]quinolones), and mcr genes (conferring resistance to polymyxins). Although the spread of carbapenemase genes has been mainly recognized in the human sector but poorly recognized in animals, colistin resistance in E. coli seems rather to be related to the use of colistin in veterinary medicine on a global scale. For the other resistance traits, their cross-transfer between the human and animal sectors still remains controversial even though genomic investigations indicate that extended-spectrum β-lactamase producers encountered in animals are distinct from those affecting humans. In addition, E. coli of animal origin often also show resistances to other-mostly older-antimicrobial agents, including tetracyclines, phenicols, sulfonamides, trimethoprim, and fosfomycin. Plasmids, especially multiresistance plasmids, but also other mobile genetic elements, such as transposons and gene cassettes in class 1 and class 2 integrons, seem to play a major role in the dissemination of resistance genes. Of note, coselection and persistence of resistances to critically important antimicrobial agents in human medicine also occurs through the massive use of antimicrobial agents in veterinary medicine, such as tetracyclines or sulfonamides, as long as all those determinants are located on the same genetic elements.

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.

High Rate of Association of 16S rRNA Methylases and Carbapenemases in Enterobacteriaceae Recovered from Hospitalized Children in Angola.

Acquired 16S rRNA methylases (RMTases) conferring pan-drug resistance to aminoglycosides were searched among enterobacterial isolates recovered in Angola. A total of 36 hospitalized children were screened for rectal colonization using the Superaminoglycoside selective medium. Twenty-two pan-aminoglycoside-resistant enterobacterial isolates were recovered, all of which produced RMTases, i.e., RmtB, ArmA, and RmtC. Highly diverse genetic backgrounds were identified for Escherichia coli and Klebsiella pneumoniae isolates, most of which coproduced carbapenemases NDM-1 or NDM-5, respectively.

A culture medium for screening 16S rRNA methylase-producing pan-aminoglycoside resistant Gram-negative bacteria

The amikacin plus gentamicin-containing SuperAminoglycoside medium was developed for screening multiple-aminoglycoside resistance in Gram-negative bacteria (Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). It was evaluated using aminoglycoside-susceptible (n=12) and aminoglycoside-resistant (n=59) Gram-negative isolates, including 16S rRNA methylase producers (n=20). Its sensitivity and specificity of detection were, respectively, of 95% and 96% for detecting multiple aminoglycoside-resistant methylase producers.