30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC.

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine analog to trap the complex in a postcatalytic state to enable determination of a global 3.0 Å cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder-to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to resensitize bacterial pathogens to aminoglycosides.

Proteomic assay for rapid characterisation of Staphylococcus aureus antimicrobial resistance mechanisms directly from blood cultures.

PURPOSE: Staphylococcus aureus is one of the most common pathogens causing bloodstream infection. A rapid characterisation of resistance to methicillin and, occasionally, to aminoglycosides for particular indications, is therefore crucial to quickly adapt the treatment and improve the clinical outcomes of septic patients. Among analytical technologies, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a promising tool to detect resistance mechanisms in clinical samples. METHODS: A rapid proteomic method was developed to detect and quantify the most clinically relevant antimicrobial resistance effectors in S. aureus in the context of sepsis: PBP2a, PBP2c, APH(3')-III, ANT(4')-I, and AAC(6')-APH(2''), directly from positive blood cultures and in less than 70 min including a 30-min cefoxitin-induction step. The method was tested on spiked blood culture bottles inoculated with 124 S.aureus, accounting for the known genomic diversity of SCCmec types and the genetic background of the strains. RESULTS: This method provided 99% agreement for PBP2a (n = 98/99 strains) detection. Agreement was 100% for PBP2c (n = 5/5), APH(3')-III (n = 16/16), and ANT(4')-I (n = 20/20), and 94% for AAC(6')-APH(2'') (n = 16/17). Across the entire strain collection, 100% negative agreement was reported for each of the 5 resistance proteins. Additionally, relative quantification of ANT(4')-I expression allowed to discriminate kanamycin-susceptible and -resistant strains, in all strains harbouring the ant(4')-Ia gene. CONCLUSION: The LC-MS/MS method presented herein demonstrates its ability to provide a reliable determination of S. aureus resistance mechanisms, directly from positive blood cultures and in a short turnaround time, as required in clinical laboratories.

Genetic background of aminoglycoside-modifying enzymes in various genetic lineages of clinical aminoglycosides-resistant E. coli and K. pneumoniae isolates in Tunisia.

AIMS: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated. MATERIALS AND METHODS: A total of 105 aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycoside compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes [aph(3´)-Ia, aph(3') IIa, aph(3´)-VIa, ant(2″)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, rmtA, rmtB, rmtC, armA, and npmA] and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by multilocus sequence typing (MLST) for representative isolates. RESULTS: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%), and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%), followed by aac(6')-Ib (36.2%) and aph(3')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by 1 and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae (ST11/ST13). CONCLUSIONS: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse ß-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.

Escherichia coli with increased aminoglycoside resistance due to an amino acid substitution at position 85 of HemC.

OBJECTIVE: The mechanism of aminoglycoside resistance due to abnormal hemin synthesis remains unclear. We investigate an Escherichia coli strain with a single amino acid substitution at position 85 of HemC. METHODS: An aminoglycoside-resistant Escherichia coli DH5α was selected by passaging in Lysogeny Broth (LB) medium containing amikacin. Whole genome sequencing was performed to determine the genetic profile of the strain. An isogenic strain of E. coli DH5α was created. Growth rates, drug susceptibilities and expressions of the heme synthetic genes were compared between the original strain and the isogenic strain. RESULTS: Whole genome sequencing revealed a nucleotide substitution at position 254 of hemC from adenine (A) to thymine (T), resulting in an amino acid substitution at position 85 of HemC from histidine (H) to leucine (L). There were no mutations in other heme synthetic genes, including hemA, hemB, hemC, hemD, hemE, hemF, hemG, hemH, hemL, hemN, hemX and hemY. The isogenic strain of E. coli DH5α with H85L in HemC was less susceptible to aminoglycosides, and its growth was slower than that of E. coli DH5α before passage. Quantitative real-time PCR showed that the expression of hemA was higher and the expressions of hemL, hemG and hemX lower in the isogenic strain than before passage. CONCLUSION: This is the first report of aminoglycoside resistance due to an amino acid substitution in HemC. These findings suggested that mutations in the heme synthetic genes may indirectly affect the growth rates of E. coli strains and their susceptibilities to aminoglycosides.

Contribution of different mechanisms to aminoglycoside resistance in clinical isolates of Acinetobacter baumannii.

The antibiotics overuse for infection treatment was the sparkle in the spreading of multi-drug resistance Acinetobacter baumannii in hospitals. In our study, we evaluated the contribution of the aminoglycoside resistance mechanisms of A. baumannii to the resistance surge in some selected Egyptian hospitals with a checkerboard assay application to retrieve the aminoglycoside activity. The resistance profile analysis of collected 200 A. baumannii isolates revealed a multidrug-resistant pattern with limited susceptibilities to aminoglycosides. Analysis of the prevalence of aminoglycoside-modifying enzyme (AMEs) genes revealed the presence of the six AMEs genes either singly or in combination in selected isolates and aph (3)-VIa gene was the predominant one. At the same time, four efflux pump genes of AdeABC and AdeKJL family showed significant (P < 0.001) up-regulation levels. Moreover, the implementation of combination strategy showed fourteen synergistic activities against two high-level aminoglycoside-resistance (HLAR) A. baumannii isolates. The findings highlighted the alarming levels of aminoglycoside resistance in A. baumannii isolates, which proved that a common enzymatic modification mechanism acts synergistically with decreased antibiotic accumulation in acquiring aminoglycoside resistance. Additionally, the study provides useful information for the promising synergistic combination therapy that reduces the therapeutic dose of aminoglycosides used and subsequently increases their clinical application.

Pseudomonas aeruginosa Phosphate Transporter PitA (PA4292) Controls Susceptibility to Aminoglycoside Antibiotics by Regulating the Proton Motive Force.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes nosocomial infections in immunocompromised patients. ß-lactam and aminoglycoside antibiotics are commonly used in the treatment of P. aeruginosa infections. Previously, we found that mutation in a PA4292 gene increases bacterial resistance to ß-lactam antibiotics. In this study, we demonstrated that mutation in PA4292 increases bacterial susceptibility to aminoglycoside antibiotics. We further found enhanced uptake of tobramycin by the ΔPA4292 mutant, which might be due to an increase of proton motive force (PMF). Sequence analysis revealed PA4292 is homologous to the Escherichia coli phosphate transporter PitA. Mutation of PA4292 indeed reduces intracellular phosphate concentration. We thus named PA4292 as pitA. Although the PMF is enhanced in the ΔpitA mutant, the intracellular ATP concentration is lower than that in the isogenic wild-type strain PA14, which might be due to lack of the ATP synthesis substrate phosphate. Overexpression of the phosphate transporter complex genes pstSCAB in the ΔpitA mutant restores the intracellular phosphate concentration, PMF, ATP synthesis, and aminoglycosides resistance. In addition, growth of wild-type PA14 in a low-phosphate medium resulted in higher PMF and aminoglycoside susceptibility compared to cells grown in a high-phosphate medium. Overall, our results demonstrate the roles of PitA in phosphate transportation and reveal the relationship between intracellular phosphate and aminoglycoside susceptibility.

Functional and Structural Characterization of Acquired 16S rRNA Methyltransferase NpmB1 Conferring Pan-Aminoglycoside Resistance.

Posttranslational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such a function. Here, we present the function and structure of NpmB1, whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides, including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single-amino-acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the ß6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

Diffusion and Characterization of Pseudomonas aeruginosa Aminoglycoside Resistance in an Italian Regional Cystic Fibrosis Centre.

AIMS: Extensively-drug-resistant Pseudomonas aeruginosa constitutes a serious threat to patients suffering from Cystic Fibrosis (CF). In these patients, P. aeruginosa lung infection is commonly treated with aminoglycosides, but treatments are largely unsuccessful due a variety of resistance mechanisms. Here we investigate the prevalence of resistance to gentamicin, amikacin and tobramycin and the main aminoglycoside resistance genes found in P. aeruginosa strains isolated at a regional CF centre. RESULTS: A total number of 147 randomly selected P. aeruginosa strains isolated from respiratory samples sent by the Marche regional Cystic Fibrosis Centre to the Microbiology lab, were included in this study. Of these, 78 (53%) were resistant to at least one of the three aminoglycosides tested and 27% were resistant to all three antibiotics, suggesting a major involvement of a chromosome-encoded mechanism, likely MexXY-OprM efflux pump overexpression. A specific pathogenic clone (found in 7/78 of the aminoglycoside resistant strains) carrying ant(2″)-Ia was isolated over time from the same patient, suggesting a role for this additional resistance gene in the antibiotic unresponsiveness of CF patients. CONCLUSIONS: The MexXY-OprM efflux pump is confirmed as the resistance determinant involved most frequently in P. aeruginosa aminoglycoside resistance of CF lung infections, followed by the ant(2″)-Ia-encoded adenylyltransferase. The latter may prove to be a novel target for new antimicrobial combinations against P. aeruginosa.

Large-scale pattern of resistance genes and bacterial community in the tap water along the middle and low reaches of the Yangtze River.

Antibiotic and metal resistance genes (ARGs and MRGs) in tap water are of great public health concern. However, very fewer studies focused on the relationship between resistance genes and opportunistic pathogens in tap water. In this study, the diversity and abundance of resistance genes and bacterial community from tap water at a large-scale along the middle and lower reaches of the Yangtze River were investigated. The total relative abundances of ARGs and MRGs were 2.95 × 10-3-1.22 × 10-1 and 1.93 × 10-3-1.20 × 10-1 copies/16S rRNA, respectively. The blaTEM and merP detected were major ARG and MRG subtypes, respectively. Mobile genetic elements (Intl1 and tnpA) showed significant correlations with the abundance of ARGs. Heavy metals also played a vital role in the co-selection of ARGs. Surprisingly, there were still eight opportunistic pathogens in tap water, among which Escherichia coli, Helicobacter pylori, Mycoplasma pneumoniae, and Porphyromonas gingivalis were the potential host of ARGs and MRGs. Escherichia coli had the highest abundance, while Bacillus anthracis had the highest detected frequency (100%), a widespread opportunistic pathogen in tap water.

Expression of the MexXY Aminoglycoside Efflux Pump and Presence of an Aminoglycoside-Modifying Enzyme in Clinical Pseudomonas aeruginosa Isolates Are Highly Correlated.

The impact of MexXY efflux pump expression on aminoglycoside resistance in clinical Pseudomonas aeruginosa isolates has been debated. In this study, we found that, in general, elevated mexXY gene expression levels in clinical P. aeruginosa isolates confer to slight increases in aminoglycoside MIC levels; however, those levels rarely lead to clinically relevant resistance phenotypes. The main driver of resistance in the clinical isolates studied here was the acquisition of aminoglycoside-modifying enzymes (AMEs). Nevertheless, acquisition of an AME was strongly associated with mexY overexpression. In line with this observation, we demonstrate that the introduction of a gentamicin acetyltransferase confers to full gentamicin resistance levels in a P. aeruginosa type strain only if the MexXY efflux pump was active. We discuss that increased mexXY activity in clinical AME-harboring P. aeruginosa isolates might affect ion fluxes at the bacterial cell membrane and thus might play a role in the establishment of enhanced fitness that extends beyond aminoglycoside resistance.

Assessment of Tedizolid In Vitro Activity and Resistance Mechanisms against a Collection of Enterococcus spp. Causing Invasive Infections, Including Isolates Requiring an Optimized Dosing Strategy for Daptomycin from U.S. and European Medical Centers, 2016 to 2018.

High-level aminoglycoside resistance was noted in 30.0% of Enterococcus faecalis and 25.2% of Enterococcus faecium isolates. Only 3.3% and 2.1% of E. faecalis isolates had elevated daptomycin MIC (≥2 mg/liter) and vancomycin resistance, respectively. In contrast, 37.4% to 40.3% of E. faecium isolates exhibited these phenotypes. Tedizolid inhibited 98.9% to 100.0% of enterococci causing serious invasive infections, including resistant subsets. Oxazolidinone resistance was mainly driven by G2576T; however, optrA and poxtA genes were also detected, including poxtA in the United States and Turkey.

Prevalence of Aminoglycoside Resistance Genes and Molecular Characterization of a Novel Gene, aac(3)-IIg, among Clinical Isolates of the Enterobacter cloacae Complex from a Chinese Teaching Hospital.

Members of the Enterobacter cloacae complex are important opportunistic human pathogens capable of causing a wide variety of infections. During recent decades, aminoglycoside-resistant E. cloacae complex isolates have increasingly been reported and have become a major concern. Here, we employed high-throughput sequencing in combination with specific PCR assays to investigate the prevalence of aminoglycoside resistance genes among 170 isolates of the E. cloacae complex collected from a teaching hospital in Wenzhou, China. A total of 12 known genes [aphA-1, strA, strB, aac(6')-IIc, aadA2, aac(3)-IId, aadB, aadA1, rmtB, armA, aadA5, and aac(6')-Ie-aph(2'')-Ia] and 1 novel gene [aac(3)-IIg] were identified, with aphA-1 (71.18%), strA (55.29%), and strB (52.35%) being the most prevalent, and aac(3)-IIg was detected with a positive rate of 21.76% (37/170). The aac(3)-IIg gene was 810 bp in length and encoded a protein that shared 72 to 78% identities with previously known AAC(3)-II aminoglycoside 3-N-acetyltransferases. The MICs of gentamicin and tobramycin were 512 µg/ml and 64 µg/ml, respectively, when aac(3)-IIg was cloned into Escherichia coli DH5α. All aac(3)-IIg-positive isolates exerted broad aminoglycoside resistance profiles, mediated by the coexistence of multiple resistance genes. Moreover, aminoglycoside resistance and resistance genes were found to be transferable in most strains (24/37). Nevertheless, pulsed-field gel electrophoresis (PFGE) and dendrogram analysis showed clonal diversity among these isolates. S1 nuclease PFGE, Southern hybridization, and whole-genome sequencing indicated that aac(3)-IIg was located on transferable as well as nontransferable plasmids of various sizes. The analysis of the genetic environment suggested that aac(3)-IIg is embedded within a class 1 integron, with IS26 playing an important role in its mobility.

Detection of antibiotic resistance profiles and aminoglycoside-modifying enzyme (AME) genes in high-level aminoglycoside-resistant (HLAR) enterococci isolated from raw milk and traditional cheeses in Turkey.

The aim of this study was isolation and identification of the high-level aminoglycoside-resistant (HLAR) enterococci in raw milk and dairy products and to analyze their antibiotic resistance and the presence of aminoglycoside-modifying enzyme (AME) genes. A total of 59 HLAR enterococci were isolated from raw milk and traditional cheese samples. Thirty-nine of the 59 HLAR enterococci were isolated on streptomycin-containing agar medium, while the other 20 HLAR strains were isolated on gentamicin containing agar medium. The 59 HLAR enterococci were identified as 26 E. faecalis (44.07%), 18 E. faecium (30.51%), 13 E. durans (22.03%), and two E. gallinarum (3.39%) by species-specific PCR. Disk diffusion tests showed that teicoplanin were the most effective antibiotics used in this study, while 89.83% of isolates were found to be resistant to tetracycline. High rates of multiple antibiotic resistance were detected in HLAR isolates. Minimum inhibitory concentration (MIC) values of HLAR enterococci against streptomycin and gentamicin were found in the range of 64 to > 4096 µg/mL. Forty-seven (79.66%) of the 59 HLAR enterococci were found to be both high-level streptomycin-resistant (HLSR) and high-level gentamicin-resistant (HLGR) by MIC tests. However, no correlation was found between the results of the disk diffusion and MIC tests for gentamicin and streptomycin in some HLAR strains. The aph(3')-IIIa (94.92%) was found to be most prevalent AME gene followed by ant(4')-Ia (45.76%), ant(6')-Ia (20.34%) and aph(2'')-Ic (10.17%). None of the isolates contained the aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib or aph(2'')-Id genes. None of the AME-encoding genes were identified in E. durans RG20.1, E. faecalis RG22.4, or RG26.1. In conclusion, HLAR enterococci strains isolated in this study may act as reservoirs in the dissemination of antibiotic resistance genes.

High incidence of virulence determinants, aminoglycoside and vancomycin resistance in enterococci isolated from hospitalized patients in Northwest Iran.

BACKGROUND: Multidrug resistant (MDR) enterococci are important nosocomial pathogens causing serious problem in hospitalized patients. The aim of present study was to investigate the frequency of high-level aminoglycoside-resistant and vancomycin-resistant enterococci (VRE) and virulence encoding genes in enterococci isolated from hospitalized patients. METHODS: A total of 100 enterococci isolated from urine samples of hospitalized patients with symptomatic urinary tract infections were investigated for antimicrobial susceptibility, the frequency of aminoglycoside and vancomycin resistance genes (including aac (6')-Ie-aph (2")-Ia, aph (3')-IIIa, ant (4')-Ia, aph (2")-Ic, aph (2")-Ib, aph (2")-Id, ant (3″)-III, ant (6')-Ia, vanA, vanB and vanC) and virulence encoding genes (including gelE, PAI, esp, ace, cyl, hyl and sprE). RESULTS: Enterococcus faecalis species was identified as predominant enterococci (69%), followed by "other" Enterococcus species (21%) and E. faecium (10%). Ninety three percent of isolates were resistant to one or more antimicrobial agents, with the most frequent resistance found against tetracycline (86%), ciprofloxacin (73%) and quinupristin-dalfopristin (53%). Gentamicin and streptomycin resistance were detected in 50 and 34% of isolates, respectively. The most prevalent aminoglycoside resistance genes were ant (3″)-III (78%) and aph (3')-IIIa (67%). Vancomycin resistance was detected in 21% of isolates. All E. faecium isolates carried vanA gene, whereas, the vanB gene was not detected in Enterococcus species. The most frequent virulence gene was ace (88.6%), followed by esp (67.1%), PAI (45.5%) and sprE (41.7%). CONCLUSION: Our study revealed the high frequency of gentamycin resistance and VRE in E. faecium isolates, with a high prevalence and heterogeneity of virulence and resistance genes. Due to high frequency of MDR enterococci, it seems that the appropriate surveillance and control measures are essential to prevent the emergence and transmission of these isolates in hospitals.

ClpA and HtpX Proteases Are Involved in Intrinsic Aminoglycoside Resistance of Stenotrophomonas maltophilia and Are Potential Aminoglycoside Adjuvant Targets.

The linkage of the protease-chaperon system, SmeYZ pump, and aminoglycoside resistance was assessed in Stenotrophomonas maltophilia The clpA, clpS, clpP, and htpX genes were upregulated in response to kanamycin exposure. Of these, clpA and htpX were the primary determinants responsible for intrinsic aminoglycoside (AG) resistance. Inactivation of clpA and htpX compromised protease-mediated intrinsic aminoglycoside resistance and weakened SmeYZ pump-mediated aminoglycoside resistance, signifying HtpX and ClpA as potential AG adjuvant targets for treatment of S. maltophilia infections.

Molecular Epidemiology of Emerging blaOXA-23-Like- and blaOXA-24-Like-Carrying Acinetobacter baumannii in Taiwan.

The rate of recovery of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates has increased significantly in recent decades in Taiwan. This study investigated the molecular epidemiology of CRAB with a focus on the mechanisms of resistance and spread in isolates with blaOXA-23-like or blaOXA-24-like All 555 CRAB isolates in our multicenter collection, which were recovered from 2002 to 2010, were tested for the presence of class A, B, and D carbapenemase genes. All isolates with blaOXA-23-like or blaOXA-24-like were subjected to pulsed-field gel electrophoresis, and 82 isolates (60 isolates with blaOXA-23-like and 22 isolates with blaOXA-24-like) were selected for multilocus sequence typing to determine the sequence type (ST) and clonal group (CG) and for detection of additional ß-lactamase and aminoglycoside resistance genes. The flanking regions of carbapenem and aminoglycoside resistance genes were identified by PCR mapping and sequencing. The localization of blaOXA was determined by S1 nuclease and I-CeuI assays. The numbers of CRAB isolates carrying blaOXA-23-like or blaOXA-24-like, especially those carrying blaOXA-23-like, increased significantly from 2008 onward. The blaOXA-23-like gene was carried by antibiotic resistance genomic island 1 (AbGRI1)-type structures located on plasmids and/or the chromosome in isolates of different STs (CG92 and novel CG786), whereas blaOXA-24-like was carried on plasmids in CRAB isolates of limited STs (CG92). No class A or B carbapenemase genes were identified. Multiple aminoglycoside resistance genes coexisted in CRAB. Tn6180-borne armA was found in 74 (90.2%) CRAB isolates, and 58 (70.7%) isolates had Tn6179 upstream, constituting AbGRI3. blaTEM was present in 38 (46.3%) of the CRAB isolates tested, with 35 (92.1%) isolates containing blaTEM in AbGRI2-type structures, and 61% of ampC genes had ISAba1 upstream. We conclude that the dissemination and spread of a few dominant lineages of CRAB containing various resistance island structures occurred in Taiwan.

Molecular Mechanisms of Intrinsic Streptomycin Resistance in Mycobacterium abscessus.

Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen Mycobacterium abscessus We recently identified two aminoglycoside-acetylating genes [aac(2') and eis2] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. M. abscessus BLASTP analysis identified MAB_2385 as a close homologue of the 3″-O-phosphotransferase [APH(3″)] from the opportunistic pathogen Mycobacterium fortuitum as a putative streptomycin resistance determinant. Heterologous expression of MAB_2385 in Mycobacterium smegmatis increased the streptomycin MIC, while the gene deletion mutant M. abscessus ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in M. abscessus ΔMAB_2385. This demonstrates that MAB_2385 encodes a specific and prime innate streptomycin resistance determinant in M. abscessus We further explored the feasibility of applying rpsL-based streptomycin counterselection to generate gene deletion mutants in M. abscessus Spontaneous streptomycin-resistant mutants of M. abscessus ΔMAB_2385 were selected, and we demonstrated that the wild-type rpsL is dominant over the mutated rpsLK43R in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an rpsL-based counterselection method in M. abscessus.

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants.

A whole-genome sequencing (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant Campylobacter jejuni and Campylobacter coli isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [aph(2″)-Ii1 and aph(2″)-Ii2 ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in Campylobacter: a lincosamide nucleotidyltransferase gene [lnu(G)], likely associated with lincomycin resistance, and two resistance enzyme genes (spw and apmA) similar to those found in Staphylococcus aureus, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a C. coli isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in Campylobacter and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing.

Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan.

This study describes highly aminoglycoside-resistant Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates obtained from an inpatient in Okinawa, Japan, with no known record of traveling overseas. The minimum inhibitory concentrations of amikacin and arbekacin against these strains were >1024 µg/ml. Whole-genome sequencing analysis revealed that these isolates harbored armA, which encodes a 16S rRNA methylase, ArmA, that confers pan-aminoglycoside resistance. This is the second report of K. pneumoniae harboring armA and the first report of K. oxytoca harboring a 16S rRNA methylase encoding gene in Japan.

ArmA and RmtB Were the Predominant 16S RMTase Genes Responsible for Aminoglycoside-resistant Isolates in Korea.

Pathogenic gram-negatives that produce 16S ribosomal RNA methyltransferases (16S RMTases) have already been distributed all over the world. To investigate the predominance of aminoglycoside resistance associated with 16S RMTases in Korea, we collected a total of 222 amikacin resistant Gram-negative clinical isolates from patient specimens between 1999 and 2015 from three hospital banks across Korea. ArmA and rmtB were the predominant 16S RMTase genes responsible for aminoglycoside-resistant isolates circulating in Korean community settings although only one rmtA-producing isolate was detected in 2006.