rpsJ Gene Research Articles

Sequence specificity defines the effectiveness of PPMOs targeting Pseudomonas aeruginosa.

Development of new therapeutics against antibiotic resistant pathogenic bacteria is recognized as a priority across the globe. We have reported using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) as species-specific antibiotics. The oligo sequences, 11 bases are designed to be complementary to specific essential genes near the Shine-Dalgarno site and inhibit translation. Here, we analyzed target specificity and the impact of genetic mutations on lead PPMOs targeting the rpsJ or acpP gene of Pseudomonas aeruginosa. Mutants in P. aeruginosa PAO1 were generated with four, two, or one base-pair mutations within the 11-base target sequence of the rpsJ gene. All mutants exhibited increased MICs compared to wild-type PAO1 when treated with the RpsJ PPMO, and the increase in the MICs was proportional to the number of base-pair mutations. Among single base-pair mutants, mutations in the middle of the sequence were more impactful than mutations in 5' or 3' end of the sequence. The increased MICs shown by the rpsJ mutants could be reversed by PPMOs designed to target the mutated rpsJ sequence. BALB/c mice infected intratracheally with mutants demonstrated increased lung burden when treated with RpsJ PPMO compared to wild-type PAO1-infected mice treated with RpsJ PPMO. Treating mice with a PPMOs designed to specifically target the mutant sequence was more effective against these mutant strains. These experiments confirm target specificity of two lead P. aeruginosa PPMOs and illustrate one potential mechanism of resistance that could emerge from an antisense approach.

Exploring the third-generation tetracycline resistance of multidrug-resistant livestock-associated methicillin-resistant Staphylococcus aureus ST9 across healthcare settings in China.

The overuse of antibiotics in livestock is contributing to the burden of antimicrobial resistance in humans, representing a One Health challenge. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has recently become a growing concern, and ST9 is the major LA-MRSA lineage in China and has emerged in clinical settings. Antimicrobial susceptibility testing was used to evaluate the tetracycline resistance of ST9 MRSA collections, and gene cloning experiments were performed to explore the resistance mechanisms. Whole-genome sequencing and comparative genomics were used to analyse the genetic features of clinical ST9 isolates. A phylogenetic tree was constructed to investigate the relationship of human- and livestock-derived ST9 isolates. Clinical ST9 isolates were found to possess several types of resistance genes and resistance-related mutations and were multidrug-resistant. Notably, all clinical ST9 isolates were resistant to third-generation tetracyclines. Cloning experiments showed that both the acquisition of the tetracycline resistance gene tet(L)/tet(63) and a mutation in the rpsJ gene contributed to third-generation tetracycline resistance. Phylogenetic analysis showed that the ST9 isolates collected in healthcare systems were probably transmitted from livestock. The ST9 lineage underwent multiple interspecies recombination events and gained many resistance elements. Furthermore, the resistance to third-generation tetracyclines may have evolved under tetracycline pressure in livestock. The evolution of ST9 MRSA in livestock and transmission of this clone between humans and livestock highlight the importance of establishing control strategies with the One Health approach to reduce the burden of antibiotic resistance.

Analysis of Efflux Pump System and Other Drug Resistance Related Gene Mutations in Tigecycline-Resistant Acinetobacter baumannii.

The isolation of tigecycline-resistant Acinetobacter baumannii in recent years has brought great difficulties to clinical prevention and treatment. To explore the effect of efflux pump system and other resistance related gene mutations on tigecycline resistance in Acinetobacter baumannii. Fluorescence quantitative PCR was used to detect the expression levels of major efflux pump genes (adeB, adeJ, and adeG) in extensive drug-resistant Acinetobacter baumannii. The minimum inhibitory concentration (MIC) of tigecycline was detected by the broth microdilution testing and efflux pump inhibition experiment to assess the role of efflux pump in tigecycline resistance of Acinetobacter baumannii. Efflux pump regulatory genes (adeR and adeS) and tigecycline resistance related genes (rpsJ, trm, and plsC) were amplified by PCR and sequenced. By sequence alignment, tigecycline sensitive and tigecycline-insensitive Acinetobacter baumannii were compared with standard strains to analyze the presence of mutations in these genes. The relative expression of adeB in the tigecycline-insensitive Acinetobacter baumannii was significantly higher than that in the tigecycline sensitive Acinetobacter baumannii (114.70 (89.53-157.43) vs 86.12 (27.23-129.34), P = 0.025). When efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added, the percentage of tigecycline-insensitive Acinetobacter baumannii with tigecycline MIC decreased was significantly higher than that of tigecycline-sensitive Acinetobacter baumannii (10/13 (76.9%) vs 26/59 (44.1%)), P = 0.032); the relative expression of adeB in the MIC decreased group was significantly higher than that in the MIC unchanged group (110.29 (63.62-147.15) vs 50.06 (26.10-122.59), P = 0.02); The relative expression levels of efflux pumps adeG and adeJ did not increase significantly, and there was no significant difference between these groups. One adeR point mutation (Gly232Ala) and eight adeS point mutations (Ala97Thr, Leu105Phe, Leu172Pro, Arg195Gln, Gln203Leu, Tyr303Phe, Lys315Asn, Gly319Ser) were newly detected. Consistent mutations in trm and plsC genes were detected in both tigecycline-insensitive and tigecycline-sensitive Acinetobacter baumannii, but no mutation in rpsJ gene was detected in them. Tigecycline-insensitive Acinetobacter baumannii efflux pump adeABC overexpression was an important mechanism for tigecycline resistance, and the mutations of efflux pump regulator genes (adeR and adeS) are responsible for adeABC overexpression. The effect of trm, plsC, and rpsJ gene mutations on the development of tigecycline resistance in Acinetobacter baumannii remains controversial.

Hydrogel Droplet Microarray for Genotyping Antimicrobial Resistance Determinants in Neisseria gonorrhoeae Isolates.

A multiplex assay based on a low-density hydrogel microarray was developed to identify genomic substitutions in N. gonorrhoeae that determine resistance to the currently recommended treatment agents ceftriaxone and azithromycin and the previously used drugs penicillin, tetracycline, and ciprofloxacin. The microarray identifies 74 drug resistance determinants in the N. gonorrhoeae penA, ponA, porB, gyrA, parC, rpsJ, mtrR, blaTEM, tetM, and 23S rRNA genes. The hydrogel elements were formed by automated dispensing of nanoliter-volume droplets followed by UV-induced copolymerization of NH2-containing oligonucleotides with gel-forming monomers. Polybutylene terephthalate plates without special modifications were used as microarray substrates. Sequences and concentrations of immobilized oligonucleotides, gel composition, and hybridization conditions were carefully selected, and the median discrimination ratio ranged from 2.8 to 29.4, allowing unambiguous identification of single-nucleotide substitutions. The mutation identification results in a control sample of 180 N. gonorrhoeae isolates were completely consistent with the Sanger sequencing results. In total, 648 clinical N. gonorrhoeae isolates obtained in Russia during the last 5 years were analyzed and genotyped using these microarrays. The results allowed us to draw conclusions about the present situation with antimicrobial susceptibility of N. gonorrhoeae in Russia and demonstrated the possibility of using hydrogel microarrays to control the spread of antibiotic resistance.

Characterization of Tigecycline Resistance Among Tigecycline Non-susceptible Klebsiella pneumoniae Isolates From Humans, Food-Producing Animals, and in vitro Selection Assay.

Emergence of extensively drug-resistant isolates of Klebsiella pneumoniae has prompted increased reliance on the last-resort antibiotics such as tigecycline (TGC) for treating infections caused by these pathogens. Consumption of human antibiotics in the food production industry has been found to contribute to the current antibiotic resistance crisis. In the current study, we aimed to investigate the mechanisms of TGC resistance among 18 TGC-non-susceptible (resistant or intermediate) K. pneumoniae (TGC-NSKP) isolates obtained from human (n = 5), food animals (n = 7), and in vitro selection experiment (n = 6). Isolates were genotyped by multilocus sequence typing (MLST). ramR, acrR, rpsJ, tetA, and mgrB (for colistin resistance) genes were sequenced. The presence of tetX, tetX1, and carbapenemase genes was examined by PCR. Susceptibility to different classes of antibiotics was evaluated by disc diffusion and broth macrodilution methods. The expression level of acrB was quantified by RT-qPCR assay. The 12 TGC-NSKP isolates [minimum inhibitory concentrations (MICs) = 4–32 mg/l] belonged to 10 distinct sequence types including ST37 (n = 2), ST11, ST15, ST45, ST1326 (animal isolates); ST147 (n = 2, human and animal isolates); and ST16, ST377, ST893, and ST2935 (human isolates). Co-resistance to TGC and colistin was identified among 57 and 40% of animal and human isolates, respectively. All human TGC-NSKP isolates carried carbapenemase genes (blaOXA–48, blaNDM–1, and blaNDM–5). tetX/X1 genes were not detected in any isolates. About 83% of TGC-NSKP isolates (n = 15) carried ramR and/or acrR alterations including missense/nonsense mutations (A19V, L44Q, I141T, G180D, A28T, R114L, T119S, Y59stop, and Q122stop), insertions (positions +205 and +343), or deletions (position +205) for ramR, and R90G substitution or frameshift mutations for acrR. In one isolate ramR amplicon was not detected using all primers used in this study. Among seven colistin-resistant isolates, five harbored inactivated/mutated MgrB due to premature termination by nonsense mutations, insertion of IS elements, and frameshift mutations. All isolates revealed wild-type RpsJ and TetA (if present). Increased expression of acrB gene was detected among all resistant isolates, with the in vitro selected mutants showing the highest values. A combination of RamR and AcrR alterations was involved in TGC non-susceptibility in the majority of studied isolates.

Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli.

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Molecular characterization of carbapenem-resistant Enterobacteriaceae and emergence of tigecycline non-susceptible strains in the Henan province in China: a multicentrer study.

Introduction Carbapenem-resistant Enterobacteriaceae (CRE) have been responsible for nosocomial outbreaks worldwide and have become endemic in several countries.Hypothesis/Gap Statement To better understand the epidemiological trends and characteristics of CRE in the Henan province.Aim We assessed the molecular epidemiological characteristics of 305 CRE strains isolated from patients in 19 secondary or tertiary hospitals in ten areas of the Henan province in China.Methodology A total of 305 CRE isolates were subjected to multiple tests, including in vitro antimicrobial susceptibility testing, PCR for carbapenemase genes bla KPC, bla NDM, bla IMP, bla VIM, bla OXA-48-like. Tigecycline-resistant genes ramR, oqxR, acrR, tetA, rpsJ, tetX, tetM, tetL were analysed in five tigecycline non-susceptible carbapenem-resistant Klebsiella pneumoniae isolates (TNSCRKP). Additionally, multilocus sequence typing (MLST) was performed for carbapenem-resistant K. pneumoniae (CRKP).Results The most common CRE species were K. pneumoniae (234, 77 %), Escherichia coli (36, 12 %) and Enterobacter cloacae (13, 4 %). All strains exhibited multi-drug resistance. Overall, 97 % (295/305) and 97 % (297/305) of the isolates were susceptible to polymyxin B and tigecycline, respectively. A total of 89 % (271/305) of the CRE isolates were carbapenemase gene-positive, including 70 % bla KPC, 13 % bla NDM, 6 % bla IMP, and 1 % combined bla KPC/bla NDM genes. K. pneumoniae carbapenemase (KPC) was the predominant carbapenemase in K. pneumoniae (87 %), whereas NDM and IMP were frequent in E. coli (53 %) and E. cloacae (69 %), respectively. Mutations in the ramR, tetA, and rpsJ genes were detected in five TNSCRKP. Moreover, 15 unique sequence types were detected, with ST11 (74 %), ST15 (9 %) and ST2237 (5 %) being dominant among K. pneumoniae strains.Conclusion A high proportion of CRE strains were carbapenemase-positive, and five carbapenem-resistant K. pneumonia isolates were tigecycline non-susceptible, indicating a need for the ongoing surveillance of CRE and effective measures for the prevention of CRE infections.

In vitro activity of eravacycline and mechanisms of resistance in enterococci

Eravacycline (ERC), the first fluorocycline, is a new tetracycline with superior activity to tigecycline (TGC) against many bacterial species. This work aimed to determine the in vitro activity of ERC compared with other tetracyclines against enterococcal clinical isolates and to analyse corresponding resistance mechanisms. A collection of 60 enterococcal strains was studied: 54 epidemiologically unrelated clinical isolates (46 Enterococcus faecium and 8 Enterococcus faecalis) including 42 vancomycin-resistant enterococci (VRE) (33 vanA and 9 vanB), 3 in vitro TGC-resistant mutants (E. faecium AusTig, HMtig1 and HMtig2) and 3 reference wild-type strains (E. faecium Aus0004 and HM1070, E. faecalis ATCC 29212). In vitro susceptibility was determined using Etest strips (for ERC) or by broth microdilution (for TGC, doxycycline, minocycline and tetracycline). Resistance genes [tet(M), tet(L), tet(O) and tet(S)] were screened by PCR for TGC- and/or ERC-resistant strains as well as sequencing of the rpsJ gene (encoding ribosomal protein S10). MIC50/90 values were 0.016/0.08, ≤0.03/0.5, 4/32, 8/16 and 32/>32 mg/L for ERC, TGC, doxycycline, minocycline and tetracycline, respectively. According to EUCAST guidelines, nine strains were categorised as resistant to TGC (MIC, 0.5-8 mg/L), including four E. faecium vanA(+) strains also resistant to ERC (MIC, 0.19-1.5 mg/L). These four strains all possessed at least one mutation in rpsJ and two tet determinants: tet(M)+tet(L) (n=2); and tet(M)+tet(S) (n=2). Although ERC has excellent in vitro activity against enterococci (including VRE), emergence of resistance is possible due to combined mechanisms (rpsJ mutations+tet genes).

Change of Hypermucoviscosity in the Development of Tigecycline Resistance in Hypervirulent Klebsiella pneumoniae Sequence Type 23 Strains.

In this study, we developed tigecycline resistance in Klebsiella pneumoniae ST23 strains in vitro and investigated the change in virulence associated with hypermucoviscosity. In vitro-induced tigecycline-resistant (TGC-IR) K. pneumoniae mutants were obtained from three tigecycline-susceptible (TGC-S) strains, belonging to ST23 and serotype K1, by culturing in media with tigecycline in a stepwise manner. An antimicrobial susceptibility test, string test, mucoviscosity assay, and capsular polysaccharide (CPS) quantification were performed. Biofilm formation and serum resistance were evaluated, and survival rates of bacterial strains in fruit flies and macrophages were measured. Alterations of rpsJ, ramR, soxR, acrR, and marR genes were investigated and the expression levels of ramA and efflux pump genes were evaluated. The hypermucoviscosity phenotype was dramatically decreased in the TGC-IR mutants. Reduced CPS production in TGC-IR mutants was also identified. Increased resistance to most other antimicrobial agents was found in TGC-IR mutants. In addition, the TGC-IR mutants exhibited reduced biofilm formation, low serum resistance, and decreased survival rates within fruit flies and macrophages. Our study shows that development of tigecycline resistance in hypervirulent K. pneumoniae strains result in defects in virulence associated with hypermucoviscosity.

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

BackgroundThe emergence and transmission of tigecycline‐ and carbapenem‐resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem‐resistant K pneumoniae (CRKP) isolates.MethodsForty‐five non‐duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (MLST), and pulsed‐field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection and mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes, which are related to tigecycline resistance. The expression levels of efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by quantitative real‐time PCR.ResultsThe resistance rate to tigecycline in CRKP isolates was 37.8% (17/45). K pneumoniae ST307 was a predominant clone type (70.6%, 12/17) among the TCRKP isolates. The expression levels of acrB (P < .001) and marA (P = .009) were significantly higher in the tigecycline‐resistant group than in the tigecycline‐intermediate and tigecycline‐susceptible groups. Increased expression of acrB was associated with marA expression (r = 0.59, P = .013).ConclusionsWe found that the activated MarA‐induced overexpression of AcrAB efflux pump plays an important role in the emergence of tigecycline resistance in CRKP isolates.

Rapid Emergence of a Pandrug-Resistant Klebsiella pneumoniae ST11 Isolate in an Inpatient in a Teaching Hospital in China After Treatment with Multiple Broad-Spectrum Antibiotics.

PurposePandrug-resistant K. pneumoniae is still rare in China, and the lack of studies limits our understanding of the emergence mechanism of pandrug-resistant K. pneumoniae. Here, we report the rapid emergence of a pandrug-resistant K. pneumoniae ST11 strain in an inpatient after treatment with multiple broad-spectrum antibiotics in China.Patients and MethodsK. pneumoniae strain KP65 was isolated from a 55-year-old male patient hospitalized in the Department of Intensive Care Unit (ICU) of a teaching hospital in China. Antimicrobial susceptibility testing was conducted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The complete genome sequence of the strain was determined using the Illumina NovaSeq 6000 platform and long-read MinION sequencer. Genomic features and resistance mechanisms of the strain were comprehensively analysed using various bioinformatics approaches.ResultsK. pneumoniae strain KP65 was found to be resistant to all antibiotics tested, including beta-lactams, aminoglycosides, quinolones, tigecycline and colistin. Seven resistance genes were identified in the genome. The carbapenem-resistant gene blaKPC-2 and extended-spectrum β-lactamase (ESBL)-producing gene blaCTX-M-65 are located on the IncFII-type plasmid pKPC-2-KP65. No mcr genes were detected in the genome, but an IS5 insertion element was found at position 117 of the mgrB gene. Regarding the rpsJ gene, single-base substitution, G169C, leading to the amino acid substitution V57L was also identified. According to in silico MLST analysis, K. pneumoniae KP65 belongs to sequence type ST11. The closest relative of K. pneumoniae KP65 is another ST11 K. pneumoniae strain, which was isolated from a bloodstream infection in Hangzhou, differing by only 53 cgMLST loci.ConclusionUnder the selective pressure of antibiotics, the KPC-2-producing K. pneumoniae ST11 strain can easily evolve pandrug resistance through chromosomal mutations. More attention is required to monitor the prevalence of the KPC-2-producing K. pneumoniae ST11 strain in China.

Biological microchip for assessing tetracycline-resistance in &lt;i&gt;Neisseria gonorrhoeae&lt;/i&gt; clinical isolates in Russian Federation

A total of 399 Neisseria gonorrhoeae clinical isolates collected in different regions of the Russian Federation in 20152017 were analyzed for tetracycline susceptibility and genetic markers of resistance. Drug susceptibility testing was performed by serial dilution method in agar and minimum inhibitory concentration (MIC) was measured according to the Russian “Guidelines for microbial susceptibility testing for antibacterial agents No. 4.2.1890-04”. Tetracycline resistance determinants were studied by using hydrogel microarray with immobilized oligonucleotide probes able to identify a series of chromosomal mutations and detect plasmid tetM gene. Different resistance determinants were found in 193 isolates (48.4%). Mutation in codon 57 in the rpsJ gene (41.2%) was most common that decreases tetracycline affinity to ribosome 30S subunit, mainly due to Val57Met substitution both as a point mutation as well as in combination with others. Mutations in the rpsJ gene were found in strains with the intermediate tetracycline susceptibility. Mutations in the porB gene (lower tetracycline influx) held the se cond place in prevalence pattern (23.1%); the Gly120Lys substitution usually led to emergence of tetracycline resistance either as a point mutation or in combination with other substitutions. Substitutions of Gly120 for other residues (Asp, Asn, and Thr) and Ala121 for Asp, Asn, and Gly had much less effect on resistance level. The –35 delA deletion in the promoter region of mtrR gene (increased expression of MtrC-MtrD-MtrE efflux pump) was observed in 11.3% strains. The tetM gene was found in 27 strains including 17 American and 10 Dutch type tetM determinants. Evolutionary tree was constructed for the tetM genes with the estimation of their homology with similar genes in genera Streptococcus, Enterococcus and Mycoplasma. Mutations in chromosomal genes resulted in increase of tetracycline MIC up to 2–4 mg/L; 4 mg/L MIC was observed in case of simultaneous presence of several mutations. Strains bearing tetM gene-containing plasmid showed extremely high resistance level: MIC ≥ 8 mg/L (64 mg/L for the two samples). Thus, long-lasting withdrawal of tetracycline use for treatment of gonococcal infections in Russia (since 2003) resulted in decreased percentage of resistant strains (including strains with intermediate susceptibility) from 75% down to 45.4%. However, currently tetracycline resistance in Russia remains elevated that is explained by the presence of different resistance determinants in the half of isolates under study.

Tigecycline resistance caused by rpsJ evolution in a 59-year-old male patient infected with KPC-producing Klebsiella pneumoniae during tigecycline treatment

Carbapenem resistant Klebsiella pneumoniae (CRKP) nosocomial infection increased rapidly in recent years. By far, the anti-infection drugs for CRKP infection are limited. Tigecycline is one of the last resort treatments for CRKP infections. In this study, curative effect of tigecycline therapy was monitored in a 59-year-old male patient infected with KPC-producing K. pneumoniae. Consecutive clonal consistent K. pneumoniae isolates were cultured during tigecycline treatment. Whole genome sequencing of the isolates was performed, and bioinformatics analysis was further performed. Five isolates, four of which were susceptible and one resistant were collected. All of the isolates belong to Sequence Type 11 (ST11), and harbouring 11 gene sequences relevant to antibiotic resistance including blaKPC-2. One amino acid substitution V57L in rpsJ was identified in the tigecycline resistant isolates. Subsequent transformation experiment confirmed the contribution of the rpsJ variant (V57L) to reduced tigecycline susceptibility. To our knowledge, this study is the first report to provide direct in vivo evidence that evolution in the rpsJ gene can lead to tigecycline resistance in patients infected with KPC-producing K. pneumoniae during tigecycline treatment. This finding serves as a therapeutic warning as the rpsJ gene is on the chromosome of CRKP strains. Under selective pressure from tigecycline, the rpsJ mutation may occur and lead to tigecycline resistant.

Antimicrobial susceptibility of bifidobacteria from probiotic milk products and determination of the genetic basis of tetracycline resistance in Enterococcus species after in vitro conjugation with Bifidobacterium animalis subsp. lactis

The study aimed at investigating the antimicrobial resistance (AMR) profiles of bifidobacteria from fermented milk products sold in the UK that are claimed to have beneficial effects on humans, and assess the potential transferability of AMR to other bacteria.Isolates of different Bifidobacterium species were screened for their susceptibility to 24 antimicrobials and the presence of AMR genes [tet(M), tet(L), tet(S), tet(Q), tet(K), tet(O), tet(W), aph(3″)-I, ant(2″)-I, aph(3″)-III, strA, strB, aadA, aadE, erm(A), erm(B), and erm(C)]. The ability of Bifidobacterium animalis subsp. lactis isolates to transfer the tet(W) gene to Enterococcus faecalis JH2-2 and Enterococcus faecium BM4105 was investigated by conjugation. Potential E. faecalis transconjugants (PETs) were analysed by whole genome sequencing (WGS) for the presence of tet(W), other DNA fragments from the donor and single-nucleotide polymorphisms (SNPs). A high level of resistance to aminoglycosides and a moderate level of resistance to tetracycline were observed, while susceptibility to macrolides, vancomycin, chloramphenicol and beta-lactams was determined. Only the tet(W) gene was detected in all B. animalis subsp. lactis isolates. Some PETs exhibited a significant tetracycline MIC increase from 1 to up to 16 mg/L. WGS did not detect tet(W) in the PETs, but SNPs in the rpsJ gene encoding the ribosomal protein S10, which is part of the 30S ribosomal subunit and contains a proposed tetracycline binding site. Substitutions (Tyr-58→Asp, Tyr-58→Cys, Asp-60→Tyr, Tyr-58→Ser, Ser-61→Tyr, Ala-54→Glu, Asp-60→Tyr) and an insertion of Thr between Thr55 and His56 were detected. No E. faecium potential transconjugants were recovered.B. animalis subsp. lactis isolates did not transfer the tet(W) gene to the Enterococcus species. The majority of the PETs had mutations within or near the ribosomal protein S10 vertex loop suggesting that mutations in the rpsJ gene confer tetracycline resistance to E. faecalis JH2-2.

Genotype Diversity in the Local Arkhangelsk (Russia) Neisseria gonorrhoeae Population: Emergence Mechanisms and Relation with Antimicrobial Resistance

132 clinical isolates of N. gonorrhoeae collected in Arkhangelsk region during 2014–2016 were analyzed in accordance with the NG-MAST protocol (Neisseria gonorrhoeae Multi Antigen Sequence Typing). A diversity of the analyzed strains was represented by 53 NG-MAST sequence-types; 30 of them were new registered due to earlier unknown either porB and tbpB sequences or their combinations. Three pathways of the diversity formation are supposed: long-term circulation of some NG-MAST-types with sequential transmission via the “sexual chains” (40.1% of the total population); introduction of new molecular types (27.3%); appearance of point mutations in porB and tbpB genes with the emergence of previously unknown genotypic variants of N. gonorrhoeae (32.6%). Emerged NG-MAST types inherited the mutational profile in penA, ponA, rpsJ, gyrA, and parC genes followed by conservation of antimicrobial resistance in the local N. gonorrhoeae population, whereas these determinants do not play a significant role in contemporary gonococci molecular evolution.

Tetracycline resistance of Neisseria gonorrhoeae in Russia, 2015–2017

The objective of this study was to estimate the tetracycline resistance level in the modern population of Neisseria gonorrhoeae in the Russian Federation, where this drug was removed from the treatment regimen for gonococcal infections in 2003. A total of 401 isolates collected between 2015 and 2017 were analyzed for genetic markers (chromosomal porB, rpsJ and mtrR gene mutations and the plasmid-located tetM gene) involved in tetracycline resistance. Antibiotic susceptibility testing revealed that 19% of the strains were tetracycline resistant (MIC > 1 mg/L) and that 10% of the strains had intermediate susceptibility (0.5 < MIC ≤ 1 mg/L). Various combinations of mutations identified in the rpsJ (Val57Met/Leu), porB (Gly120Lys/Asp/Asn/Thr and Ala121/Asp/Asn/Gly), and mtrR (−35 del A) genes resulted in MIC increases of up to 1.47 mg/L (geometric mean value). The presence of the tetM gene was detected in 29 strains, including 18 tetM genes of the American type and 11 of the Dutch type. The tetM gene was associated with a strong increase in resistance (MIC > 8 mg/L). One N. gonorrhoeae isolate was found to carry a defective tetM gene with an AG deletion at position 1239–1240, а new stop codon was introduced that caused a defect in TetM protein synthesis and decrease in the tetracycline resistance. Phylogenetic trees constructed using N. gonorrhoeae NG-MAST and tetM loci were compared. Complex relationship was observed between the N. gonorrhoeae sequence type and the tetM plasmid type. Partial recovery of N. gonorrhoeae tetracycline susceptibility was observed relative to the proportion of isolates with resistance detected ten years ago (75%). However, the current levels of tetracycline resistance still preclude the renewed use of these drugs for gonococcal infection therapy.

Oligonucleotide Microchip for the Identification of Infectious Agents of Reproductive System with Simultaneous Analysis of Determinants of Resistance to Antimicrobial Substances.

We developed a multiplexed DNA microarray-based assay allowing identification of 12 causative agents of reproductive tract infections with the simultaneous detection of 47 genetic determinants of resistance to antimicrobial substances. The microarray was tested on 93 isolates of Neisseria gonorrhoeae, 32 isolates of Treponema pallidum and 29 samples of Ureaplasma spp./Mycoplasma spp. The N. gonorrhoeae isolates had multiple mutations in the penA, ponA, rpsJ, gyrA, parC, and mtrR genes; their prognostic value significantly increased when combinations of mutations were detected. In the analyzed T. pallidum isolates, single A2058G substitution in the 23S rRNA gene responsible for macrolide resistance was found. DNA sequences of Ureaplasma spp./Mycoplasma spp. were determined as wild type, which was not fully consistent with the results of analysis of their antimicrobial susceptibility.

Drug resistance mutations and susceptibility phenotypes of Neisseria gonorrhoeae isolates in Russia

Steady growth in the degree of antimicrobial resistance in Neisseria gonorrhoeae calls for the control of the spreading of resistance mutations. Here we present the data describing drug resistance mutations, the results of antimicrobial susceptibility tests, and molecular genotypes of 128 recent N. gonorrhoeae isolates collected across 9 regions of the Russian Federation. The mutations in chromosome genes penA, ponA, rpsJ, gyrA, parC, which determine the susceptibility of N. gonorrhoeae to penicillins, tetracyclines, and fluoroquinolones were detected by multiplex amplification followed by hybridization on a hydrogel microarray. The most frequent mutation was an insertion of an aspartate at position 345 of penA gene (76.6%), whereas mutations Leu421Pro in ponA gene, Val57Met in rpsJ gene, Ser91Phe in gyrA gene, Asp95Gly in gyrA gene, and Ser87Arg in parC gene were detected in 32.8-36.7% of strains. One third of studied N. gonorrhoeae isolates harbored multiple drug resistance mutations in bacterial chromosome, resulting in the bimodal distribution of mutation profiles and related patterns of antimicrobial susceptibility. The spread of multiple resistance could be explained by the vertical transfer of the mutations resulting in the clonality of the N. gonorrhoeae population.

Step-Wise Increase in Tigecycline Resistance in Klebsiella pneumoniae Associated with Mutations in ramR, lon and rpsJ.

Klebsiella pneumoniae is a gram-negative bacterium that causes numerous diseases, including pneumonia and urinary tract infections. An increase in multidrug resistance has complicated the treatment of these bacterial infections, and although tigecycline shows activity against a broad spectrum of bacteria, resistant strains have emerged. In this study, the whole genomes of two clinical and six laboratory-evolved strains were sequenced to identify putative mutations related to tigecycline resistance. Of seven tigecycline-resistant strains, seven (100%) had ramR mutations, five (71.4%) had lon mutations, one (14.2%) had a ramA mutation, and one (14.2%) had an rpsJ mutation. A higher fitness cost was observed in the laboratory-evolved strains but not in the clinical strains. A transcriptome analysis demonstrated high expression of the ramR operon and acrA in all tigecycline-resistant strains. Genes involved in nitrogen metabolism were induced in the laboratory-evolved strains compared with the wild-type and clinical strains, and this difference in nitrogen metabolism reflected the variation between the laboratory-evolved and the clinical strains. Complementation experiments showed that both the wild-type ramR and the lon genes could partially restore the tigecycline sensitivity of K. pneumoniae. We believe that this manuscript describes the first construct of a lon mutant in K. pneumoniae, which allowed confirmation of its association with tigecycline resistance. Our findings illustrate the importance of the ramR operon and the lon and rpsJ genes in K. pneumoniae resistance to tigecycline.

Molecular epidemiology of drug-resistant Neisseria gonorrhoeae in Russia (Current Status, 2015).

BackgroundThe widespread distribution of Neisseria gonorrhoeae strains that are resistant to previously used and clinically implemented antibiotics is a significant global public health problem. In line with WHO standards, the national Gonococcal Antimicrobial Surveillance Programme (RU-GASP) has been in existence in Russia since 2004; herein, the current status (2015) is described, including associations between N. gonorrhoeae antimicrobial susceptibility, primary genetic resistance determinants and specific strain sequence types.MethodsA total of 124 N. gonorrhoeae strains obtained from 9 regions in Russia in 2015 were examined using N. gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST), an antimicrobial susceptibility test according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria and an oligonucleotide microarray for the identification of mutations in the penA, ponA, rpsJ, gyrA and parC genes responsible for penicillin G, tetracycline, and fluoroquinolone resistance. Genogroup (G) isolates were evaluated based on their porB and tbpB sequence types (STs).ResultsNG-MAST analysis showed a diversified population of N. gonorrhoeae in Russia with 58 sequence types, 35 of which were described for the first time. The STs 807, 1544, 1993, 5714, 9476 and 12531, which were typical for some Russian Federation regions and several countries of the former Soviet Union, were represented by five or more isolates. The internationally widespread ST 1407 was represented by a single strain in the present study. Division into genogroups facilitated an exploration of the associations between N. gonorrhoeae sequence type, antimicrobial resistance spectra and genetic resistance determinant contents. Preliminarily susceptible (G-807, G-12531) and resistant (G-5714, G-9476) genogroups were revealed. The variability in the most frequently observed STs and genogroups in each participating region indicated geographically restricted antimicrobial susceptibility in N. gonorrhoeae populations.ConclusionsResistance or intermediate susceptibility to previously recommended antimicrobials, such as penicillin G (60.5 %), ciprofloxacin (41.1 %) and tetracycline (25 %), is common in the N. gonorrhoeae population. Based on previous reports and current data, ceftriaxone and spectinomycin should be recommended for first-line empiric antimicrobial monotherapy for gonorrhoea in Russia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1688-7) contains supplementary material, which is available to authorized users.