Macrolide, Lincosamide And Streptogramin B Resistance Research Articles

Distribution of Macrolide, Lincosamide, and Streptogramin B and Detection of erm Genes in Staphylococcus aureus from Wounds in Uyo, Nigeria

The increasing resistance to macrolide, lincosamide, and streptogramin B (MLSB) among methicillin-resistant Staphylococcus aureus (MRSA) is a challenge. The study determined the constitutive and inducible MLSB resistance and erm genes in S. aureus from wounds using the erythromycin-clindamycin D-zone test and a multiplex polymerase chain reaction assay, respectively. Of the 260 patients recruited in the study, S. aureus was isolated from the wounds of 102 patients, giving a prevalence rate of 39.2%. Of the 102 S. aureus isolates, 32.4% were methicillin-sensitive S. aureus (MSSA), and 67.6% were MRSA. The prevalence of S. aureus in wounds was higher in females (39.8%) than in males (38.4%). The highest prevalence of S. aureus was found in divorce patients (59.1%) and those residing in urban areas (39.7%). There was no statistical difference between the occurrence of MRSA and MSSA in wounds based on the sex (p = 0.97), age (p = 0.08), and marital status (p = 0.41) of the patients. All (100%) MRSA were resistant to Cefoxitin, and chloramphenicol, while ˂ 50% of MRSA were resistant to Tetracycline, Ciprofloxacin, and Gentamycin. Of the 69 MRSA isolates from the wounds, 21.7% and 26.1% were iMLSB and cMLSB phenotypes, respectively. Nine (9) MSSA were iMLSB phenotypes, and eight MSSA were cMLSB phenotypes. Among the 12 representative isolates, three (3) MRSA and one (1) MSSA isolate possessed the ermC gene. This study has revealed that screening tests for iMLSB-resistant S. aureus strains are critical for the therapeutic management of wound infections caused by S. aureus.

Helcococcus kunzii methyltransferase Erm(47) responsible for MLSB resistance is induced by diverse ribosome-targeting antibiotics.

To determine the mechanism of induction of erm(47) and its atypical expression in the Gram-positive opportunistic pathogen Helcococcus kunzii, where it confers resistance to a subset of clinically important macrolide, lincosamide and streptogramin B (MLSB) antibiotics. The resistant H. kunzii clinical isolate UCN99 was challenged with subinhibitory concentrations of a wide range of ribosome-targeting drugs. The methylation status of the H. kunzii ribosomal RNA at the MLSB binding site was then determined using an MS approach and was correlated with any increase in resistance to the drugs. The H. kunzii erm(47) gene encodes a monomethyltransferase. Expression is induced by subinhibitory concentrations of the macrolide erythromycin, as is common for many erm genes, and surprisingly also by 16-membered macrolide, lincosamide, streptogramin, ketolide, chloramphenicol and linezolid antibiotics, all of which target the 50S ribosomal subunit. No induction was detected with spectinomycin, which targets the 30S subunit. The structure of the erm(47) leader sequence functions as a hair trigger for the induction mechanism that expresses resistance. Consequently, translation of the erm(47) mRNA is tripped by MLSB compounds and also by drugs that target the 50S ribosomal subunit outside the MLSB site. Expression of erm(47) thus extends previous assumptions about how erm genes can be induced.

Analysis of Resistance to Macrolide-Lincosamide-Streptogramin B Among mecA-Positive Staphylococcus Aureus Isolates.

ObjectivesGenetic determinants conferring resistance to macrolide, lincosamide, and streptogramin B (MLSB) via ribosomal modification such as, erm, msrA/B and ereA/B genes are distributed in bacteria. The main goals of this work were to evaluate the dissemination of MLSB resistance phenotypes and genotypes in methicillin-resistant Staphylococcus aureus (MRSA) isolates collected from clinical samples.MethodsA total of 106 MRSA isolates were studied. Isolates were recovered from 3 hospitals in Tehran between May 2016 to July 2017. The prevalence of MLSB-resistant strains were determined by D-test, and then M-PCR was performed to identify genes encoding resistance to macrolides, lincosamides, and streptogramins in the tested isolates.ResultsThe frequency of constitutive resistance MLSB, inducible resistance MLSB and MSB resistance were 56.2%, 22.9%, and 16.6%, respectively. Of 11 isolates with the inducible resistance MLSB phenotype, ermC, ermB, ermA and ereA were positive in 81.8%, 63.6%, 54.5% and 18.2% of these isolates, respectively. In isolates with the constitutive resistance MLSB phenotype, the prevalence of ermA, ermB, ermC, msrA, msrB, ereA and ereB were 25.9%, 18.5%, 44.4%, 0.0%, 0.0%, 11.1% and 0.0%, respectively.ConclusionClindamycin is commonly administered in severe MRSA infections depending upon the antimicrobial susceptibility findings. This study showed that the D-test should be used as an obligatory method in routine disk diffusion assay to detect inducible clindamycin resistance in MRSA so that effective antibiotic treatment can be provided.

English

Widespread use of Macrolide, lincosamide and Streptogramin B (MLSB) family of antibiotics in the treatment of Staphylococcus aureus (S. aureus) has led to an increased resistance to MLSB antibiotics. The purpose of this study was to determine the frequency of MLSB resistance among clinical isolates of methicillin sensitive S. aureus (MSSA) and Methicillin resistant S. aureus (MRSA) from Mansoura University Children Hospital (MUCH), Egypt, phenotypically by using D‑test and genotypically by detection of erm genes by PCR. Different microbiological samples were collected under complete aseptic condition from patients in MUCH according to the site of infection over a period of 9 months from March 2016 to November 2016. S. aureus isolates were identified using standard microbiological methods. MRSA was detected by growth on oxacillin screen agar plate and cefoxitin disk screen test. Antimicrobial susceptibility of the isolates was determined by Kirby-Bauer disk diffusion method. S. aureus isolates that were found to be erythromycin resistant were further studied for inducible clindamycin resistance using D-zone test according to CLSI recommendations. erm genes in S. aureus isolates were detected by PCR. Among 230 S. aureus isolates, 164 were MSSA (71.3%) and 66 were MRSA (28.7%). Twenty-five MSSA (15.2%), and 37 MRSA (56.1%) isolates were erythromycin resistant. Constitutive MLSB phenotype (cMLSB) (30.3 and 4.2%) and inducible MLSB phenotype (iMLSB) (22.7 and 7.9%) were observed in MRSA and MSSA, respectively by D-zone test. The rate of iMLSB phenotype and cMLSB phenotype in MRSA was significantly higher than in MSSA isolates. The frequency of ermA, ermB and ermC genes were 72.9, 5.4 and 13.5% in MRSA isolates and 60, 4 and 12% in MSSA isolates, respectively. Screening test for of iMLSB‑resistant strains is very important by double disk diffusion test (D‑test). This phenotypic test is simple, accessible and reliable method that can be done in every laboratory and research facility, without the need of costly genetic tests. Since the treatment of patients infected with S. aureus with iMLSB phenotype with clindamycin can lead to the expansion of constitutive resistance (cMLSB) and therapy failure. Key words: Clindamycin, cMLSB, erm genes, iMLSB phenotype, MRSA, MSSA, S. aureus.

Phenotypic and Genotypic Study of Inducible Clindamycin Resistance in Clinical Isolates of Staphylococcus aureus in Tabriz, Northwest Iran

Background: Resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics is mediated by erm and msrA genes in Staphylococcus aureus. The expression of these genes can lead to three phenotypes, namely constitutive resistance (cMLSB), inducible resistance (iMLSB), which are resistant to macrolide, lincosamide, and streptogramin B antibiotics, and MSB phenotype, which is resistant only to macrolide and streptogramin B. Inducible clindamycin resistance is an important concern because it is not detected in routine laboratory tests. Objectives: The aim of this study was to determine the frequency of MLSB phenotypes and genotypes among 215 clinical isolates of S. aureus and then, examine their resistance to antibacterial agents, which is recommended for methicillin-resistant S. aureus (MRSA) isolates. Methods: Two hundred and fifteen non-repetitive clinical isolates of S. aureus were collected. Resistance to antibacterial agents was determined by disk diffusion and E-test methods. Susceptibility to clindamycin and erythromycin was tested by D-test. All isolates were screened by PCR for the presence of nucA, mecA, ermA, ermB, ermC, and msrA genes. Results: The prevalence of iMLSB, cMLSB and MSB phenotypes among all the isolates was determined as 10.69%, 34.42%, and 0%, respectively. In our study, iMLSB was prevalent more in methicillin susceptible S. aureus (MSSA) (11.71%) than MRSA (9.19%) isolates (P = 0.557). In contrast, the rate of cMLSB was significantly higher in MRSA (79.31%) than MSSA (3.90%) isolates (P = 0.000). No MSB phenotype was detected in our study. The most prevalent genes were ermC and ermA with 39% and 21.5% frequencies, respectively. Six isolates showed D phenotype, while the PCR results of erm genes were negative. All 215 isolates of S. aureus were negative for the presence of ermB and msrA genes. Conclusions: The rate of iMLSB in S. aureus isolates is relatively high in the Northwest Iran. Since isolates with inducible resistance may mutate and change to constitutive resistance, to prevent clinical treatment failure, D-test should be performed along with routine antibiotic susceptibility tests. In this study, ermC gene was the predominant genetic determinant for the expression of MLSB resistance. This predominance is probably due to the spread of distinctive clones (which carry ermC gene) in our region.

English

Clindamycin has long been an option for treating both methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant S. aureus (MRSA) infections. So, it is utmost important to perform the susceptibility test for erythromycin and clindamycin. And, there is concern on use of this antibiotic in the presence of erythromycin resistance because of the possibility of induction of cross-resistance among members of macrolide, lincosamide and streptogramin B (MLSB) group. During August 2011 to May 2012, a total of 207 isolates of S. aureus were isolated and among which 29.47% (61) isolates were confirmed as MRSA by cefoxitin (30 µg) disc. All the isolates were further processed for MLSB resistance test by double disc diffusion test of erythromycin (2 µg) and clindamycin (15 µg) at a distance of 15 and 22 mm between them. This study result show 12.56% (26) and 14.49% (30) of inducible macrolide-lincosamide-streptogramin B phenotype (iMLSB) resistance type at 22 and 15 mm disc distance, respectively, showing 15 mm disc distance is potential than 22 mm and 17.39% (36) of cMLSB resistance type. Similarly, both iMLSB and cMLSB are greater in MRSA than MSSA and constitutes 18.05 (11) and 36.06% (22), respectively. Thus, this study concludes that D-test should be used as a mandatory method and is more potential in 15 mm disc apart. Key words: Staphylococcus aureus, methicillin resistant S. aureus (MRSA), methicillin sensitive S. aureus (MSSA), inducible macrolide-lincosamide-streptogramin B phenotype (iMLSB), cMLSB, D-test.

Characterization ofEnterococcus faeciumwith Macrolide Resistance and Reduced Susceptibility to Quinupristin/Dalfopristin in a Japanese Hospital: Detection of Extensive Diversity inerm(B)-Regulator Regions

Cross-resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics is mainly mediated by the erm (erythromycin ribosome methylation) genes that encode 23S rRNA methylases in enterococi, and various mechanisms are involved in the streptogramin B resistance. Prevalence of MLSB resistance and its genetic mechanisms were analyzed for a total of 159 strains of Enterococcus faecium isolated from clinical specimens in a university hospital in Japan from 1997 to 2006. Resistance to erythromycin (EM) and clindamycin was detected in 88.1% and 89.9% of all the strains examined, respectively, and expression of resistance was totally constitutive. Although none of the strain was resistant to quinupristin/dalfopristin (Q/D), 28 strains (17.6%) showed intermediate resistance to Q/D (MIC: 2 μg/ml). The erm(B) gene was detected in 139 strains (87.4%), and msrC was found in all the strains examined, whereas no other known MLSB resistance genes were identified. The erm(B) regulator region (RR) containing a coding region of the leader peptide was classified into 13 genetic variations (L1-L3, M, S1-S7, D, and R genotypes) in 56 strains. However, no relatedness was identified between the erm(B) RR genotype and EM resistance, or reduced susceptibility to Q/D, although most of Q/D-intermediate strains were assigned to the L1, L2, and S1 genotypes. Q/D-intermediate strains were classified into five multiple-locus variable-number tandem-repeat analysis (MLVA) types, including four types of clonal complex (CC)-C1, five sequence types (STs), including four STs of CC-17, and several resistance gene/virulence factor profiles. The present study revealed the occurrence of Q/D-intermediate E. faecium, which are composed of heterogeneous strains in Japan, and more genetic diversity in the erm(B) RRs than those reported previously.

Klinik Staphylococcus İzolatlarında Makrolid, Linkozamid ve Streptogramin B Grubu Antibiyotiklere Direnç Gelişimine Neden Olan erm ve msr Genlerinin Araştırılması

Objective: Cross-resistance is an important issue for macrolide, lincosamide and streptogramin B (MLSB) antibiotics. The erm genes alter their ribosomal binding site by encoding ribosomal methylases. Phenotypic presentation of erm-mediated resistance can be inducible (iMLSB) or constitutive (cMLSB). Expression of msr genes which encode active efflux pumps confers the MSB phenotype. In this study, we investigated the frequency of MLSB resistance phenotypes and the presence of erm and msr genes in clinical Staphylococcus isolates. Material and Methods: The frequency of MLSB resistance phenotypes were investigated using D-zone test in 731 clinical Staphylococcus strains. The presence of erm and msr genes was investigated by polymerase chain reaction in macrolide-resistant strains. Results: Of the investigated isolates, 37.3% had iMLSB, 35.8% had cMLSB, and 26.9% had MSB phenotypes. Among studied, 45.9% of the strains carried ermC, 15.5% carried ermA, and 4.2% carried ermA and ermC genes. Phenotypic presentation of 51.4% of the erm gene carriers were iMLSB and 48.6% were cMLSB. Of the MSB phenotype strains, 73.3% carried the msrA+msrB gene combination and 3.3% carried msrB alone. Various erm and msr gene combinations were determined in 13.7% of the isolates of which 54.3% expressed iMLSB or cMLSB phenotypes and 45.7% expressed the MSB phenotype. MSB phenotype and gene combination frequencies were more in coagulase-negative staphylococci (CoNS). Conclusion: Investigating genes conferring resistance to lincosamides is important for reducing the risk of treatment failure especially for erythromycin resistant, clindamycin susceptible strains. Due to the increasing resistance problem in staphylococcal infections, clinicians must be aware of resistance development while prescribing MLSB antibiotics.

Macrolide, Lincosamide, and Streptogramin B–Constitutive-Type Resistance inCorynebacterium pseudodiphtheriticumIsolated from Upper Respiratory Tract Specimens

Corynebacterium pseudodiphtheriticum is commonly found in normal upper respiratory tract flora in humans. In certain conditions it can cause the opportunistic infections, especially in immunocompromised patients. In certain strains of Corynebacterium sp., the macrolide, lincosamide, and streptogramin B (MLSb) resistance mechanism related to the presence of the erm(X) gene was discovered; hence, the need appeared for further investigation to confirm the existence of this gene among C. pseudodiphtheriticum. About 58 strains of C. pseudodiphtheriticum were used in this study. All strains were isolated from the nasal mucous membrane of patients with upper respiratory tract infection symptoms. Among the tested strains 52 were erythromycin resistant, and only 6 were erythromycin sensitive. The tested strains showed a very high percentage (89.7%) of the phenotype MLSb-constitutive resistance mechanism. The MLSb-inducible resistance among the tested strains was not observed. Association of the MLSb mechanism with resistance to chloramphenicol, trimethoprim/sulfamethoxazole and chloramphenicol, and trimethoprim/sulfamethoxazole was observed in 12.1%, 15.5%, and 44.8% tested strains, respectively. Among all isolates with the phenotype MLSb resistance, the presence of the erm(X) gene was confirmed by the polymerase chain reaction method. The results suggest that C. pseudodiphtheriticum with the MLSb-constitutive-type resistance can play a significant role in crossing this mechanism with other Corynebacterium sp., which colonize the nasal and throat mucous membrane.

Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N(6)-monomethyltransferases or N(6),N(6)-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLS(B)) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLS(B) and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin.

Macrolide resistance based on the Erm-mediated rRNA methylation.

Macrolide, lincosamide and streptogramin B (MLSB) antibiotics are extensively used for the treatment of wide variety of clinically important Gram-positive bacteria. MLSB antibiotics inhibit protein biosynthesis by targeting the peptidyl transferase centre within the 50S ribosomal subunit. The most widespread mechanism of bacterial resistance to MLSB antibiotics, reported early after their introduction into clinical practice is the modification of the target site exhibited by a family of rRNA methyltransferases designated Erm. Using S-adenosyl-L-methionine, Erm enzymes catalyze mono- or dimethylation of a specific adenine residue in the 23S rRNA. The methyl group sterically hinders the MLSB binding site and disrupts the hydrogen bonding between the macrolides and the rRNA, thus rendering bacteria resistant. This review summarizes the current understanding of Erm-mediated resistance, in light of high-resolution structural data of bacterial ribosome and with specific focus on the results of recent genetic, biochemical and structural studies of Erm methyltransferases and their cognate rRNA substrate. Although many features of MLSB resistance remain indistinct, the present knowledge can now serve as the guidance for development of both new antimicrobial drugs and potential inhibitors of Erm enzymes, hence providing a new lead to solve the urgent problem of the macrolide resistance based on the ribosome methylation.

Evaluation of macrolide and lincosamide antibiotics for plasmid maintenance in low pHClostridium acetobutylicumATCC 824 fermentations

Clarithromycin, midecamycin and tylosin effectively provided selective pressure for maintenance of a macrolide, lincosamide and streptogramin B resistance (MLSr) plasmid in Clostridium acetobutylicum ATCC 824 at pH values as low as 4.5, whereas erythromycin, oleandomycin, spiramycin, lincomycin and clindamycin did not. Furthermore, clarithromycin, midecamycin and tylosin were not degraded by late stationary-phase ATCC 824 fermentation fluids. Solvent formation, but not acid formation. was inhibited by tylosin but not by clarithromycin in an MLSr plasmid containing strain. Clarithromycin should therefore be useful for the maintenance of selective pressure for MLSr plasmids in low pH recombinant C. acetobutylicum ATCC 824 fermentations.

Isolation and characterization of sporulation lacZ fusion mutants of Bacillus megaterium

Summary: A derivative of Bacillus megaterium QM B1551 cured of all seven resident plasmids was mutated to Lac–2. Transposon Tn917 carrying lacZ and cat was then used to isolate four spo: :lacZ-cat fusion mutants by screening for colonies expressing the fusion in stationary phase. The sporulation frequencies of the mutants ranged from 10–7 to 10–2. Macrolide, lincosamide and streptogramin B resistance (MLSr) of the mutants cotransduced 100% with the sporulation defect and all four mutations mapped near trp by transduction in the order: trp–hisH–spo. All four mutants isolated were defective early in sporulation since β-galactosidase could be detected between zero and 2 h after the end of exponential growth. Electron microscopy of two of the mutants expressing the enzyme at t1–t2 revealed a defect prior to or just at the beginning of septum formation in one, and after completion of septum formation in the other. Little or no synthesis of dipicolinic acid, glucose dehydrogenase or alkaline phosphatase was detected in the mutants, but each was neutral protease positive. These results show that the mutations were in at least two early genes expressed before glucose dehydrogenase production. This study represents the first genetic characterization of sporulation mutants in B. megaterium and also demonstrates that gene fusion technology can be used in this species.