Genetic diversity of macrolides resistant Staphylococcus aureus clinical isolates and the potential synergistic effect of vitamins, C and K3.

BACKGROUND: Macrolide antibiotics have been extensively used for the treatment of Staphylococcus aureus infections. However, the emergence of macrolide-resistant strains of S. aureus has become a major concern for public health. The molecular mechanisms underlying macrolide resistance in S. aureus are complex and diverse, involving both target site modification and efflux pump systems. In this study, we aim to overcome the molecular diversity of macrolide resistance mechanisms in S. aureus by identifying common molecular targets that could be exploited for the development of novel therapeutics. METHODS: About 300 Staphylococcus aureus different isolates were recovered and purified from 921 clinical specimen including urine (88), blood (156), sputum (264), nasal swabs (168), pus (181) and bone (39) collected from different departments in Tanta University Hospital. Macrolide resistant isolates were detected and tested for Multi Drug Resistant (MDR). Gel electrophoresis was performed after the D test and PCR reaction for erm(A), (B), (C), msr(A), and mph(C) genes. Finally, we tried different combinations of Erythromycin or Azithromycin antibiotics with either vitamin K3 or vitamin C. RESULTS: Macrolide resistance S. aureus isolates exhibited 7 major resistance patterns according to number of resistance markers and each pattern included sub patterns or subgroups. The PCR amplified products of different erm genes; analysis recorded different phenotypes of the Staphylococcus aureus isolates according to their different genotypes. In addition, our new tested combinations of Erythromycin and vitamin C, Erythromycin, and vitamin K3, Azithromycin and vitamin C and Azithromycin and vitamin K3 showed significant antibacterial effect when using every antibiotic alone. Our findings provide new insights into the molecular mechanisms of macrolide resistance in S. aureus and offer potential strategies for the development of novel protocols to overcome this emerging public health threat.

Molecular and Clinical Features of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus in Tertiary Care Hospitals in South India.

Objectives: This study aimed to detect heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) among methicillin-resistant S. aureus (MRSA) isolated from healthcare-associated infections and identify staphylococcal cassette chromosome mec (SCCmec) types. Methods: This study was conducted from February 2019 to March 2020 and included patients admitted in 4 tertiary care hospitals in Karnataka, India. Isolation and identification of MRSA were done using standard bacteriological methods. Antimicrobial susceptibility testing was done using Kirby-Bauer disc diffusion; macrolide-lincosamide-streptogramin B phenotypes were identified using the D test. The minimum inhibitory concentration (MIC) of vancomycin was determined using agar dilution. hVISA were confirmed by the modified population analysis profile-area under the curve test. SCCmec types and the Panton-Valentine leukocidin (pvl) gene were detected using multiplex polymerase chain reaction. Results: Of 220 MRSA stains, 14 (6.4%) were hVISA. None of the MRSA isolates was vancomycin-intermediate or -resistant and all hVISA were susceptible to linezolid and teicoplanin. The macrolide-streptogramin B phenotype was present in 42.9% of hVISA; 92.9% of the hVISA strains had vancomycin MIC in the range of 1-2 µg/mL. Majority of the hVISA and vancomycin-susceptible MRSA were isolated from patients with skin and soft tissue infections. SCCmec III and IV were present in 50% and 35.7% of hVISA, respectively; 14.3% of the hVISA harboured SCCmec V. Conclusion: The prevalence rate of hVISA among MRSA was 6.4%. Therefore, MRSA strains should be tested for hVISA before starting vancomycin treatment. None of the isolates was vancomycin-intermediate or -resistant and all the hVISA strains were susceptible to linezolid and teicoplanin. The majority of the hVISA were isolated from patients with skin and soft tissue infections and harboured SCCmec III and IV.

Distribution of MecA and Erm Genes among Methicillin-resistant Staphylococcus Aureus with Inducible Resistance to Clindamycin.

BACKGROUND: The emergence of Methicillin-resistant Staphylococcus aureus and its ability to confer cross-resistance to macrolide-lincosamide-streptogramin B has complicated the treatment against it. Gene-based studies among phenotypic methicillin-resistant isolates with inducible resistance to clindamycin are less available in Nepal. This work was undertaken to detect the mecA and erm genes among such phenotypes isolated from clinical samples. METHODS: S. aureus isolated from different clinical samples was identified by standard microbiological procedures (Gram-staining, colony morphology, and different biochemical tests). Methicillin-resistant and inducible resistant to clindamycin phenotypes were detected by using cefoxitin disc (30 µg) and a double disk diffusion test according to the Clinical and Laboratory Standards Institute guidelines and mecA and erm genes were detected by polymerase chain reaction. RESULTS: Among 120 S. aureus isolates, 51.67% (n=62) were MRSA, and the prevalence of inducibly-resistant, constitutively-resistant and Macrolide-Streptogramin phenotypes were 15.83% (n=19), 28.33% (n=34) and 15.83% (n=19) respectively. While 35.84% (n=43) of isolates showed sensitivity to both antibiotics, erythromycin and clindamycin. Out of 14 inducibly-resistant phenotypes, 57.14% (n=8) were found carrying ermC and 28.57% (n=4) phenotypes contained both ermA and ermC. All phenotypes were positive for the mecA gene. CONCLUSIONS: Macrolides-Lincosamide-Streptogramin B resistance was predominant among methicillin-resistant S. aureus. While all isolates with inducible clindamycin resistance harbored mecA gene, most of them also harbored ermC gene. The higher prevalence of inducible-resistant to clindamycin indicated the need for rational use of antimicrobial agents.

Novel macrolide-lincosamide-streptogramin B resistance gene erm(56) in Trueperella pyogenes.

Whole-genome sequence analysis of a macrolide, lincosamide, streptogramin B (MLSB)-resistant Trueperella pyogenes from a dog revealed a new 23S ribosomal RNA methylase gene erm(56). Expression of the cloned erm(56) confers resistance to MLSB in T. pyogenes and Escherichia coli. The erm(56) gene was flanked by two IS6100 integrated on the chromosome next to a sul1-containing class 1 integron. GenBank query revealed additional erm(56)-containing elements in another T. pyogenes and in Rothia nasimurium from livestock. IMPORTANCE A novel 23S ribosomal RNA methylase gene erm(56) flanked by insertion sequence IS6100 was identified in a Trueperella pyogenes isolated from the abscess of a dog and was also present in another T. pyogenes and in Rothia nasimurium from livestock. It was shown to confer resistance to macrolide, lincosamide, streptogramin B antibiotics in T. pyogenes and E. coli, indicating functionality in both Gram-positive and Gram-negative bacteria. The detection of erm(56) on different elements in unrelated bacteria from different animal sources and geographical origins suggests that it has been independently acquired and likely selected by the use of antibiotics in animals.

The antimicrobial activity of cethromycin against Staphylococcus aureus and compared with erythromycin and telithromycin.

BACKGROUND: This study aims to explore the antibacterial activity of cethromycin against Staphylococcus aureus (S. aureus), and its relationship with multilocus sequence typing (MLST), erythromycin ribosomal methylase (erm) genes and macrolide-lincosamide-streptogramin B (MLSB) phenotypes of S. aureus. RESULTS: The minimum inhibitory concentrations (MICs) of cethromycin against 245 S. aureus clinical isolates ranged from 0.03125 to ≥ 8 mg/L, with the resistance of 38.8% in 121 methicillin-resistant S. aureus (MRSA). This study also found that cethromycin had strong antibacterial activity against S. aureus, with the MIC ≤ 0.5 mg/L in 55.4% of MRSA and 60.5% of methicillin-sensitive S. aureus (MSSA), respectively. The main MLSTs of 121 MRSA were ST239 and ST59, and the resistance of ST239 isolates to cethromycin was higher than that in ST59 isolates (P = 0.034). The top five MLSTs of 124 MSSA were ST7, ST59, ST398, ST88 and ST120, but there was no difference in the resistance of MSSA to cethromycin between these STs. The resistance of ermA isolates to cethromycin was higher than that of ermB or ermC isolates in MRSA (P = 0.016 and 0.041, respectively), but the resistance of ermB or ermC isolates to cethromycin was higher than that of ermA isolates in MSSA (P = 0.019 and 0.026, respectively). The resistance of constitutive MLSB (cMLSB) phenotype isolates to cethromycin was higher than that of inducible MLSB (iMLSB) phenotype isolates in MRSA (P < 0.001) or MSSA (P = 0.036). The ermA, ermB and ermC genes was mainly found in ST239, ST59 and ST1 isolates in MRSA, respectively. Among the MSSA, the ermC gene was more detected in ST7, ST88 and ST120 isolates, but more ermB genes were detected in ST59 and ST398 isolates. The cMLSB phenotype was more common in ST239 and ST59 isolates of MRSA, and was more frequently detected in ST59, ST398, and ST120 isolates of MSSA. CONCLUSION: Cethromycin had strong antibacterial activity against S. aureus. The resistance of MRSA to cethromycin may had some clonal aggregation in ST239. The resistance of S. aureus carrying various erm genes or MLSB phenotypes to cethromycin was different.

Tn5406, a staphylococcal transposon associated with macrolide-lincosamide-streptograminb resistance in clinical isolates of Staphylococcus aureus.

PURPOSE: In this study, we aimed to investigate the occurrence of MLSb resistance in clinical isolates of Staphylococcus aureus with respect to their association with transposons. METHODS: The present study was performed with clinical isolates of S. aureus. The MLSb resistant phenotypes in the obtained isolates were determined by D zone test or double disc diffusion test as per CLSI 2020 guidelines. MLSb resistance encoding genes were detected by PCR. The genes tested were ermA, ermB, ermC, msrA, mphC, vga, vgb and lnuB. The MLSb resistant Staphylococcal isolates were selected to analyze the association of the genes with mobile genetic elements Tn554, Tn5406, Tn917, Tn6133, Tn551 by PCR based method. Primer pairs were designed using sequences from transposons and the resistance genes, respectively. RESULTS: During this study, 268 isolates of S. aureus were obtained of which 233 (86.94%) isolates exhibited different MLSb resistant phenotypes. The predominant gene among the MLSb resistant isolates was msrA followed by vgaA and mphC genes. PCR assay was employed to determine whether the genes msrA, mphC and vgaA were carried by Tn554, Tn5406, Tn917, Tn6133, Tn551 transposons. PCR amplification with the designed primer pairs revealed vgaA gene being part of Tn5406. CONCLUSION: The presence of Tn5406 in all the vgaA harboring isolates highlights its potential of spread across isolates. Moreover, the co-existence of different MLSb resistance encoding genes observed in the study shows that the combination of genes involved in different mechanism mediated the nature of MLSb resistance.

Prevalence of Inducible Macrolide, Lincosamide, and Streptogramin B (inducible MLSB) Resistance in Clindamycin-Susceptible Staphylococcus aureus at Okayama University Hospital.

Inducible resistance to the macrolide, lincosamide, and streptogramin B (iMLSB) antibiotic family is a latent mechanism for antimicrobial resistance in Staphylococcus aureus. We here investigated the frequency and genotypic profiles of iMLSB resistance in clindamycin (CLDM)-susceptible S. aureus isolated in Okayama University Hospital from June 2020 to June 2021. We phenotypically screened the iMLSB resistance via D-zone test and performed PCR testing for the erythromycin ribosomal methylase (erm) genes: ermA and ermC. Among 432 CLDM-susceptible S. aureus isolates, 138 (31.9%) exhibited an iMLSB-resistance phenotype, with methicillinresistant S. aureus isolates (MRSA; 61 isolates: 58.6%) exhibiting higher positivity than methicillin-sensitive S. aureus isolates (MSSA; 77 isolates: 23.5%) (p<0.001). Male patients had a higher frequency of iMLSB resistance than females (OR [95%CI]: 1.8 [1.2-2.8]; p=0.007). Genotypically, ermA predominated in both MSSA (70.1%) and MRSA (86.9%) compared to ermC (14.3% in MSSA and 11.5% in MRSA). A single strain of MRSA possessed both ermA and ermC, while 12 (15.6%) MSSA isolates were negative for both ermA and ermC, suggesting the presence of other genetic mechanisms. Collectively, these results show that approximately 33% of CLDM-susceptible S. aureus isolates at our university hospital exhibited iMLSB resistance, predominantly caused by ermA in both MSSA and MRSA.

Characterization of Streptococcus pneumoniae Macrolide Resistance and Its Mechanism in Northeast China over a 20-Year Period.

Due to the resistance of Streptococcus pneumoniae to ß-lactams, macrolides, and tetracyclines, treatment alternatives have become increasingly limited worldwide. We aim to describe the characterization of erythromycin-resistant S. pneumoniae (ERSP) strains in northeastern China over a period of 20 years. A total of 1,240 ERSP strains were collected and classified into five groups based on the ages of the patients. Etest strips and Kirby-Bauer disk diffusion were performed for drug susceptibility testing. The capsule swelling test was used for capsule typing. The phenotype of drug resistance was detected by the erythromycin and clindamycin double-disk method. The ermB, ermTR, mefA, and tetM genes were detected by PCR. Among the 1,240 ERSP strains, 510 were invasive isolates, and 730 were noninvasive isolates. The results of drug susceptibility testing showed that the rates of resistance to penicillin, amoxicillin, cefotaxime, ceftriaxone, meropenem, tetracycline, trimethoprim-sulfamethoxazole, and chloramphenicol varied among the different age groups. 19F, 19A, 23F, 14, and 6B were the serotypes that were commonly found among ERSP strains. Among all strains, 99.03% (1,228/1,240) exhibited an MLSB (macrolide-lincosamide-streptogramin B) resistance phenotype, of which 1,221 strains displayed a constitutive MLSB (cMLSB) phenotype and 7 strains showed an inducible MLSB (iMLSB) phenotype. All of these strains carried the ermB gene. In contrast, only 0.97% of strains of M phenotypes were found to carry the mefA gene. Both the ermB and mefA genes were detected in 704 strains that exhibited multidrug resistance, whereas the ermTR gene was not detected. Furthermore, 1,185 tetracycline-resistant strains were found to carry the tetM gene. Macrolide antimicrobial drugs should be used cautiously for the empirical treatment of S. pneumoniae infections. IMPORTANCE This study presents a retrospective analysis using 1,240 clinical erythromycin-resistant Streptococcus pneumoniae (ERSP) isolates collected in northeastern China between January 2000 and December 2019. The serotype distribution, corresponding vaccine coverage, as well as resistance phenotypes, genes, and mechanisms to macrolide and tetracycline of these isolates were systematically described, analyzed, and discussed. We hope that this study will inform clinicians in their respective regions when selecting antimicrobial agents. We also hope that this study is useful for researchers in related fields. Finally, we emphasize in this study that vaccination is the best preventive measure for S. pneumoniae infection considering its resistance to commonly used antibiotics. The determination of the S. pneumoniae serotype distribution also provides valuable empirical evidence for local health authorities when introducing appropriate vaccines in a specific area.

NMR characterization of streptogramin B and L-156,587, a non-synergistic pair of the streptogramin family antibiotic complexes produced inductively by a combined culture of Streptomyces albogriseolus and Tsukamurella pulmonis.

The complete 1 H and 13 C NMR characterization of streptogramin B (1), the major component of a clinically important synergistic antibiotic complex, was presented for the first time, along with those of L-156,587 (2), a dehydrated congener of streptogramin A (3). Compounds 1 and 2 were not synergistic and produced by Streptomyces albogriseolus in co-culture with Tsukamurella pulmonis, which poses a question on the adaptive significance of the induced production of this antibiotic pair.

Distribution of Macrolide-Lincosamide-Streptogramin B Antibiotics Resistance Genes in Clinical Isolates of Staphylococci.

BACKGROUND: Staphylococci are posing threat due to increasing trend of antimicrobial resistance particularly methicillin. Macrolide lincosamide streptogramin B (MLSB) family of antibiotics is commonly used to treat such infections. This study was aimed to determine the prevalence of inducible clindamycin resistance and observation of erm and msr genes among Staphylococci isolated from tertiary care hospital of Nepal during July 2017 to March 2018. METHODS: Staphylococci from different clinical specimens were identified and antibiotic susceptibility profile was assessed following Kirby Bauer disc diffusion method. The double disc diffusion or D-zone test as outlined in CLSI document M100-S24 was performed to examine inducible clindamycin resistant isolates. Multiplex PCR was performed for detection of erm and msr gene in isolates using specific primers for ermA, ermB, ermC, msrA and msrB genes. RESULTS: Of the 60 Staphylococci isolates, 39 (65%) were S. aureus and 21 (35%) were coagulase negative Staphylococci (CNS) with 25 (64%) and 15 (71%) representing methicillin resistant S. aureus and CNS respectively. Constitutive and inducible MLSB phenotype was observed among 24 (40%) and 14 (23%) isolates respectively by D test. The most prevalent resistant gene was ermC (37%) followed by msrB (12%), ermB (10%) and msrA (10%). None of the isolates were found to possess ermA gene. CONCLUSIONS: The presence of constitutive and inducible MLSB as well as resistant genes among Staphylococci necessitates detection of such isolates to minimize treatment failure. The result from this study may help elucidate the predominant resistant characteristics in clinical Staphylococci isolated from tertiary care hospital of Nepal.

Macrolide-lincosamide-streptogramin B resistance phenotypes and their associated genotypes in Staphylococcus aureus isolates from a tertiary level public hospital of Uruguay / Fenotipos de resistencia a macrólidos lincosamidas-estreptograminas B y sus genotipos asociados en Staphylococcus aureus aislados en un hospital público de nivel terciario en Uruguay

Abstract This study was undertaken to investígate the resistance phenotypes to macrolide-lincosamide-streptogramin B (MLSb) antibiotics and their associated genotypes in isolates of Staphylococcus aureus. We analyzed one hundred, consecutive, non-duplicate isolates (methicillin-susceptible MSSA, n = 53 and methicillin-resistant MRSA, n =47) obtained from var-ious clinical samples between July 2012 to December 2013. The resistance profile to MLSb antibiotics was determined by phenotypic methods and the resistance genes were detected by PCR assays. All of the isolates were subjected to pulsed-field gel electrophoresis (SmaI-PFGE). The overall prevalence of resistance to MLSb antibiotics was 38% and the resistance phenotype distribution was as follows: cMLSb, 22%; iMLSB, 10%; MSb, 5% and L, 1%. We detected ermA, ermC, ermB and mrsA/B genes in these resistant isolates. The single ermA gene was commonly observed mainly in those with a cMLSb R phenotype, whereas the combination ermA and ermC was more commonly observed in isolates with inducible expression. The patterns of SmaI-PFGE suggest a great genetic diversity in both MRSA and MSSA resistant to MLSb antibiotics. The results demonstrate the local presence of S. aureus resistant to MLSb antibiotics and its most frequently described responsible genes. Some of these isolates, especially those with the iMLSB phenotype, may be associated with therapeutic failure. Therefore, efforts should be directed to the correct detection of all MLSb resistant isolates using appropriate laboratory tests. PFGE results reveal a wide spread of resistance genes rather than the circulation of S. aureus clones resistant to MLSb antibiotics.

Resumen Los objetivos de este estudio fueron investigar en Staphylococcus aureus la presencia de fenotipos resistentes a los antibióticos macrólidos, lincosamidas y estreptograminas tipoB (MLSb) y conocer sus genotipos responsables. Analizamos 100 aislamientos consecutivos, no duplicados (53 sensibles a meticilina [MSSA] y 47 resistentes a meticilina [MRSA]), obtenidos entre 2012 y 2013 a partir de diferentes muestras clínicas. El perfil de resistencia a los antibióticos MLSb fue determinado por métodos fenotípicos y los genes de resistencia se detectaron por PCR. Todos los aislamientos fueron comparados por SmaI-PFGE. La prevalencia global de resistencia a los antibióticos MLSB fue del 38% y la distribución de los fenotipos de resistencia fue la siguiente: cMLSB, 22%; iMLSB, 10%; MSB, 5%; L, 1%. Se detectaron los genes ermA, ermC y mrsA/B en los aislamientos resistentes. El gen ermA se observó, sobre todo, en aislamientos con fenotipo resistente constitutivo R (cMLSB), mientras que la combinación ermA y ermC se detectó principalmente en aislamientos con resistencia inducible (iMLSB). Los patrones de Smal-PFGE sugieren una gran diversidad genética en los aislamientos resistentes a los antibióticos MLSb, tanto MRSA como MSSA. Los resultados demuestran la presencia local de S. aureus resistentes a los antibióticos MLSB y de sus genes responsables más frecuentemente descritos. Estos cultivos, especialmente aquellos con fenotipo resistente iMLSB, pueden asociarse con fallas terapéuticas. Por lo tanto, los esfuerzos deben dirigirse a la correcta detección de todos los cultivos resistentes a MLSB utilizando pruebas de laboratorio adecuadas. Los resultados de Smal-PFGE sugieren una amplia diseminación de genes de resistencia, más que la circulación de clones resistentes a los antibióticos MLSB.

The novel and transferable erm(51) gene confers macrolides, lincosamides and streptogramins B (MLSB ) resistance to clonal Rhodococcus equi in the environment.

The use of mass antimicrobial treatment has been linked to the emergence of antimicrobial resistance in human and animal pathogens. Using whole-genome single-molecule real-time (SMRT) sequencing, we characterized genomic variability of multidrug-resistant Rhodococcus equi isolated from soil samples from 100 farms endemic for R. equi infections in Kentucky. We discovered the novel erm(51)-encoding resistance to MLSB in R. equi isolates from soil of horse-breeding farms. Erm(51) is inserted in a transposon (TnErm51) that is associated with a putative conjugative plasmid (pRErm51), a mobilizable plasmid (pMobErm51), or both enabling horizontal gene transfer to susceptible organisms and conferring high levels of resistance against MLSB in vitro. This new resistant genotype also carries a previously unidentified rpoB mutation conferring resistance to rifampicin. Isolates carrying both vapA and erm(51) were rarely found, indicating either a recent acquisition of erm(51) and/or impaired survival when isolates carry both genes. Isolates carrying erm(51) are closely related genetically and were likely selected by antimicrobial exposure in the environment.

Solithromycin Can Specifically Induce Macrolide-Lincosamide-Streptogramin B Resistance.

Objectives: Solithromycin is a fluoroketolide that is considered to be a noninducing antibiotic for macrolide-lincosamide-streptogramin B resistance mediated by erm genes. The exact activity of solithromycin to induce erm gene expression remains to be determined. Materials and Methods: The potential of solithromycin to induce erm(A), erm(C), and erm(B) gene expression was examined using a lacZ reporter assay, double-disk diffusion test, and determination of the minimal inhibitory concentration after incubation with subinhibitory concentration of different antibiotics. Results: Neither solithromycin nor the ketolides telithromycin and cethromycin induced erm(A) or erm(C) gene expression. However, solithromycin could significantly induce erm(B) gene expression at levels greater than that seen for cethromycin and clindamycin, but less than that for erythromycin, rokitamycin, and telithromycin. Conclusion: Solithromycin does not induce erm(A) and erm(C) gene expression, but does induce erm(B) gene expression, although to a weaker extent than that seen for macrolides.

Macrolide-lincosamide-streptogramin B resistance phenotypes and their associated genotypes in Staphylococcus aureus isolates from a tertiary level public hospital of Uruguay.

This study was undertaken to investigate the resistance phenotypes to macrolide-lincosamide-streptogramin B (MLSB) antibiotics and their associated genotypes in isolates of Staphylococcus aureus. We analyzed one hundred, consecutive, non-duplicate isolates (methicillin-susceptible MSSA, n=53 and methicillin-resistant MRSA, n=47) obtained from various clinical samples between July 2012 to December 2013. The resistance profile to MLSB antibiotics was determined by phenotypic methods and the resistance genes were detected by PCR assays. All of the isolates were subjected to pulsed-field gel electrophoresis (SmaI-PFGE). The overall prevalence of resistance to MLSB antibiotics was 38% and the resistance phenotype distribution was as follows: cMLSB, 22%; iMLSB, 10%; MSB, 5% and L, 1%. We detected ermA, ermC, ermB and mrsA/B genes in these resistant isolates. The single ermA gene was commonly observed mainly in those with a cMLSB R phenotype, whereas the combination ermA and ermC was more commonly observed in isolates with inducible expression. The patterns of SmaI-PFGE suggest a great genetic diversity in both MRSA and MSSA resistant to MLSB antibiotics. The results demonstrate the local presence of S. aureus resistant to MLSB antibiotics and its most frequently described responsible genes. Some of these isolates, especially those with the iMLSB phenotype, may be associated with therapeutic failure. Therefore, efforts should be directed to the correct detection of all MLSB resistant isolates using appropriate laboratory tests. PFGE results reveal a wide spread of resistance genes rather than the circulation of S. aureus clones resistant to MLSB antibiotics.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among methicillin-resistant Staphylococcus aureus strains isolated from burn patients.

The increasing resistance to macrolide, lincosamide, and streptogramin B agents among methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide problem for the health community. This study aimed to investigate the prevalence of ermA, ermB, ermC, and msrA in MRSA strains isolated from burn patients in Ahvaz, southwest of Iran. A total of 76 isolates of S. aureus were collected from January to May 2017 from Taleghani Burn Hospital in Ahvaz. Among 76 S. aureus strains collected, 60 (78.9%) isolates were MRSA. The antimicrobial susceptibility testing for MRSA showed extreme high resistance rate to clarithromycin (100%) and azithromycin (100%), followed by erythromycin (98.3%). The PCR assay revealed that the frequency rates of msrA, ermA, and ermC genes were 23 (38.3%), 28 (46.7%), and 22 (36.7%), respectively. In addition, none of the MRSA isolates had the ermB gene. Because of the high prevalence of macrolide and lincosamide resistance found in MRSA isolates from infections of burn patients in Ahvaz, southwest of Iran, it is recommended that local periodic survey be performed for controlling the dissemination of antimicrobial resistance.

Expression of resistance genes instead of gene abundance are correlated with trace levels of antibiotics in urban surface waters.

In this study, antibiotic resistance to macrolide-lincosamide-streptogramin B (MLSB) antibiotics in total microbial community in surface water in a coastal urban city was measured using a modified fluorescence in situ hybridization (FISH) technique. This FISH technique quantified the rate of antibiotic resistance to MLSB antibiotics through targeting methylation site of A2058 of 23S rRNAs resulting from expressed erythromycin ribosome methylation (erm) genes. Correlations between the rates of MLSB resistance measured by FISH and macrolide concentrations was stronger than that between the relative abundance of erm genes and macrolide concentrations, especially in residential areas where the main detected antibiotics were macrolides. These results suggest that trace levels of antibiotics in environmental waters, which was as low as 40 ng L-1, may still play important roles in the development and spread of antibiotic resistance. Additionally, methylation as a result of erm gene expression, instead of erm gene abundance, was a better indicator of selective pressure of trace level macrolides. The rates of MLSB resistance varied significantly among land use types, suggesting that anthropogenic activities are important factors to select for erm gene expression in the environment. Microbial community analysis of representative surface water samples showed that relatively high rates of MLSB resistance were observed in Alphaproteobacteria (42%), Acidobacteria (36%), Bacteroidaceae (32%), Chloroflexi (27%), and Betaproteobacteria (20.2%).

Staphylococcus aureus with an erm-mediated constitutive macrolide-lincosamide-streptogramin B resistance phenotype has reduced susceptibility to the new ketolide, solithromycin.

BACKGROUND: Solithromycin, the fourth generation of ketolides, has been demonstrated potent antibacterial effect against commonly-isolated gram-positive strains. However, Staphylococcus aureus (S. aureus) strains with a higher solithromycin MIC have already been emerged, the mechanism of which is unknown. METHODS: Antimicrobial susceptibility test was performed on 266 strains of S. aureus. The antibiotic resistance phenotype of erm-positive strain was determined by D-zone test. Spontaneous mutation frequency analysis was performed to compare the risk levels for solithromycin resistance among different strains. Efflux pumps and mutational analysis of ribosomal fragments as well as erm(B) gene domains were detected. Quantitative reverse transcription polymerase chain reaction was conducted to compare the transcriptional expression of the erm gene between the constitutive macrolide-lincosamide-streptogramin B (cMLSB)- and inducible MLSB (iMLSB)-phenotypes. RESULTS: In the erm-positive S. aureus strains, the minimum inhibitory concentration (MIC)50/90 of solithromycin (2/> 16 mg/L) was significantly higher than that in the erm-negative strains (0.125/0.25 mg/L). Of note, the MIC50 value of the strains with iMLSB (0.25 mg/L) was significantly lower than that of the strains with cMLSB (4 mg/L). A comparison among strains demonstrated that the median mutational frequency in isolates with cMLSB (> 1.2 × 10- 4) was approximately > 57-fold and > 3333-fold higher than that in iMLSB strains (2.1 × 10- 6) and in erythromycin-sensitive strains (3.6 × 10- 8), respectively. The differential antibiotic in vitro activity against strains between cMLSB and iMLSB could not be explained by efflux pump carriers or genetic mutations in the test genes. The expression of the erm genes in strains with cMLSB did not differ from that in strains with iMLSB. CONCLUSIONS: The reduced susceptibility to solithromycin by S. aureus was associated with the cMLSB resistance phenotype mediated by erm.