High-level Macrolide Resistance Research Articles

A Review of the Impact of Streptococcal Infections and Antimicrobial Resistance on Human Health.

Streptococcus pneumoniae, Streptococcus pyogenes (GAS), and Streptococcus agalactiae (GBS) are bacteria that can cause a range of infections, some of them life-threatening. This review examines the spread of antibiotic resistance and its mechanisms against antibiotics for streptococcal infections. Data on high-level penicillin-resistant invasive pneumococci have been found in Brazil (42.8%) and Japan (77%). The resistance is caused by mutations in genes that encode penicillin-binding proteins. Similarly, GAS and GBS strains reported from Asia, the USA, and Africa have undergone similar transformations in PBPs. Resistance to major alternatives of penicillins, macrolides, and lincosamides has become widespread among pneumococci and streptococci, especially in Asia (70-95%). The combination of several emm types with erm(B) is associated with the development of high-level macrolide resistance in GAS. Major mechanisms are ribosomal target modifications encoded by erm genes, ribosomal alterations, and active efflux pumps that regulate antibiotic entry due to mefA/E and msrD genes. Tetracycline resistance for streptococci in different countries varied from 22.4% in the USA to 83.7/100% in China, due to tet genes. Combined tetracycline/macrolide resistance is usually linked with the insertion of ermB into the transposon carrying tetM. New quinolone resistance is increasing by between 11.5 and 47.9% in Asia and Europe. The mechanism of quinolone resistance is based on mutations in gyrA/B, determinants for DNA gyrase, or parC/E encoding topoisomerase IV. The results for antibiotic resistance are alarming, and urgently call for increased monitoring of this problem and precautionary measures for control to prevent the spread of resistant mutant strains.

Macrolide-resistant Mycoplasma pneumoniae in an Iranian Pediatric Sample with Community-Acquired Pneumonia

Background: To the best of our knowledge, the prevalence of macrolide-resistant Mycoplasma pneumoniae (MRM) in Iranian children has not been investigated. Objectives: The present study aimed to evaluate the prevalence of MRM in Iranian children with community-acquired pneumonia (CAP). Methods: A total of 222 children with CAP, aged 3 - 15 years, who were hospitalized in 10 different children's hospitals, were enrolled in this study. Mycoplasmas were detected using the polymerase chain reaction (PCR) assay. The severity of CAP was evaluated according to the Infectious Diseases Society of America (IDSA) guidelines. The level of C-reactive protein (CRP) was also measured by the particle-enhanced turbidimetric immunoassay. Additionally, the chest X-rays of children with CAP were recorded and sent to a radiologist for further evaluation. Results: Twenty-one children (9.4%) diagnosed with CAP also had M. pneumoniae infection, 17 (77.27%) of whom were positive for A2063G transition and high-level macrolide resistance. The severity of CAP (P ≥ 0.99), CRP level (0.07), and chest X-ray changes (P = 0.08) were not significantly different between children with MRM pneumonia and those with macrolide-susceptible M. pneumoniae. Conclusions: The prevalence of high-level MRM pneumonia in children is high in Iran, similar to other Asian countries. However, this type of Mycoplasma infection was not associated with the severity of CAP and did not have significant effects on chest X-ray (CXR) changes or the CRP level in the patients.

Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible Streptococcus pneumoniae isolates from people living with HIV in Dar es Salaam, Tanzania

BackgroundOver one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing.MethodsWe assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP.ResultsFifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLSB phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic correlates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes.ConclusionThe erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLSB in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon.

1321. Increased Prevalence of Group A Streptococcus (GAS) High Level Macrolide Resistance During the COVID-19 Pandemic

Abstract Background COVID-19 affected the epidemiology of many respiratory pathogens including GAS. Assessing genetic heterogeneity (emm type, antimicrobial resistance, virulence factors) can inform treatment recommendations and targets for potential GAS vaccines. We assessed GAS clinical antibiotic susceptibility and performed whole genome sequencing (WGS) among pediatric pharyngeal isolates from 2020–2022. Methods From 1/2020–3/2022 we collected throat swabs in pediatric clinics and EDs from children aged 3-18 years in Chicago, IL; Atlanta, GA; Portland, OR; and Phoenix, AZ: 1) with acute GAS pharyngitis and 2) among a convenience sample of asymptomatic children to assess for GAS colonization. Swabs were plated on blood agar. E-tests were used to assess clinical susceptibility to erythromycin (ERY) and ciprofloxacin (CIP). emm type and antimicrobial resistance genes (ERY, Clindamycin (CLI), and fluoroquinolones) were assessed by WGS. Results 1144 pharyngeal swabs were collected: 359/684 (52%) from children with GAS pharyngitis by rapid test and 20/460 (4.3%) from asymptomatic children yielded GAS on culture. Phenotypic resistance: 55/364 (15%) tested isolates were ERY resistant and 5/364 (1.4%) CIP resistant. The proportion of isolates with ERY resistance increased significantly from 2020 (6%) to 2021–2022 (25%) (χ2 = 23.70, p&amp;lt; .00001) (Figure 1). MICs were high among ERY resistant GAS (Table 1). Genotypic resistance: Of 304 sequenced GAS isolates 40/304 (13%) were ERY resistant, 35/304 (11.5%) were both ERY resistant and CLI (inducible or constitutive) resistant, and 4/304 (1.3%) fluoroquinolone resistant. ermB (62%) was the most common gene for ERY resistance and constitutive CLI resistance, followed by ermTR (23%) and ermT (11%) both conferring inducible CLI resistance. Among the 20 isolates from asymptomatic children no ERY, CLI, or CIP resistance occurred, and no resistance genes were identified. emm types 11, 9, 77, 58 and 94 were associated with ERY and CLI resistance. Conclusion ERY resistance increased from 2020–2022. The high rate of CLI resistance among ERY resistant GAS was associated with erm genes. These results are important to inform treatment recommendations for GAS pharyngitis and targets for vaccine development that can reduce antimicrobial-resistant GAS disease. Disclosures All Authors: No reported disclosures.

Detection of highly macrolide-resistant Legionella pneumophila strains from a hotel water network using systematic whole-genome sequencing.

Implementation of an antibiotic resistance detection tool in Legionella daily surveillance at the French National Reference Centre for Legionella. Systematic WGS of Legionella pneumophila isolates and bioinformatics detection of specific mutations linked to antibiotic resistance. Phenotypic validation of antibiotic resistance detected by WGS was performed by the broth microdilution method. More than 3000 L. pneumophila strains were screened for antibiotic resistance. A macrolide resistance-associated A2052G mutation in the 23S rRNA gene was identified in the genome of eight isolates from a hotel water network. High-level macrolide resistance (i.e. MICs of 1024-2048 mg/L for azithromycin and erythromycin) with no cross-resistance to other antimicrobials was phenotypically confirmed by antimicrobial susceptibility testing for the eight isolates. Systematic WGS of L. pneumophila is a powerful tool for first-line high-throughput screening of antibiotic resistance before phenotypic validation.

Successful Intra- but Not Inter-species Recombination of msr(D) in Neisseria subflava.

Resistance acquisition via natural transformation is a common process in the Neisseria genus. Transformation has played an important role in the emergence of resistance to many antimicrobials in Neisseria gonorrhoeae and Neisseria meningitidis. In a previous study, we found that currently circulating isolates of Neisseria subflava had acquired an msr(D) gene that has been found to result in macrolide resistance in other bacteria but never found in Neisseria species before. To determine if this resistance mechanism is transferable among Neisseria species, we assessed if we could transform the msr(D) gene into other commensal and pathogenic Neisseria under low dose azithromycin pressure. Intraspecies recombination in commensal N. subflava was confirmed with PCR and resulted in high-level macrolide resistance. Whole-genome sequencing of these transformed strains identified the complete uptake of the msr(D) integration fragment. Sequence analysis showed that a large fragment of DNA (5 and 12 kb) was transferred through a single horizontal gene transfer event. Furthermore, uptake of the msr(D) gene had no apparent fitness cost. Interspecies transformation of msr(D) from N. subflava to N. gonorrhoeae was, however, not successful.

High-Level Macrolide Resistance Due to the Mega Element [mef(E)/mel] in Streptococcus pneumoniae

Transferable genetic elements conferring macrolide resistance in Streptococcus pneumoniae can encode the efflux pump and ribosomal protection protein, mef(E)/mel, in an operon of the macrolide efflux genetic assembly (Mega) element- or induce ribosomal methylation through a methyltransferase encoded by erm(B). During the past 30 years, strains that contain Mega or erm(B) or both elements on Tn2010 and other Tn916-like composite mobile genetic elements have emerged and expanded globally. In this study, we identify and define pneumococcal isolates with unusually high-level macrolide resistance (MICs > 16 μg/ml) due to the presence of the Mega element [mef(E)/mel] alone. High-level resistance due to mef(E)/mel was associated with at least two specific genomic insertions of the Mega element, designated Mega-2.IVa and Mega-2.IVc. Genome analyses revealed that these strains do not possess erm(B) or known ribosomal mutations. Deletion of mef(E)/mel in these isolates eliminated macrolide resistance. We also found that Mef(E) and Mel of Tn2010-containing pneumococci were functional but the high-level of macrolide resistance was due to Erm(B). Using in vitro competition experiments in the presence of macrolides, high-level macrolide-resistant S. pneumoniae conferred by either Mega-2.IVa or erm(B), had a growth fitness advantage over the lower-level, mef(E)/mel-mediated macrolide-resistant S. pneumoniae phenotypes. These data indicate the ability of S. pneumoniae to generate high-level macrolide resistance by macrolide efflux/ribosomal protection [Mef(E)/Mel] and that high-level resistance regardless of mechanism provides a fitness advantage in the presence of macrolides.

Novel mechanism responsible for high-level macrolide resistance in Moraxella catarrhalis.

BackgroundHigh-level macrolide-resistant Moraxella catarrhalis strains have been isolated; however, the underlying mechanism has not been well elucidated. We investigated the role of mutations in the 23S rRNA gene and the L4 and L22 ribosomal proteins using spontaneous erythromycin-resistant mutants and transformants.Materials and methodsThe erythromycin-susceptible M. catarrhalis ATCC25238 and clinical isolate Mc19 were used as parental strains. To obtain spontaneous erythromycin-resistant mutants, in vitro stepwise selection was performed using brain–heart infusion agar plates containing various concentrations of erythromycin. The role of the mutations identified in the spontaneous mutants was validated using transformation experiments.ResultsWe obtained two spontaneous mutants with high-level resistance to erythromycin, S25-32-af10 and S19-256-af10, from ATCC25238 and Mc19, respectively. S25-32-af10 exhibited mutations of Q61R in L4 and Insertion98SRADRIS in L22. S19-256-af10 exhibited three C2611T-mutated alleles in the 23S rRNA gene and G65A in L4. Transformants with single mutations identified in S25-32-af10 or S19-256-af10 showed higher erythromycin and azithromycin minimum inhibitory concentrations (MICs) than those of each parental strain. However, transformants with multiple mutations identified in S25-32-af10 or S19-256-af10 showed macrolide MICs similar to those of each parental strain.ConclusionOur results provide the first evidence suggesting that Q61R in L4 and Insertion98SRADRIS in L22 are involved in the synergistic acquisition of high-level resistance to both 14- and 15-member macrolides, and that C2611T in the 23S rRNA gene and G65A in L4 also synergistically contribute toward conferring high-level 14-member macrolide resistance to M. catarrhalis.

Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila.

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

Epidemiological analysis of pneumococcal serotype 19A in healthy children following PCV7 vaccination.

After the introduction of conjugate vaccines, a strong rearrangement of pneumococcal serotypes was observed globally. Probably most concerning was the emergence of serotype 19A, which has not only high invasive disease potential, but also high antibiotic resistance. In the current study we focused on the increased prevalence of serotype 19A after the PCV vaccination rate became widely used in Hungary. A total of 2262 children aged 3-6 years were screened for pneumococcus carriage using nasal swabs. Children were divided into two groups according to the vaccination rates, low level (group 1) vs. high level (group 2). While the carriage rate did not change over time (average 32·9%), the serotype distribution differed greatly in the two groups. The prevalence of serotype 19A increased >eightfold. Almost all 19A isolates had high-level macrolide resistance and elevated penicillin minimum inhibitory concentrations. Genotyping methods revealed that these new 19A isolates are different from the previously frequent Hungary19A-6 PMEN clone. Both the carriage rate and the overall penicillin and macrolide resistance remained stable over time, but while several serotypes were represented in group 1, serotype 19A alone was clearly dominant in group 2.

Rapid assay of A2058T-mutated 23S rRNA allelic profiles associated with high-level macrolide resistance in Moraxella catarrhalis.

We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.

Novel macrolide-resistance genes, mef(C) and mph(G), carried by plasmids from Vibrio and Photobacterium isolated from sediment and seawater of a coastal aquaculture site.

The aim of this study was to determine whether mef(C) and mph(G), originally found on the transferable multi-drug plasmid pAQU1 from Photobacterium damselae subsp. damselae isolated from seawater of a fish farm, are responsible for conferring macrolide resistance. Since these genes are localized head-to-tail on pAQU1 and only four nucleotides exist between them, the single- and combination-effect of these genes was examined. When mph(G) alone was introduced to Escherichia coli, the minimum inhibitory concentrations (MICs) against erythromycin, clarithromycin and azithromycin increased, whereas introduction of mef(C) alone did not influence macrolide susceptibility. Introduction of both mef(C) and mph(G) dramatically increased the MICs to the same three macrolides, i.e. >512 μg ml(-1) , >512 μg ml(-1) and 128 μg ml(-1) respectively. These results suggest that the macrolide phosphotransferase encoded by mph(G) is essential for macrolide resistance, while the efflux pump encoded by mef(C) is required for high-level macrolide resistance. The tandem-pair arrangements of the mef(C) and mph(G) genes were conserved on plasmids ranging in size from 240 to 350 kb of the 22 erythromycin-resistant strains belonging to Vibrio and Photobacterium obtained from the fish farm. Sixteen of 22 plasmids ranged in size from 300 to 350 kb. This is the first report of novel macrolide resistance genes originating from a marine bacterium. In this study, mef(C) and mph(G) were found to be novel macrolide-resistance genes, and this is the first report of macrolide-resistance genes originating from a marine bacterium. These genes may be responsible for previously reported cases of the emergence of erythromycin-resistant bacteria in aquaculture sites by an unknown mechanism. The introduction of the tandem arrangement of the mef(C) and mph(G) genes in Escherichia coli increased the MICs to erythromycin, clarithromycin and azithromycin, suggesting a novel mechanism conferring high-level macrolide resistance via combined expression of the efflux pump and macrolide phosphotransferase.

100 Macrolide resistance is common among members of the CF airway microbiota

Objectives Chronic use of azithromycin has been associated with increased macrolide resistance in recognised CF pathogens. The aims of this study were to determine 1.macrolide resistance amongst other members of the CF airway microbiota 2.if resistance is associated with current patient prescription of azithromycin. Methods The susceptibility (MIC) of CF respiratory isolates (n = 26: Streptococcus, n=8; Rothia, n=5; Veillonella, n=5; Prevotella, n=4; Actinomyces, n=3; Fusobacterium , n=1) to azithromycin, clarithromycin and erythromycin was determined by Etest®. Isolates were separated into two groups depending on whether they were isolated from CF patients currently prescribed or not currently prescribed azithromycin and macrolide susceptibility was compared between the groups using the Mann–Whitney test. Results High-level macrolide resistance (>256 mg/mL) was common among the isolates tested: azithromycin, n=10/26 (38%); clarithromycin, n=6/26 (23%); erythromycin, n=11/26 (42%). Isolates from CF patients currently prescribed azithromycin (n = 9) had significantly higher MICs for the three macrolide antibiotics compared to isolates from patients not prescribed azithromycin (n = 17) [Table]. TableAzithromycin prescription versus susceptibilityMIC50 (mg/mL)PCurrent prescription of azithromycin (n = 9)No current prescription of azithromycin (n = 17)Azithromycin>25620.005Clarithromycin470.50.001Erythromycin>25640.01 Conclusion Resistance to three different macrolide antibiotics was common in bacteria present in the CF airway microbiota and was associated with current azithromycin prescription in CF patients. Work supported by a Wellcome Trust (FA) Vacation Scholarship.

Prevalence and molecular analysis of macrolide-resistant Moraxella catarrhalis clinical isolates in Japan, following emergence of the highly macrolide-resistant strain NSH1 in 2011.

Although Moraxella catarrhalis is known to be susceptible to macrolides, highly macrolide-resistant M. catarrhalis isolates have recently been reported in Japan and China. In this study, we investigated the prevalence of macrolide-resistant M. catarrhalis isolates in Tokyo and Chiba, Japan, and studied the mechanisms underlying their resistance. Specifically, we determined the susceptibility of 593 clinical isolates (collected between December 2011 and May 2014) to erythromycin, using the disk diffusion method. For isolates with erythromycin resistance, we identified the MICs of seven antimicrobial agents, including macrolides, and used PFGE to analyse the clonal spread. We also performed sequencing analysis to investigate macrolide-resistance targets. Thirteen isolates (2.2 %) were found to be resistant to erythromycin, showing a high MIC90 to erythromycin, clarithromycin, clindamycin and azithromycin. However, those isolates, in addition to 156 randomly selected erythromycin-susceptible strains, were susceptible to amoxicillin-clavulanate, cefixime and levofloxacin. The 13 highly macrolide-resistant isolates were classified into 10 clades and harboured three or four A2058T-mutated 23S rRNA alleles. Three highly macrolide-resistant isolates also exhibited mutations in ribosomal proteins L4 (V27A and R161C) and L22 (K68T). To the best of our knowledge, we have demonstrated for the first time that, whilst the prevalence of macrolide-resistant M. catarrhalis isolates is low in clinical settings in Japan, genetically diverse isolates with high-level macrolide resistance due to the acquisition of an A2058T mutation in the 23S rRNA have already spread. Our study therefore lays the basis for epidemiological studies of macrolide-resistant M. catarrhalis clinical isolates.

High-Level Macrolide-Resistant Moraxella catarrhalis and Development of an Allele-Specific PCR Assay for Detection of 23S rRNA Gene A2330T Mutation: A Three-Year Study at a Chinese Tertiary Hospital.

Previous studies indicate that macrolide resistance in Moraxella catarrhalis isolates is less common in adults than in children. However, few studies have investigated M. catarrhalis macrolide resistance mechanisms in adult patients. In this study, 124 M. catarrhalis isolates were collected from adult patients in a Chinese tertiary hospital, between 2010 and 2013, and investigated for antimicrobial resistance. We found that only seven isolates were macrolide resistant and all exhibited high-level macrolide resistance (minimum inhibitory concentrations >256 μg/ml). Multilocus sequence typing (MLST) suggested that M. catarrhalis has a diverse population; in particular, both pulsed-field gel electrophoresis and MLST revealed that all the seven high-level macrolide-resistant M. catarrhalis belonged to different clones. A 934-bp 23S rRNA gene sequencing showed that only nine isolates (including all the seven macrolide-resistant isolates) had mutations within the studied region, and only the seven macrolide-resistant isolates had mutation of A2330T. No other known macrolide-resistance determinant genes (ermA, ermB, mefA, or mefE) were detected. These findings support previous studies in children on M. catarrhalis macrolide-resistant isolates and suggest that the 23S rRNA gene A2330T mutation is responsible for the high M. catarrhalis macrolide resistance. The findings prompted us to successfully develop a simple allele-specific polymerase chain reaction assay for high-level macrolide-resistant 23S rRNA gene A2330T mutation for future clinical and further surveillance use.

Identification of ribosomal RNA methyltransferase gene ermF in Riemerella anatipestifer

Riemerella anatipestifer is a major bacterial pathogen of waterfowl, globally responsible for avian septicaemic disease. As chemotherapy is the predominant method for the prevention and treatment of R. anatipestifer infection in poultry, the widespread use of antibiotics has favoured the emergence of antibiotic-resistant strains. However, little is known about R. anatipestifer susceptibility to macrolide antibiotics and its resistance mechanism. We report for the first time the identification of a macrolide resistance mechanism in R. anatipestifer that is mediated by the ribosomal RNA methyltransferase ermF. We identified the presence of the ermF gene in 64/206 (31%) R. anatipestifer isolates from different regions in China. An ermF deletion strain was constructed to investigate the function of the ermF gene on the resistance to high levels of macrolides. The ermF mutant strain showed significantly decreased resistance to macrolide and lincosamide, exhibiting 1024-, 1024-, 4- and >2048-fold reduction in the minimum inhibitory concentrations for erythromycin, azithromycin, tylosin and lincomycin, respectively. Furthermore, functional analysis of ermF expression in E. coli XL1-blue showed that the R. anatipestifer ermF gene was functional in E. coli XL1-blue and conferred resistance to high levels of erythromycin (100 µg/ml), supporting the hypothesis that the ermF gene is associated with high-level macrolide resistance. Our work suggests that ribosomal RNA modification mediated by the ermF methyltransferase is the predominant mechanism of resistance to erythromycin in R. anatipestifer isolates.

Single-nucleotide polymorphism PCR for the detection of Mycoplasma pneumoniae and determination of macrolide resistance in respiratory samples

The aim of this study was to develop a single-nucleotide polymorphism (SNP) PCR assay to be performed directly on respiratory samples for the simultaneous detection of Mycoplasma pneumoniae and its 23S rRNA gene mutations, which are responsible for macrolide resistance. For multiplex SNP PCR, two outer primers for amplification of the 23S rRNA gene and two mutant-specific primers for the discrimination of single base changes were designed. A total of 73M. pneumoniae-positive samples and 100M. pneumoniae-negative samples were analyzed using this assay. By SNP PCR, we detected two mutations conferring high-level macrolide resistance in 22 samples (A2063G from 20 and A2064G from 2 samples); these results are identical to those produced by the 23S rRNA gene sequencing of M. pneumoniae-positive samples. Thus, this assay can be used as a practical method for the simultaneous detection of M. pneumoniae and mutations associated with macrolide resistance directly from respiratory samples.

Antimicrobial Susceptibilities and Treatment Options for Pediatric Mycoplasma pneumoniae Infections - Does Macrolide Resistance Matter?

Mycoplasma pneumoniae is an important pathogen of the upper and lower respiratory tracts of children and adults. Historically, it has been susceptible in vitro to macrolides, tetracyclines, and fluoroquinolones. Standardized methods for performance and interpretation of in vitro susceptibility tests have now been published for M. pneumoniae and mechanisms of acquired resistance to macrolides have been shown to be due to mutations in the 23S rRNA gene. Emergence and widespread dissemination of clinically significant high-level macrolide resistance in Asia and documentation of its occurrence to a lesser extent in several European countries and the United States during the past decade is worrisome since treatment options for children are limited. This article summarizes the current status of antimicrobial susceptibility testing, mechanisms of antimicrobial resistance, epidemiology of macrolide resistance and treatment alternatives for pediatric infections caused by M. pneumoniae. Keywords: Mycoplasma pneumoniae, antimicrobial susceptibility, treatment, antibiotic resistance, macrolide.

Report of ribosomal RNA methylase gene erm(B) in multidrug-resistant Campylobacter coli

Campylobacter is a major foodborne enteric pathogen and macrolides are the drug of choice for the clinical therapy of campylobacteriosis. Macrolide resistance among Campylobacter compromises clinical treatment, is associated with adverse health events and is a significant public health concern. Here, we report the first identification of a horizontally transferrable macrolide resistance mechanism in porcine Campylobacter coli ZC113 that is mediated by a ribosomal RNA methylase, Erm(B). Horizontal transfer of a macrolide resistance determinant between C. coli and Campylobacter jejuni was performed by natural transformation. Whole-genome sequencing was initially used to identify the ribosomal methylase-encoding gene erm(B) in Campylobacter. Cloning of erm(B) into C. jejuni NCTC 11168 was performed to evaluate whether the erm(B) gene is responsible for high-level macrolide resistance in Campylobacter. The erm(B) gene was identified in ZC113, conferred high-level resistance to macrolides and was associated with a chromosomal multidrug-resistant genomic island (MDRGI). The MDRGI probably originated from Gram-positive bacteria and was horizontally transferred between C. coli and C. jejuni via natural transformation. Furthermore, the erm(B)-positive isolate ZC113 was resistant to all clinically important antibiotics used for treating campylobacteriosis and is essentially multidrug-resistant Campylobacter. To the best of our knowledge, this is the first report of a horizontally transferable macrolide resistance mechanism in thermophilic Campylobacter. Surveillance of erm(B) and its associated MDRGI in both C. coli and C. jejuni is urgently warranted.

Antimicrobial susceptibility and mechanisms of high-level macrolide resistance in clinical isolates of Moraxella nonliquefaciens

We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing β-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ≥128 mg l(-1)) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P<0.0001). Thirty-two isolates with high-level macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.