Macrolide Resistance Mutations Research Articles

Mycoplasma pneumoniae Macrolide Resistance in Children in Central Ohio Detected by Sequencing

Background Mycoplasma pneumoniae(Mp) is one of the most common causes of LRTI in school aged children, with estimates of up to 20% of cases of community-acquired pneumonia (CAP). Macrolides are the drug of choice for treating Mp infections, but in the last 2 decades there has been emergence of resistance worldwide. Reported rates in USA vary from 3.5 up to 10.8%, but rates in Ohio are unknown. Currently there is no well-established, standardized method for detecting macrolide resistance and antibiotic treatment is empiric because of these limitations. Our goal was to determine the frequency of macrolide resistance in children with Mp respiratory infections in central Ohio.MethodsA convenience sample of Mp positive PCR identified by our laboratory-developed assay, obtained from both inpatient and outpatient settings, were selected. These were then cultured using Remel’s SP4 glucose broth and then incubated at 35°C until isolates grew, or for a maximum of 4 weeks. All samples that yield positive cultures were then sequenced for the domain V of the 23S rRNA (nucleotide 1937–2154, accession no. X68422) using Sanger methods and sequences were compared with corresponding region of wildtype reference strain (ATCC 15322).ResultsFor the period of October 2015-March 2017, culture was attempted in 433 PCR-positive samples, and 347 (80%) yield an isolate. Sequencing was performed in those 347 samples and was successful in 334 (96%). A macrolide resistance mutation was detected in 10 samples (3%). From the 10 mutations detected, 7 (70%) were the A2063G mutation, and 3 (30%) were A2064G. We also detected a deletion in A2065 of unknown significance in 3 samples (0.9%).ConclusionIn this pediatric population, culture was successful in 80% of PCR positive samples, thus providing isolates for sequencing to be able to assess our local resistance. Our overall Mp resistance was 3%, suggesting that empiric treatment with macrolides is still appropriate in Central Ohio. Additional studies are needed to correlate the presence of these resistance mutations with phenotypic susceptibility testing and clinical outcomes.Disclosures O. Ramilo, Abbvie: Board Member, Consulting fee; Regeneron: Board Member, Consulting fee; Janssen: Board Member and Investigator, Consulting fee and Research grant; NIH: Grant Investigator, Research grant; A. Leber, BioFIre Dia gnostics: Research Contractor and Scientific Advisor, Research support, Speaker honorarium and Travel expenses

Clinical Characteristics of Macrolide-Resistant Mycoplasma pneumoniae from Children in Jeju.

Mycoplasma pneumoniae is the major pathogen of community-acquired pneumonia in children. The prevalence of macrolide-resistant M. pneumoniae (MRMP) is important owing to the limited alternative therapies for children. We analyzed 111 M. pneumoniae obtained from 107 children admitted for lower respiratory tract infection at Jeju National University Hospital between 2010 and 2015. Macrolide resistance of M. pneumoniae was searched for using polymerase chain reaction (PCR) and sequencing. Of 107 clinical M. pneumoniae, 11 (10.3%) carried macrolide resistance mutations in the 23S rRNA gene. All macrolide resistance mutations were A2063G transitions. We found an acquired A2063G mutation of M. pneumoniae from a patient during macrolide treatment. Patients' characteristics and clinical severity did not differ between those with MRMP and macrolide-sensitive M. pneumoniae, with the exception of frequent pleural effusion in the MRMP group. The prevalence of MRMP (10.3%) in Jeju Island was relatively lower than those of surrounding countries in East Asia. Previous antimicrobial usage and timing of diagnostic test should be considered when determining of macrolide resistance of M. pneumoniae.

Development of an endpoint genotyping assay to detect the Mycoplasma pneumoniae 23S rRNA gene and distinguish the existence of macrolide resistance-associated mutations at position 2063

The prevalence of macrolide-resistant Mycoplasma pneumoniae harboring a mutation in the 23S rRNA gene is increasing, and rapid detection assays are needed for clinical management. We developed an endpoint genotyping assay to detect the M. pneumoniae 23S rRNA gene and determine the existence of macrolide resistance-associated mutations at position 2063 (A2063G, A2063T and A2063C mutations). This A2063B genotyping assay detected more than 50 copies/reaction of the M. pneumoniae gene in every nucleotide mutation at position 2063. Of 42 clinical specimens, 3 were positive without mutation, 6 were positive with the A2063G mutation, and 33 were negative. The results were confirmed using nested PCR with the sequencing of the M. pneumoniae 23S rRNA gene, and a high sensitivity (90%), specificity (100%), and coincidence ratio (kappa coefficient=0.93) were obtained. Therefore, the A2063B genotyping assay is useful for the rapid discrimination of macrolide resistance mutations at position 2063.

Treponema pallidum pallidum Genotypes and Macrolide Resistance Status in Syphilitic Lesions among Patients at 2 Sexually Transmitted Infection Clinics in Lima, Peru.

We report the circulating genotypes and the frequency of macrolide-resistance patterns among Treponema pallidum pallidum DNA isolated from syphilitic lesions from patients who attended 2 sexual health clinics in Lima, Peru. We implemented and used a molecular typing scheme to describe local T. pallidum pallidum strains. Among 14 specimens, subtype 14d/f was the most prevalent strain in 7 fully typed T. pallidum DNA specimens obtained from men who have sex with men and transgender women presenting with chancre-like lesions. No macrolide-resistance mutations were found in T. pallidum DNA from 10 lesions.

O031 Pelvic Inflammatory Disease (PID), Mycoplasma genitalium and macrolide resistance in England

Background/introduction Mycoplasma genitalium (MG) is increasingly implicated in PID pathogenesis with many studies showing MG is as common as chlamydia in high-risk women. Current PID treatment guidelines specify antibiotics with low efficacy against MG. Increasing reports of macrolide resistance suggests first line treatment for MG (azithromycin) may have limitations. Aim(s)/objectives To document rates of MG in a cohort of women with acute PID, and the proportion with baseline macrolide resistance. Methods As part of a multicentre, open-label, non-inferiority RCT comparing ofloxacin/metronidazole (arm-1) with azithromycin 1g day-1; 500 mg od day 2–5, metronidazole/ceftriaxone (arm-2), samples were collected for baseline chlamydia, gonorrhoea and mycoplasma infection. Microbiological cure rates were documented at 6–8 weeks. Positive MG specimens were examined for macrolide resistance using a 23S rRNA PCR. Results 313 women were recruited, median age 25. Preliminary results showed chlamydia was confirmed in 9.5%, MG in 8.2% and gonorrhoea in 0.4%. Of the 16 samples available for resistance testing, 9 (56%) had macrolide resistance mutations (A2058G/T, A2059G/C) at baseline. The reference laboratory received test-of-cure samples for only 8 patients with MG, of which 6 were negative, however 2 remained positive, both with A2059G nucleotide substitutions. Further results will be presented. Discussion/conclusion MG infection was nearly as common as chlamydia in this cohort. Failure of patients to return at 6–8 weeks affected our ability to properly assess test-of-cure rates. Baseline macrolide resistance was unexpectedly high and impacted negatively on treatment success.

Syphilis epidemiology in 1994-2013, molecular epidemiological strain typing and determination of macrolide resistance in Treponema pallidum in 2013-2014 in Tuva Republic, Russia.

The incidence of syphilis in the Tuva Republic (geographical centre of Asia), Russia has been exceedingly high historically. No detailed examinations and no molecular investigations of Treponema pallidum strains transmitted in the Tuva Republic, or in general, in Russia, were published internationally. We examined the syphilis epidemiology in 1994-2013, and the molecular epidemiology and macrolide resistance in T. pallidum strains in 2013-2014 in the Tuva Republic. Among 95 mainly primary or secondary syphilis patients, the arp, tpr, tp0548 and 23S rRNA genes in 85 polA gene-positive genital ulcer specimens were characterized. The syphilis incidence in Tuva Republic peaked in 1998 (1562), however declined to 177 in 2013. Among the 70 (82%) completely genotyped specimens, six molecular strain types were found. Strain type 14d/f accounted for 91%, but also 14c/f, 14d/g, 14b/f, 14i/f, 9d/f, and 4d/f were identified. Two (2.4%) specimens contained the 23S rRNA A2058G macrolide resistance mutation. This is the first internationally published typing study regarding T. pallidum in Russia, performed in the Tuva Republic with the highest syphilis incidence in Russia. The two molecular strain types 4d/f and 9d/f have previously been described only in Eastern and Northern China and for the first time, macrolide-resistant syphilis was described in Russia.

005.4 Multiplex assay for simultaneous detection ofmycoplasma genitaliumand macrolide resistance using pass mnazyme QPCR

Introduction Treatment of M. genitalium (Mg) infection with single dose 1 g macrolide antibiotic, azithromycin, is routinely utilised in clinical practice however this has been associated with emergence of macrolide resistance and ineffective cure rates. Mutations at two positions, 2058 and 2059 (E. coli numbering) in the Mg 23S rRNA gene have been associated with macrolide resistance. Simultaneous detection of Mg and mutations associated with azithromycin failure could be used to offer rapid delivery of effective second line regimens. This study evaluates a combined diagnostic-resistance assay for potential use in clinical settings. Methods Clinical samples diagnosed with Mg were evaluated with a combined-diagnostic resistance assay that employs novel “Primer Assisted Sequence Switching” (PASS) primers coupled with Multi-component Nucleic Acid enzyme” (MNAzyme) detection. PASS primers selectively amplify target sequences resulting in enhanced specificity for mutant over wild-type and MNAzymes allow for efficient detection and discrimination of multiple mutations simultaneously. Multiplexed PASS MNAzyme qPCR was evaluated by comparison to previously screened clinical samples for Mg (MgPa gene) and 23S mutations using Sanger sequencing and HRM analysis. Results Using artificial templates, this assay was able to detect mutation templates ranging from 10–10,240 copies/reaction, with an associated Mg detection limit comparable to existing assays used in routine diagnostics. Preliminary screening of DNA from 24 clinical samples revealed Mg detection range of 3–300,000 copies/reaction. This assay was able to detect the correct mutation in 21/24 cases (87.5%), however was not readily able to assign a mutation at the lowest concentrations tested, an issue present in all rapid mutation screening tests for Mg. Conclusion Multiplexed PASS MNAzyme qPCR offers the ability for simultaneous detection of Mg and macrolide resistance mutations. This assay offers considerable advantages in clinical settings with rapid identification of macrolide resistant strains and the ability to implement effective second line agents without delay. Disclosure of interest statement SpeeDx is the developer and manufacturer of the assay evaluated in this study.

005.5 Treponema pallidumstrain-types in australia and association with macrolide resistance

Introduction Greater understanding of the molecular epidemiology of Trepomema pallidum has the potential to enhance control measures. The aim of this study was to determine the strain-types of T. pallidum in Sydney, Australia, and investigate clinical and epidemiological associations. Methods Stored T. pallidum DNA from samples between 2004–2011were catagorised into strain-types using analysis of the acidic repeat protein (arp), T.pallidum repeat sub- family (tpr) genes, and sequence analysis of the TP0548 gene as described by Marra (2010). Associations between strain-type, the macrolide resistance mutation A2058G, and clinical and demographic characteristics were further investigated. Results 194 samples from 187 patients were successfully strain-typed into at least 19 separate strains. The predominant strains were 14d/g (91/194; 47%), and 14d/f (18/194; 9%).14d/g remained the commonest strain throughout the study period, and was associated with the A2058G mutation (90/91 vs 70/103 non-14d/g strains: OR 42.4; 95% CI 5.7–317.9 p < 0.001). Clinical information was available for 91 samples. 90 were male, of whom 89 reported sex with other men. 21/48 (44%) strains from HIV negative patients were strain 14d/g vs 20/43 (47%) from those HIV positive (OR 1.1; 95% CI 0.5–2.5 p = 0.79). When acquisition was reported as being outside Australia 2/13 (15%) cases were strain 14d/g vs 39/78 (50%) of those reporting sex only within Australia (OR 0.18; 95% CI 0.04–0.87 p = 0.033.) Both cases of neurosyphilis were attributable to TP0548 gene sequence “f”. Conclusion This is the first time that enhanced strain-typing has been used to define the epidemiology of Treponema pallidum infections in the Asia-Pacific region. The most common strain (14d/g) was associated with macrolide resistance and acquisition within Australia. Despite the diversity of strains, the lack of association with HIV status suggests sexual networks between HIV negative and HIV positive men overlap. Disclosure of interest statement Phillip Read received an RACP Novartis Sexual Health Research Scholarship to part fund this research.

P05.13 Prevalence and anatomical distribution ofmycoplasma genitaliummacrolide resistance markers from subjects enrolled in a multi-centre us clinical study

<h3>Introduction</h3> This study evaluated the prevalence and anatomical distribution of <i>Mycoplasma genitalium</i> (Mgen) 23s rRNA mutations (23s-MTs) conferring macrolide resistance among male and female subjects enrolled in a prospective multi-site US clinical study. <h3>Methods</h3> Specimens obtained from symptomatic and asymptomatic men and women enrolled from 7 diverse US clinical sites, including obstetrics and gynaecology, family planning, public health, and sexually transmitted disease clinics, were tested using a research TMA assay for Mgen (Hologic, Inc.) on the DTS System or TIGRIS DTS System. Samples positive for Mgen by TMA were evaluated by nested PCR or RT-PCR and Sanger sequencing of Mgen 23S rRNA to identify the presence of macrolide resistance mutations at position 2058 (A2058G, A2058C, A2058T) or position 2059 (A2059G). <h3>Results</h3> Of 50 male subjects with Mgen 23s rRNA sequence results, 21 (42%) contained 23s-MTs. Slightly more 23s-MTs were found in urethral swabs vs male urine samples (44.8% vs 36.7%, respectively). For female subjects, 65 of 128 (50.8%) harboured 23s-MTs, with higher prevalence found in vaginal swab samples (50.2%) compared to urine (46%), Thinprep liquid Pap (41.7%), and endocervical swabs (37.8%). Sequencing of samples collected from anatomically distinct female urogenital sites (vagina, cervix, urine) revealed 18 of 35 (51.4%) subjects had a unique complement of Mgen 23s-MT and/or wild-type sequences at each anatomic site. For male subjects with both urine and urethral swab samples, 3 of 9 (33.3%) subjects had unique Mgen 23s-MT/WT sequences in each sample type. <h3>Conclusion</h3> Mgen strains harbouring 23s rRNA-mediated macrolide resistance phenotypes were highly prevalent in this US cohort of male and female subjects. Detection of different macrolide-resistant Mgen strains in samples collected from different anatomical locations suggests that previous estimates for resistance rates that relied on a single anatomical site sample collection may have underestimated the extent of Mgen macrolide resistance in the population. <h3>Disclosure of interest statement</h3> D Getman, M O’Donnell, and A Jiang are scientists employed by Hologic. S Cohen is a student at Occidental College and a summer intern at Hologic.

Macrolide Resistance in the Syphilis Spirochete, Treponema pallidum ssp. pallidum: Can We Also Expect Macrolide-Resistant Yaws Strains?

Treponema pallidum ssp. pallidum (TPA) causes over 10 million new cases of syphilis worldwide whereas T. pallidum ssp. pertenue (TPE), the causative agent of yaws, affects about 2.5 million people. Although penicillin remains the drug of choice in the treatment of syphilis, in penicillin-allergic patients, macrolides have been used in this indication since the 1950s. Failures of macrolides in syphilis treatment have been well documented in the literature and since 2000, there has been a dramatic increase in a number of clinical samples with macrolide-resistant TPA. Scarce data regarding the genetics of macrolide-resistant mutations in TPA suggest that although macrolide-resistance mutations have emerged independently several times, the increase in the proportion of TPA strains resistant to macrolides is mainly due to the spread of resistant strains, especially in developed countries. The emergence of macrolide resistance in TPA appears to require a two-step process including either A2058G or A2059G mutation in one copy of the 23S rRNA gene and a subsequent gene conversion unification of both rRNA genes. Given the enormous genetic similarity that was recently revealed between TPA and TPE strains, there is a low but reasonable risk of emergence and spread of macrolide-resistant yaws strains following azithromycin treatment.

Characterization of MDR and XDR Streptococcus pneumoniae in Canada, 2007-13.

The goal of this study was to characterize Streptococcus pneumoniae demonstrating MDR (resistant to three or more antimicrobial classes) or XDR (resistant to five or more classes) phenotypes, collected from Canada during the CANWARD 2007-13 study. From 2007 to 2013 inclusive, S. pneumoniae isolates were collected as a part of the CANWARD surveillance study. MDR and XDR isolates were subjected to PFGE, MLST, molecular detection of pneumococcal pili and macrolide resistance determinants mef(A/E) and erm(B), sequencing of PBPs 1A, 2B and 2X and comparison with Pneumococcal Molecular Epidemiology Network (PMEN) clones. Of 2129 S. pneumoniae isolates collected during the CANWARD 2007-13 study, 61 (2.9%) were found to be MDR. Of these MDR isolates, 43 (70.5%) were XDR. The most common serotypes for both MDR and XDR S. pneumoniae were 19A and 19F. Twenty-nine of 61 isolates (48%) demonstrated resistance to clarithromycin, clindamycin, doxycycline, penicillin and trimethoprim/sulfamethoxazole. All isolates possessed at least one macrolide resistance determinant and mutations in PBPs 1A, 2B and 2X. The most common clone was piliated, XDR ST320, an internationally circulating double-locus variant of Taiwan(19F)-14 (ST236). Though the rate of MDR S. pneumoniae has remained relatively stable since 2007, XDR strains have emerged in Canada. These strains are virulent, possess resistance determinants and are related to international clones.

Mass Treatment with Single-Dose Azithromycin for Yaws

BackgroundMass treatment with azithromycin is a central component of the new World Health Organization (WHO) strategy to eradicate yaws. Empirical data on the effectiveness of the strategy are required as a prerequisite for worldwide implementation of the plan.MethodsWe performed repeated clinical surveys for active yaws, serologic surveys for latent yaws, and molecular analyses to determine the cause of skin ulcers and identify macrolide-resistant mutations before and 6 and 12 months after mass treatment with azithromycin on a Papua New Guinean island on which yaws was endemic. Primary-outcome indicators were the prevalence of serologically confirmed active infectious yaws in the entire population and the prevalence of latent yaws with high-titer seroreactivity in a subgroup of children 1 to 15 years of age.ResultsAt baseline, 13,302 of 16,092 residents (82.7%) received one oral dose of azithromycin. The prevalence of active infectious yaws was reduced from 2.4% before mass treatment to 0.3% at 12 months (difference, 2.1 percentage points; P<0.001). The prevalence of high-titer latent yaws among children was reduced from 18.3% to 6.5% (difference, 11.8 percentage points; P<0.001) with a near-absence of high-titer seroreactivity in children 1 to 5 years of age. Adverse events identified within 1 week after administration of the medication occurred in approximately 17% of the participants, included nausea, diarrhea, and vomiting, and were mild in severity. No evidence of emergence of resistance to macrolides against Treponema pallidum subspecies pertenue was seen.ConclusionsThe prevalence of active and latent yaws infection fell rapidly and substantially 12 months after high-coverage mass treatment with azithromycin, with the reduction perhaps aided by subsequent activities to identify and treat new cases of yaws. Our results support the WHO strategy for the eradication of yaws. (Funded by Newcrest Mining and International SOS; YESA-13 ClinicalTrials.gov number, NCT01955252.)

Treatment efficacy, treatment failures and selection of macrolide resistance in patients with high load of Mycoplasma genitalium during treatment of male urethritis with josamycin.

BackgroundAzithromycin has been widely used for Mycoplasma genitalium treatment internationally. However, the eradication efficacy has substantially declined recent decade. In Russia, josamycin (another macrolide) is the recommended first-line treatment for M. genitalium infections, however, no data regarding treatment efficacy with josamycin and resistance in M. genitalium infections have been internationally published. We examined the M. genitalium prevalence in males attending an STI clinic in Moscow, Russia from December 2006 to January 2008, investigated treatment efficacy with josamycin in male urethritis, and monitored the M. genitalium DNA eradication dynamics and selection of macrolide resistance in M. genitalium during this treatment.MethodsMicroscopy and real-time PCRs were used to diagnose urethritis and non-viral STIs, respectively, in males (n = 320). M. genitalium positive patients were treated with recommended josamycin regimen and treatment efficacy was monitored using quantitative real-time PCR. Macrolide resistance mutations were identified using sequencing of the 23S rRNA gene.ResultsForty-seven (14.7%) males were positive for M. genitalium only and most (85.1%) of these had symptoms and signs of urethritis. Forty-six (97.9%) males agreed to participate in the treatment efficacy monitoring. All the pre-treatment M. genitalium specimens had wild-type 23S rRNA. The elimination of M. genitalium DNA was substantially faster in patients with lower pre-treatment M. genitalium load, and the total eradication rate was 43/46 (93.5%). Of the six patients with high pre-treatment M. genitalium load, three (50%) remained positive post-treatment and these positive specimens contained macrolide resistance mutations in the 23S rRNA gene, i.e., A2059G (n = 2) and A2062G (n = 1).ConclusionsM. genitalium was a frequent cause of male urethritis in Moscow, Russia. The pre-treatment M. genitalium load might be an effective predictor of eradication efficacy with macrolides (and possibly additional antimicrobials) and selection of macrolide resistance. Additional in vivo and in vitro data are crucial to support the recommendation of using josamycin as first-line treatment for M. genitalium infections in Russia. It would be valuable to develop international M. genitalium management guidelines, and quantitative diagnostic PCRs determining also M. genitalium load and resistance mutations (for macrolides and ideally also moxifloxacin) should ideally be recommended.

Gene Mutations Associated with Macrolide-resistance and p1 Gene Typing of Mycoplasma pneumoniae Isolated in Yamagata, Japan, between 2004 and 2013

To clarify the epidemiologic features of Mycoplasma pneumoniae, we examined 358 M. pneumoniae strains isolated between 2004 and 2013 in Yamagata, Japan. Analysis of macrolide-resistance-associated 23S ribosomal RNA (rRNA) domain V mutations revealed 6 kinds of mutants (81 A2063G, 43 A2063T, 1 A2063C, 1 A2064C, 4 C2617G and 1 C2617 mutation). There were only two mutants before 2009, but mutants A2063T and A2063G increased in 2009 and from 2010, respectively. The annual ratio of mutants varied from 20.4% to 76.4% between 2009 and 2013. Typing of the p1 gene revealed 4 types; 278 type 1, and 3 kinds of type 2 variant strains (10 type 2a, 5 type 2b and 65 type 2c). Type 1 strains accounted for between 85.2% and 100% of isolates from 2004 to 2011, whereas type 2 variant strains increased by 26.5% and 66.1% in 2012 and 2013, respectively. These results indicate that type 1 strains may have been replaced by type 2 variant strains in 2013. Furthermore, the ratio of type 1 strains with a 23S rRNA mutation was 65.1% in 2012 and 95.2% in 2013, but none of the type 2 variant strains had this mutation. In conclusion, type 1 strains with macrolide-resistant mutations appeared in 2006 and increased from 2009. In contrast, type 2 variant strains, which increased in 2012 and became predominant in 2013, showed no mutations.

Macrolide resistance and azithromycin failure in a Mycoplasma genitalium-infected cohort and response of azithromycin failures to alternative antibiotic regimens.

Our aim was to determine the efficacy of 1 g azithromycin and alternative antibiotic regimens in a prospective cohort of Mycoplasma genitalium-infected participants, and factors associated with azithromycin failure. Consecutive eligible M. genitalium-infected men and women attending the Melbourne Sexual Health Centre between July 2012 and June 2013 were treated with 1 g of azithromycin and retested by polymerase chain reaction (PCR) on days 14 and 28. Cure was defined as PCR negative on day 28. Cases failing azithromycin were treated with moxifloxacin, and those failing moxifloxacin were treated with pristinamycin. Pre- and posttreatment samples were assessed for macrolide resistance mutations (MRMs) by high-resolution melt analysis. Mycoplasma genitalium samples from cases failing moxifloxacin were sequenced for fluoroquinolone resistance mutations. Multivariable analysis was used to examine associations with azithromycin failure. Of 155 participants treated with 1 g azithromycin, 95 (61% [95% confidence interval {CI}, 53%-69%]) were cured. Pretreatment MRM was detected in 56 (36% [95% CI, 28%-43%]) participants, and strongly associated with treatment failure (87% [95% CI, 76%-94%]; adjusted odds ratio, 47.0 [95% CI, 17.1-129.0]). All 11 participants who had MRM detected in posttreatment samples failed azithromycin. Moxifloxacin was effective in 53(88% [95% CI, 78%-94%]) of 60 cases failing azithromycin; all failures had gyrA and parC mutations detected in pretreatment samples. Six of 7 patients failing moxifloxacin treatment received pristinamycin, and all were PCR negative 28 days after pristinamycin treatment. We report a high azithromycin failure rate (39%) in an M. genitalium-infected cohort in association with high levels of pretreatment macrolide resistance. Moxifloxacin failure occurred in 12% of patients who received moxifloxacin; all had pretreatment fluoroquinolone mutations detected. Pristinamycin was highly effective in treating macrolide- and quinolone-resistant strains.

Characteristics of Mycoplasma pneumoniae infection identified on culture in a pediatric clinic.

The appropriate choice of antibiotics against Mycoplasma pneumoniae infection has become difficult, as the prevalence of macrolide-resistant M. pneumoniae has increased. Throat swab specimens were collected from children with clinically suspected M. pneumoniae infection while visiting an outpatient clinic. Cultures for M. pneumoniae were done, and all isolates were sequenced for the presence of a mutation in 23S rRNA. Of the 80 specimens collected between February 2012 and March 2013, 27 (34%) were positive for M. pneumoniae on culture. Macrolide-resistant mutation was detected in 24 isolates (89%): 23 isolates had an A2063G transition, and one had a C2617G mutation. Both the median age and the prevalence of pneumonia were significantly higher in M. pneumoniae-positive than in M. pneumoniae-negative children (median, 7 years vs 4 years; 88.9% vs 60.4%, respectively). The percentage of serum samples with particle agglutination titer ≥ 1:160 was 69.6% in M. pneumoniae-positive cases and 17.6% in M. pneumoniae-negative cases when the serum was collected ≥ 4 days after the onset of fever. Defervescence within 72 h after the initiation of macrolides never occurred in M. pneumoniae-positive children and also did not occur in 54% of M. pneumoniae-negative children. Switching to either minocycline or tosufloxacin resulted in fever resolution within 48 h in M. pneumoniae-positive children. The described clinical and laboratory characteristics of M. pneumoniae infection may be useful in guiding appropriate treatment in an outpatient clinic.

Identification of Campylobacter jejuni and determination of point mutations associated with macrolide resistance using a multiplex TaqMan MGB real-time PCR

Campylobacter jejuni, one of the most important food-borne pathogens, develops resistance to macrolides by acquiring point mutations in the 23S rDNA gene (e.g. A2075G or A2074C) and in ribosomal protein L4 (e.g. G74D or G57D). In this study, a multiplex fluorescence real-time PCR assay, based on TaqMan minor groove binder (MGB) probes, was developed to identify C. jejuni containing mutations associated with macrolide resistance. The VS1-MGB probe was designed based on the VS1 gene and was used to identify C. jejuni. The 23S rDNA-MGB probe was designed to distinguish macrolide resistance mutations in 23S rDNA, whilst 57D-MGB and 74D-MGB were designed to detect resistance mutations in ribosomal protein L4. The specificity and accuracy of the established method were 100% identical to the classic biochemical method, mapA PCR, minimum inhibitory concentration (MIC) determination and DNA sequencing. The linear detection limitation of the method was 0.03ng/reaction genomic DNA and 3CFU/reaction bacterial colonies. In conclusion, this study provides a novel method that can specifically identify C. jejuni and accurately detect the gene mutations associated with macrolide resistance.

Development of macrolide resistance in Mycoplasma pneumoniae-infected Swedish patients treated with macrolides

The proportion of Mycoplasma pneumoniae isolates carrying point mutations in the 23S region of the genome associated with macrolide resistance has increased. We evaluated the probability of developing M. pneumoniae macrolide resistance mutations during macrolide treatment. M. pneumoniae strains from initial and follow-up oropharyngeal samples from 38 Swedish patients were tested for 23S rRNA mutations by amplifying and pyrosequencing the gene target region. The duration of symptoms and of M. pneumoniae carriage were recorded in cases with and without the macrolide resistance mutation. Macrolide resistance mutations were absent in all baseline strains. Twenty-two patients were prescribed macrolides; the longest M. pneumoniae carriage time in these patients was 7 months and the longest symptomatic period was 7.5 months. Macrolide resistance was detected after macrolide treatment in 1 patient (carriage time 3.5 months and symptomatic period 7.5 months). M. pneumoniae may develop macrolide resistance during macrolide treatment. A long duration of M. pneumoniae carriage and symptoms is common in patients regardless of the presence of resistance.

Rapid detection of Mycoplasma pneumoniae and its macrolide-resistance mutation by Cycleave PCR

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia, which is often empirically treated with macrolides such as erythromycin and azithromycin. Recent studies have found a gradual increase in the numbers of macrolide-resistant strains due to mutations in the 23S rRNA gene. A2063G is the most common mutation, followed by A2064G. We developed a Cycleave PCR method for detecting macrolide-resistant strains of M. pneumoniae. With use of this method, the resistant strains can be quickly separated from the susceptible ones. In this work, via this method, both M. pneumoniae and resistance mutants were successfully identified from 101 clinical isolates as well as from 136 nasopharyngeal specimens. The findings of this study may allow clinicians to determine the treatment plan more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.

P1 gene of Mycoplasma pneumoniae in clinical isolates collected in Beijing in 2010 and relationship between genotyping and macrolide resistance

Background Mycoplasma pneumoniae is a common pathogen that caused community-acquired pneumonia (CAP). P1 protein served as major adhesion and immunodominant protein in Mycoplasma pneumoniae, but little about P1 gene was learned and the relationship between P1 genotype and macrolide resistance has yet to be explored. Methods The DNA sequence of the entire P1 gene from 35 strains isolated from clinical specimens collected in Beijing, China, in 2010 was determined. The resulting sequences were checked for known macrolide resistance mutations, such as A2063G, A2064G, C2617G in domain V of 23S rRNA. Antibiotic susceptibility test was done to further identify macrolide resistant strains. Results Thirty-four clinical strains were type 1, and were identical to type 1 reference strain MP129. Only one clinical strain, MpYYM22, was type 2, and proved to be variant 2c. One synonymous point mutation in the P1 type 1 gene from two isolates was identified relative to the MP129 P1 sequence at nucleotide position (nt) 552 (C&gt;A), while another two isolates had missense mutations at nt 2504 (G&gt;A). This point mutation caused an amino acid change from glycine to glutamic acid. An AGT tri-nucleotide variable-number tandem repeat (VNTR), coding for serine and repeating 6-11 times, up to 15-16 times, was found in the region between the RepMP4 and RepMP2/3 elements in the 35 isolates examined. All 35 clinical strains, including MpYYM22, demonstrated macrolide resistance with the range of minimum inhibitory concentration (MIC) of erythromycin from 64 to 256 μg/ml, having an A2063G transition in domain V of the 23S rRNA gene. Conclusions P1 type 1 was the dominant type of Mycoplasma pneumoniae in Beijing in 2010, although variant 2c strains were present. More samples are needed to determine whether there is a relationship between the P1 genotype and macrolide resistance, as the 35 strains examined did not allow a conclusive result. However, the AGT tri-nucleotide VNTR may be a more informative locus for multi-locus VNTR analysis.