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Antimicrobial susceptibility testing (AST) of human mycoplasmas using microdilution is time-consuming. In this study, we compared the performance of MICRONAUT-S plates (Biocentric-Bruker) designed for AST of Ureaplasma parvum , Ureaplasma urealyticum , and Mycoplasma hominis with the results using the Clinical & Laboratory Standards Institute (CLSI) reference method. Then, we investigated the prevalence and mechanisms of resistance to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. The two methods were compared using 60 strains. For the resistance prevalence study, U. parvum -, U. urealyticum -, and M. hominis -positive clinical specimens were collected for 1 month each year in 22 French diagnostic laboratories. MICs were determined using the MICRONAUT-S plates. The tet (M) gene was screened using PCR, and fluoroquinolone resistance-associated mutations were screened using PCR and Sanger sequencing. Comparing the methods, 99.5% (679/680) MICs obtained using the MICRONAUT-S plates concurred with those obtained using the CLSI reference method. For 90 M . hominis isolates, the tetracycline, levofloxacin, and moxifloxacin resistance rates were 11.1%, 2.2%, and 2.2%, respectively, with no clindamycin resistance. For 248 U . parvum isolates, the levofloxacin and moxifloxacin resistance rates were 5.2% and 0.8%, respectively; they were 2.9% and 1.5% in 68 U . urealyticum isolates. Tetracycline resistance in U. urealyticum (11.8%) was significantly ( P < 0.001) higher than in U. parvum (1.2%). No macrolide resistance was observed. Overall, the customized MICRONAUT-S plates are a reliable, convenient tool for AST of human mycoplasmas. Tetracycline and fluoroquinolone resistance remain limited in France. However, the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires monitoring., Importance: Antimicrobial susceptibility testing of human urogenital mycoplasmas using the CLSI reference broth microdilution method is time-consuming and requires the laborious preparation of antimicrobial stock solutions. Here, we validated the use of reliable, convenient plates designed for antimicrobial susceptibility testing that allows the simultaneous determination of the MICs of eight antibiotics of interest. We then investigated the prevalence and mechanisms of resistance of each of these bacteria to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. We showed that the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires ongoing monitoring., Competing Interests: The authors declare no conflict of interest.

As macrolide resistance in Mycoplasma genitalium is increasing worldwide, macrolide resistance-associated mutations should be assessed in M. genitalium -positive specimens. New commercial kits are available for detection of macrolide resistance concurrently with M. genitalium We prospectively evaluated the handling and clinical performances of three commercial kits for detection of macrolide resistance in M. genitalium Between August and December 2018, remnants of all urogenital specimens determined to be M. genitalium positive using an in-house real-time PCR assay were prospectively collected at the French National Reference Center for Bacterial Sexually Transmitted Infections, Bordeaux University Hospital, Bordeaux, France. The internal control of each kit was added to the primary specimen before DNA extraction, and the absence of amplification inhibition associated with the addition of the three internal controls was assessed. Specimens were evaluated with four assays: the ResistancePlus MG assay (SpeeDx), the S-DiaMGRes assay (Diagenode), the RealAccurate TVMGres assay (PathoFinder), and amplification and sequencing of the 23S rRNA gene (the reference assay). Overall, 195 M. genitalium -positive specimens were assessed. The positive agreement of M. genitalium detection for each kit ranged between 94.8% and 96.4%. Among 154 specimens with M. genitalium positivity as detected by the three commercial kits and 23S rRNA sequencing data, the clinical sensitivity and specificity ranges of the three commercial kits for detecting macrolide resistance-associated mutations were 95 to 100% and 94.6 to 97.3%, respectively. The sensitivity and specificity values were similar among the kits. The launch of three easy-to-use sensitive and specific commercial kits for simultaneous detection of M. genitalium and macrolide resistance will be useful for resistance-guided therapy., (Copyright © 2020 American Society for Microbiology.)

Objectives: The aim was to compare the antimicrobial efficacy of four different grafts: a standard graft (Intergard, IG), an IG graft soaked in rifampicin (IGrif), a silver impregnated graft (Intergard Silver, IGS), and a silver + triclosan impregnated graft (Intergard Synergy, IGSy)., Methods: This was a seven day in vitro study. The IG, IGrif, IGS, and IGSy grafts were each contaminated separately with the following microorganisms: Staphylococcus epidermidis, Methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans from both clinical and American Type Culture Collection (ATCC) origins. The in vitro antimicrobial efficacy was evaluated by time to kill assays at T0, T24h, T48h, T72h, and T168h. Bactericidal activity was defined as >3 log 10 reduction factor (logRF). Additionally, Rifampicin, triclosan and silver resistance development were screened., Results: As anticipated for the non-antimicrobial IG, all microorganism strains proliferated. The IGSy and the IGS showed a seven day bactericidal efficacy (>3 logRF) for all tested microorganisms. This efficacy was confirmed at all time points for IGSy only, demonstrating faster bactericidal efficacy than IGS. The IGrif demonstrated a seven day bactericidal efficacy against the ATCC MRSA only, while showing no activity against C. albicans and ATCC E. coli. Regarding ATCC S. epidermidis, clinical MRSA and clinical E. coli, IGrif, although bactericidal at earlier time points, lost its antimicrobial efficacy at seven days leading to the emergence of rifampicin resistant mutants in four of six, two of six, and two of six assays, respectively. Mutant strains were also detected in ATCC MRSA in one of six assays. No triclosan or silver resistance has emerged at T7days., Conclusion: For all microorganisms tested, the Synergy graft combining silver with triclosan demonstrated a more sustainable and efficient seven day antimicrobial activity than the rifampicin soaked graft. The emergence of rifampicin resistant mutants suggests preference for a Synergy graft over a graft soaked in rifampicin, to prevent or treat an infection when a biological solution is not feasible., (Copyright © 2018 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the clinical performance of the Aptima Mycoplasma genitalium transcription-mediated amplification (MG-TMA) CE-marked for in vitro diagnosis (CE-IVD) assay for the detection of Mycoplasma genitalium in male and female clinical samples in comparison with the in-house real-time PCR (in-house PCR) assay routinely used in our laboratory. A total of 1,431 clinical specimens obtained from 1,235 patients were prospectively collected at the Bacteriology Department of Bordeaux University Hospital (France). Additional research-use-only Aptima M. genitalium transcription-mediated amplification (TMA) assays, Alt1-TMA and Alt2-TMA, were performed on discordant specimens to determine M. genitalium infection status. All confirmed M. genitalium -positive specimens were tested for macrolide resistance using three assays: the in-house 23S rRNA FRET PCR assay, the SpeeDx ResistancePlus MG assay and the nested reverse transcription-PCR (RT-PCR) sequencing assay. The comparison of the MG-TMA assay with the in-house PCR results showed a moderate correlation (kappa value, 0.69). The MG-TMA assay had higher clinical sensitivity compared to that of the in-house PCR assay (100% versus 59.74%, respectively) and similar specificity (99.10% versus 100%, respectively) for M. genitalium detection. In this study, the prevalence of M. genitalium infection was 5.90% (72/1,220 patients). The nested RT-PCR sequencing assay was the most sensitive but the most laborious assay for detecting macrolide-resistance-associated mutations. The prevalence of resistance was 8.33% (6/72). To our knowledge, this is the first clinical evaluation of the MG-TMA CE-IVD assay. The MG-TMA assay performed on the automated Panther system is a very sensitive and specific method for the detection of M. genitalium in clinical specimens., (Copyright © 2017 Le Roy et al.)

Remnant specimens from 601 women obtained in a cross-sectional study from rural South Africa were tested for Mycoplasma genitalium. Overall, 10.8% of women were infected with M. genitalium either in the vagina or in the rectum. Macrolide resistance, although of low prevalence, in M. genitalium is described for the first time in Sub-Saharan Africa.

Objectives: Mycoplasma genitalium is a sexually transmitted organism associated with non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. Resistance to macrolides has been recently associated with point mutations in the 23S rRNA gene. The aim of this study was to detect these mutations using a large French collection of M. genitalium-positive specimens. We evaluated whether these mutations were related to azithromycin treatment failure and whether macrolide-resistant M. genitalium may be spreading., Patients and Methods: A retrospective study conducted in France between 2003 and 2010 included 156 urogenital clinical specimens from 136 patients that were positive for M. genitalium. Mutations in domain V of M. genitalium 23S rRNA were detected using amplification and sequencing. The mutated strains were genotyped by studying single nucleotide polymorphisms in the mgpB gene., Results: We have detected macrolide resistance-associated mutations in M. genitalium since 2006 at a rate of 13.2%, ranging from 10% to 15.4% of patients per year. Nine mutations at position 2059 as well as two A2058G substitutions, one A2062T substitution and one C2038T substitution (Escherichia coli numbering) were identified in M. genitalium. These patients had treatment failure with azithromycin in 75% (6/8) of cases. For one patient, genotyping showed selection for the mutation during treatment with azithromycin., Conclusions: For the first time, we describe macrolide resistance for M. genitalium in France and demonstrate that its detection has increased since 2006. Epidemiological surveillance of M. genitalium is necessary to adapt treatments to M. genitalium infections.

A child with Job's syndrome was treated for pneumonia due to Mycoplasma pneumoniae. A mixed population of wild-type bacteria and an A2059G mutant was detected during josamycin treatment failure. The same multilocus variable-number tandem-repeat analysis (MLVA) type (MLVA type I) was isolated before and after treatment failure. The child recovered after ciprofloxacin treatment.

Mycoplasma pneumoniae is a pathogenic mycoplasma responsible for respiratory tract infections in humans, which occurs worldwide in children and adults. This article focuses on its antibiotic susceptibility profile and on the development of acquired resistance in this microorganism. The lack of a cell wall in mycoplasmas makes them intrinsically resistant to β-lactams and to all antimicrobials that target the cell wall. M. pneumoniae is susceptible to macrolides and related antibiotics, tetracyclines and fluoroquinolones. Macrolides and related antibiotics are the first-line treatment for respiratory infections caused by M. pneumoniae. However, strains with acquired resistance to macrolides have recently emerged worldwide and have been spreading in Europe, USA and A sia especially, with more than 90% of Chinese isolates resistant to erythromycin and azithromycin. This acquired resistance can be detected by PCR methods directly from respiratory specimens and is related to 23S rRNA mutations.

Three isolates of Mycoplasma amphoriforme, a new Mycoplasma species rarely described to date, were obtained from respiratory tract specimens from two children and one adult with respiratory tract infections. Molecular methods were required to distinguish them from Mycoplasma pneumoniae. MICs of macrolides, tetracyclines and fluoroquinolones were identical to those for M. pneumoniae, except for that of ciprofloxacin, which was slightly more potent against M. amphoriforme. M. amphoriforme could possibly have been involved in one case of severe respiratory infection with sepsis, but further studies are needed to specify its role as a potential respiratory tract pathogen.

Objectives: Mycoplasma pneumoniae is a common aetiological agent of community-acquired respiratory tract infections for which macrolides are the treatment of choice. In France, only two macrolide-resistant isolates were reported in 1999. In contrast, several recent data reported that macrolide-resistant M. pneumoniae isolates have been spreading since 2000 in Japan. Mutations A2058G (Escherichia coli numbering), A2058C, A2059G, A2062G, C2611A and C2611G in domain V of the 23S rRNA gene were associated in vivo or in vitro with this resistance. The aim of this study was to determine whether macrolide resistance of M. pneumoniae is emerging in France., Patients and Methods: We developed a duplex real-time PCR for the detection of the six 23S rRNA mutations associated with macrolide resistance in M. pneumoniae and a simplex real-time PCR for the identification of the A2058G mutation, the most common one. Both methods rely on fluorescence resonance energy transfer coupled to melting curve analysis and are directly applicable to clinical samples. The duplex real-time PCR assay, first validated on 40 genetically characterized M. pneumoniae strains, was then applied directly on 248 French respiratory tract clinical samples., Results: Among M. pneumoniae-positive specimens collected before 2005, no macrolide-resistant M. pneumoniae isolate was detected. In contrast, among 51 samples collected between 2005 and 2007, five (9.8%) yielded a resistant genotype, suggesting a recent increase in macrolide-resistant M. pneumoniae isolates in France., Conclusions: The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

Mycoplasma pneumoniae isolates are divided in two types based on the sequence variations in the P1 adhesin gene. The type of P1 adhesin gene of 155 clinical isolates of M. pneumoniae collected in France between 1994 and 2006 was determined by a PCR-restriction fragment length polymorphism method. Until 1995, all strains belonged to type 1. In 1996 and 1997, type 1 was still predominant, but type 2 increased. Finally, since 1998, both types were present in about the same proportion. In our study, a novel sequence of the P1 adhesin gene was described in one strain. This strain could not be classified into type 1 or 2 because of variability in both P1 gene repeat elements, RepMP4 and RepMP2/3. This new sequence was certainly issued from recombination with repetitive sequences localized outside of the P1 gene in the M. pneumoniae chromosome. Moreover, MICs of erythromycin, tetracycline, and ciprofloxacin were determined for the 155 isolates. All isolates remained susceptible to tetracycline and ciprofloxacin, but two macrolide-resistant strains, isolated from two children in 1999, were identified. They harbored an A-to-G substitution at position 2058 or 2059 (Escherichia coli numbering) in domain V of 23S rRNA, associated with resistance to macrolides, lincosamides, and ketolides. To our knowledge, this is the first description of macrolide-resistant isolates of M. pneumoniae in France, but at this time, there is no sign of recent diffusion of resistant strains.

Resistant mutants of Ureaplasma parvum were selected by serial passages of a susceptible strain in subinhibitory concentrations of different macrolides and related antibiotics (erythromycin, azithromycin, josamycin, quinupristin, quinupristin/dalfopristin, pristinamycin and telithromycin). Mechanisms of resistance were characterised by sequencing portions of genes encoding 23S rRNA and ribosomal proteins L4 and L22. Mutants with significantly increased minimum inhibitory concentrations could be selected with all the selector antibiotics, except quinupristin and pristinamycin. Mutants harboured mutations in domain V of the 23S rRNA gene at nucleotides G2056, G2057 or A2058 (Escherichia coli numbering) and in conserved portions of ribosomal proteins L4 and L22. Most of the mutations were associated with complete loss of macrolide and ketolide activity, whereas streptogramin combinations were less affected.

Objectives: Mycoplasma hominis is intrinsically resistant to 14- and 15-membered macrolides and to the ketolide telithromycin but is susceptible to josamycin, a 16-membered macrolide, and lincosamides. The aim of our study was to investigate the in vitro development of macrolide resistance in M. hominis and to study the impact of ribosomal mutations on MICs of various macrolides and related antibiotics., Methods: Selection of macrolide-resistant mutants was performed by serial passages of M. hominis PG21 in broth medium containing subinhibitory concentrations of clindamycin, pristinamycin, quinupristin/dalfopristin and telithromycin. Stepwise selection of josamycin-resistant mutants was performed onto agar medium containing increasing inhibitory concentrations of josamycin. Resistant mutants were characterized by PCR amplification and DNA sequencing of 23S rRNA, L4 and L22 ribosomal protein genes., Results: Various mutations in domain II or V of 23S rRNA were selected in the presence of each selector antibiotic and were associated with several resistance phenotypes. Josamycin was the sole antibiotic that selected for single amino acid changes in ribosomal proteins L4 and L22. Unexpectedly, the C2611U transition selected in the presence of clindamycin and the quinupristin/dalfopristin combination was associated with decreased MICs of erythromycin, azithromycin and telithromycin, leading to a loss of the intrinsic resistance of M. hominis to erythromycin and azithromycin., Conclusions: Ribosomal mutations were associated with resistance to macrolides and related antibiotics in M. hominis. Some mutants showed a loss of the intrinsic resistance to erythromycin and azithromycin.

Mycoplasma pneumoniae is a pathogenic mycoplasma responsible for respiratory tract infections in humans, occurring worldwide in children and adults. This review briefly focuses on its antibiotic susceptibility profile and on the development of acquired resistance for this microorganism. The lack of a cell wall in mycoplasmas makes them intrinsically resistant to beta-lactams and to all antimicrobials which target the cell wall. Intrinsic resistance related to specific mycoplasma species concerns essentially the acrolide-lincosamide-streptogramin-ketolide (MLSK) antibiotic group. M. pneumoniae is susceptible to all MLSK antibiotics, except to lincomycin. Among the three antibiotic classes used for the treatment of mycoplasmal infections including tetracyclines, MLSK group, and fluoroquinolones, macrolides and related antibiotics are the drug of choice for respiratory infections caused by M. pneumoniae. Both target alterations and efflux mechanisms implicated in acquired antibiotic resistance have been described in mycoplasmas either by genetic mutation or transfer of new genes carried by transposons. At present, M. pneumoniae remains greatly susceptible to antibiotics, but as this mycoplasma is difficult to isolate, the number of clinical strains tested is limited and the occurrence of acquired resistance not well documented. However some strains having acquired resistance to MLSK have been decribed in vivo and erythromycin-resistant isolates are spreading now in Japan. To date, no clinical isolates resistant to fluoroquinolones or tetracyclines have been described in the literature, but some strains having acquired resistance to both classes have been selected in vitro. Molecular diagnosis of this acquired resistance has been related to target alterations, in ribosome for macrolides and tetracyclines, or in topoisomerase II genes for fluoroquinolones.

Macrolide-resistant mutants of Mycoplasma pneumoniae were selected in vitro from the susceptible reference strain M129, by 23 to 50 serial passages in subinhibitory concentrations of macrolides and related antibiotics, erythromycin A, azithromycin, josamycin, clindamycin, quinupristin, quinupristin-dalfopristin, pristinamycin, and telithromycin. Mutants for which the MICs are increased could be selected with all antibiotics except the streptogramin B quinupristin. Portions of genes encoding 23S rRNA (domains II and V) and ribosomal proteins L4 and L22 of mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible strain M129. No mutation could be detected in domain II of 23S rRNA. Two point mutations in domain V of 23S rRNA, C2611A and A2062G, were selected in the presence of erythromycin A, azithromycin, josamycin, quinupristin-dalfopristin, and telithromycin. Mutants selected in the presence of clindamycin and telithromycin harbored a single amino acid change (H70R or H70L, respectively) in ribosomal protein L4, whereas insertions of one, two, or three adjacent glycines at position 60 (M. pneumoniae numbering) were selected in the presence of both streptogramin combinations. Telithromycin was the sole antibiotic that selected for substitutions (P112R and A114T) and deletions ((111)IPRA(114)) in ribosomal protein L22. Three sequential mutational events in 23S rRNA and in both ribosomal proteins were required to categorize the strain as resistant to the ketolide. Azithromycin and erythromycin A were the only selector antibiotics that remained active (MICs, 0.06 and 1 micro g/ml, respectively) on their mutants selected after 50 passages.

Objective: To evaluate the long-term effect of a program of rotating antibiotics on the incidence of ventilator-associated pneumonia and the susceptibilities of Gram-negative bacilli responsible for ventilator-associated pneumonia., Design: Prospective program for the surveillance of antibiotic susceptibilities of microorganisms responsible for ventilator-associated pneumonia., Setting: Academic, university-based, medical intensive care unit (16 beds)., Subjects: 2,856 mechanically ventilated patients., Interventions: A new program of antibiotic use was introduced at the end of 1996 that involved the rotation of antibiotics in empirical and therapeutic use of the treatment of ventilator-associated pneumonia. The rotation concerned the beta-lactam and aminoglycoside classes, with a rotation interval of 1 month. The use of antibiotics was monitored monthly. No preference was given to any particular antibiotic. In a previous study, the period before the introduction of this protocol (1995-1996) was compared with the period 2 yrs after (1997-1998): The results indicated a decreased incidence of ventilator-associated pneumonia, a lower incidence of potentially resistant Gram-negative bacilli, and increased sensitivities of Gram-negative bacilli, especially Pseudomonas aeruginosa and Burkholderia cepacia. After 1998, we decided to continue a routine for this rotation. The long-term effect of this program was studied by comparing the incidence of Gram-negative bacilli responsible for ventilator-associated pneumonia and their susceptibilities obtained in a third period: 1999-2001. The long-term effect (5 yrs) of such a strategy-2-yr protocol period (1997-1998) and 3-yr routine period (1999-2001)-could be evaluated., Measurements and Main Results: During the 7-yr study period, 2,856 patients were mechanically ventilated for >48 hrs. The incidence of ventilator-associated pneumonia remained significantly lower in period 3 (1999-2001): 23% (period 1, 1995-1996) vs. 15.7% (period 2, 1997-1998) vs. 16.3% (period 3, 1999-2001; p =.002). Late-onset ventilator-associated pneumonia occurred in 86.6% and 94% of cases, respectively, in periods 1 and 3 (p =.02). The decrease of the incidence of early-onset ventilator-associated pneumonia was statistically significant during the 7-yr study period: 13% vs. 9% vs. 5.9% (p =.02). Combined with a higher incidence of late-onset ventilator-associated pneumonia, the incidence of potentially resistant Gram-negative bacilli increased in period 3: 42.2% vs. 34.5% vs. 41.7% (nonsignificant), except for B. cepacia: 11.7% vs. 7.4% vs. 3.7% (p =.005). Nevertheless, the potential antibiotic-resistant Gram-negative bacilli were more sensitive to most of the beta-lactams, especially piperacillin-tazobactam and cefepime., Conclusions: Rotation of antibiotics could help to avoid ventilator-associated pneumonia. It could greatly improve the susceptibilities of the potentially antibiotic-resistant Gram-negative bacilli responsible for late-onset ventilator-associated pneumonia. This program could be applied in routine with good results 5 yrs after its introduction. Further studies, especially multiple-center trials, are necessary to confirm this result and better define the rotation type and intervals.

The mechanisms of intrinsic resistance of Mycoplasma hominis to 14- and 15-membered macrolides were investigated in comparison with those of M. pneumoniae, which is naturally susceptible to macrolides. Radiolabeled erythromycin was not accumulated by M. hominis PG21, but addition of an ABC transporter inhibitor increased the level of erythromycin uptake more than two times, suggesting the existence of an active efflux process. The affinity of [(14)C]erythromycin to ribosomes isolated from M. hominis was dramatically reduced relative to that to ribosomes isolated from M. pneumoniae. The nucleotide sequences of 23S rRNA of both ribosomal operons rrnA and rrnB and ribosomal proteins L4 and L22 of M. hominis were obtained. Compared to the sequence of M. pneumoniae, M. hominis harbored a G2057A transition in its 23S rRNA sequence, as did M. fermentans, another mycoplasma that is erythromycin resistant. An additional C2610U change was also found in the sequence of M. hominis. Moreover, two M. hominis clinical isolates with acquired resistance to 16-membered macrolides were examined for mutations in domain II and domain V of 23S rRNA and in ribosomal proteins L4 and L22. Compared to the sequence of reference strain PG21, one isolate harbored a A2059G transition and a C2611U transition in one of the two rrn operons, while the other one was mutated only at position 2059, also on the same operon. No mutation was found in the two ribosomal protein sequences. Overall, the present study is an exhaustive characterization of the intrinsic resistance of M. hominis to 14- and 15-membered macrolides and the first description of mycoplasma clinical isolates resistant to macrolide, lincosamide, and streptogramin antibiotics harboring a mutation at position 2611 in the 23S rRNA.