Multilocus variable-number tandem-repeat analysis-confirmed emergence of a macrolide resistance-associated mutation in Mycoplasma pneumoniae during macrolide therapy for interstitial pneumonia in an immunocompromised child.

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.

Detection of macrolide resistance in Mycoplasma genitalium in France.

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.

The spread of Mycoplasma pneumoniae is polyclonal in both an endemic setting in France and in an epidemic setting in Israel.

Mycoplasma pneumoniae infections occur both endemically and epidemically, and macrolide resistance has been spreading for 10 years worldwide. A substantial increased incidence of M. pneumoniae infections has been reported in several countries since 2010. Whether this increased incidence is attributed to different or to the same M. pneumoniae genotype is unknown. We have developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for the molecular typing of M. pneumoniae isolates. In this study, the MLVA typing method was modified and validated to be applicable directly to respiratory tract specimens without culture. This method was applied to 34 M. pneumoniae-positive specimens received at the Bordeaux Hospital, France, between 2007 and 2010 in an endemic setting, and to 63 M. pneumoniae-positive specimens collected during an epidemic surge of M. pneumoniae infections in 2010 in Jerusalem, Israel. The M. pneumoniae endemic spread was shown to be polyclonal in France, with 15 MLVA types identified. Strikingly, the Israeli epidemic surge was also a multi-clonal phenomenon, with 18 circulating MLVA types. The macrolide resistance-associated substitution, A2058G, was found in 22% of the Israeli patients. Macrolide-resistant M. pneumoniae belonged to four MLVA types, the MLVA type Z being the most frequent one. An association between the MLVA type Z and macrolide resistance might exist since macrolide resistance was present or generated during the course of illness in all patients infected with this MLVA type. In conclusion, the discriminatory power of the MLVA showed that the spread of M. pneumoniae strains in France in an endemic setting was polyclonal as well as the surge of M. pneumoniae infections in Israel in 2010.

Potential role of Mycoplasma hominis in interleukin (IL)-17-producing CD4+ T-cell generation via induction of IL-23 secretion by human dendritic cells.

BACKGROUND: Mycoplasma hominis, a human urogenital pathogen, is involved in genital and extragenital infections and arthritis, particularly in immunocompromised patients. The interleukin (IL) 23/T helper (Th) 17 axis is associated with inflammatory and autoimmune diseases. The aim of this study was to assess the IL-23 response to M. hominis in human dendritic cells (DCs) and the CD4(+) T-cell differentiation in response to M. hominis-infected DCs. METHODS: Human monocyte-derived DCs were cultured with phosphate-buffered saline, lipopolysaccharide, or M. hominis PG21. Cocultures with heterologous T cells were performed. Extracts from M. hominis were separated and incubated with DCs. Isolates from different clinical syndromes were tested. RESULTS: M. hominis induced the maturation of human DCs with predominant IL-23 secretion in a Toll-like receptor 2-dependent manner. The in vitro immunomodulatory capacity of M. hominis was contained in a lipoprotein-enriched fraction from the mycoplasma. M. hominis-activated DCs induced IL-17-producing CD4(+) T cells. Interestingly, clinical isolates differed in their ability to promote IL-23 secretion by DCs. CONCLUSIONS: Taken together, our findings demonstrate a major role for the IL-23/Th17 axis in the defense against M. hominis and indicate a potential role for these bacteria in inflammatory and autoimmune diseases.

Variable-number tandem-repeat markers for typing Mycobacterium intracellulare strains isolated in humans.

BACKGROUND: Mycobacterium intracellulare, a species of the Mycobacterium avium complex, may be the cause of severe lung, lymphatic node, skin and bone/joint infections, as well as bacteriemia. The goal of this work was to identify Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) markers and to study their variability in a collection of isolates of M. intracellulare collected in humans. We studied 61 isolates collected in humans between 2001 and 2008, as well as the reference strain, M. intracellulare ATCC 13950. RESULTS: We identified 45 MIRU-VNTR candidates, of which 17 corresponded to the MIRU-VNTR identified in the genome of M. intracellulare ATCC 13950. Among the 45 potential MIRU-VNTR, seven were selected for use in a MIRU-VNTR assay applied to our collection of isolates. Forty-four patterns were found by MIRU-VNTR typing and the discriminatory power of the assay was high with a Hunter-Gaston diversity index of 0.98. We do not have evidence of a particular distribution of MIRU-VNTR polymorphism according to clinical situation. CONCLUSIONS: Our results suggest that MIRU-VNTR typing could be used for molecular epidemiological studies applied to M. intracellulare.

Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas.

Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs)). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden-Meyerhoff-Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine) and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes.