The increased incidence of Mycoplasma pneumoniae in France in 2011 was polyclonal, mainly involving M. pneumoniae type 1 strains.

An increased incidence of Mycoplasma pneumoniae infections was reported in 2011 in two cities in France, Bordeaux and Caen. Two complementary molecular typing methods, PCR-RFLP on adhesin P1 and multilocus variable number tandem repeat analysis (MLVA), were used to determine whether this phenomenon was clonal. In 2011, the percentage of M. pneumoniae-positive patients doubled in both cities compared with 2010. Macrolide resistance remained stable at 8.3% of patients. Eighteen MLVA types were identified among 94 M. pneumoniae-positive specimens, demonstrating that the phenomenon was multiclonal. Types P, J, U, X and E were the most frequent and 81.6% of the strains were adhesin P1 type 1.

Method comparison for molecular typing of French and Tunisian Mycoplasma genitalium-positive specimens.

In this study, 76 French and Tunisian urogenital specimens were subjected to molecular typing by using the two main Mycoplasma genitalium molecular typing methods, the mgpB single nucleotide polymorphism (SNP) typing method and the combination analysis of a variable-number tandem-repeat (VNTR) marker in MG309 and mgpB SNP. Furthermore, we tried to develop a multiple-locus VNTR analysis (MLVA) method. The genome of M. genitalium G37(T) was analysed for VNTRs and four VNTRs were used for an MLVA. The method, applied directly on clinical specimens, was based on a genescan analysis of VNTR loci labelled with fluorescent dyes by using multiplex PCR and capillary electrophoresis. This method had a 1.00 diversity index (DI) while the mgpB SNP typing and the combination of MG309 and mgpB SNPs had DIs of 0.853 and 0.989, respectively. However, among the sets of two concurrent specimens, taken at the same time from the urogenital tracts of 12 patients, only nine had matching MLVA profiles, while the two other methods gave identical profiles for all specimens amplified, except for one set. Moreover, eight new sequence types were described with the mgpB SNP typing method. The three molecular typing methods revealed a genetic heterogeneity, suggesting that M. genitalium was endemic in France and Tunisia and that the infections were not due to the clonal dissemination of one strain. Comparison of the typing results obtained with the three methods showed that the MLVA assay seemed too discriminatory to be used in future studies of sexual networks of M. genitalium infection. According to the discriminatory power and the feasibility of each mgpB-based method, we recommend that the mgpB analysis be used for general epidemiological studies and that the combination of MG309-STR and mgpB SNP methods should be used for sexual-network studies of M. genitalium infection.

Detection and susceptibility testing of Mycoplasma amphoriforme isolates from patients with respiratory tract infections.

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.

Development of multiple-locus variable-number tandem-repeat analysis for molecular typing of Mycoplasma pneumoniae.

In this study we report on the development of a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The genomic content of M. pneumoniae M129 was analyzed for VNTRs, and 5 of the 17 VNTRs identified were selected for use in an MLVA assay. The method was based on a GeneScan analysis of VNTR loci labeled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This approach was applied to a collection of 265 isolates from various European countries, Japan, and Tunisia; and 26 distinct VNTR types were found. The VNTR assay was compared to the P1 adhesin PCR-restriction fragment length polymorphism (RFLP) typing method and showed a far better resolution than the P1 PCR-RFLP method. The discriminatory power of MLVA (Hunter-Gaston diversity index [HGDI], 0.915) for the 265 isolates was significantly higher than that of the P1 PCR-RFLP method (HGDI, 0.511). However, there was a correlation between the typing results obtained by MLVA and the P1 gene PCR-RFLP method. The potential value of MLVA of M. pneumoniae as an epidemiological tool is discussed, and the use of the VNTR markers in further investigations of the potential use of MLVA in outbreaks of M. pneumoniae infections is proposed.

Tetracycline resistance in Ureaplasma spp. and Mycoplasma hominis: prevalence in Bordeaux, France, from 1999 to 2002 and description of two tet(M)-positive isolates of M. hominis susceptible to tetracyclines.

Twenty-four of 128 clinical isolates of Mycoplasma hominis and 6 of 276 clinical isolates of Ureaplasma spp. from Bordeaux, France (1999 to 2002), were resistant to tetracycline and harbored the tet(M) gene. For M. hominis, we also found an increase in tetracycline resistance and two tet(M)-positive isolates that were susceptible to tetracyclines.

First report of macrolide-resistant strains and description of a novel nucleotide sequence variation in the P1 adhesin gene in Mycoplasma pneumoniae clinical strains isolated in France over 12 years.

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.

Moisturizing lotions can increase transdermal absorption of the herbicide 2,4-dichlorophenoxacetic acid across hairless mouse skin.

Moisturizing lotions can be an effective treatment for occupationally induced dry skin. These compounds are designed to be hygroscopic and retain water to keep the stratum corneum hydrated, while at the same time enhancing the horny layer to prevent increases in transepidermal water loss (TEWL). Skin hydration levels, however, are known to influence barrier properties. The purpose of this work was to compare skin moisture levels induced by four commercially available moisturizing lotions with their capacity as transdermal penetration enhancers using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as a model chemical. Further, the effect of moisturizing the skin after washing with sodium lauryl sulfate (SLS) on transdermal absorption was determined. Skin moisture levels were also measured noninvasively and were correlated to penetration enhancement. Hairless mouse skin was pretreated with commercially available moisturizing lotions either with or without SLS washing and in vitro permeability studies were performed with the herbicide 2,4-D. The data demonstrate that pretreatment with three of the four lotions tested increased the transdermal absorption of 2,4-D as evidenced by cumulative penetration or faster lag times (p < 0.05). Skin moisture levels correlated with the penetration enhancement capabilities of the lotion. Washing the skin with 5% SDS increased the transdermal absorption of 2,4-D (p < 0.05) and application of moisturizing lotions increased the absorption further. In summary moisturizing lotions may influence transdermal penetration of the skin, with the more effective moisturizers having a greater effect on 2,4-D absorption.

Differential contribution of kainate receptors to excitatory postsynaptic currents in superficial layer neurons of the rat medial entorhinal cortex.

Although in situ hybridization studies have revealed the presence of kainate receptor (KAR) mRNA in neurons of the rat medial entorhinal cortex (mEC), the functional presence and roles of these receptors are only beginning to be examined. To address this deficiency, whole cell voltage clamp recordings of locally evoked excitatory postsynaptic currents (EPSCs) were made from mEC layer II and III neurons in combined entorhinal cortex-hippocampal brain slices. Three types of neurons were identified by their electroresponsive membrane properties, locations, and morphologies: stellate-like "Sag" neurons in layer II (S), pyramidal-like "No Sag" neurons in layer III (NS), and "Intermediate Sag" neurons with varied morphologies and locations (IS). Non-NMDA EPSCs in these neurons were composed of two components, and the slow decay component in NS neurons had larger amplitudes and contributed more to the combined EPSC than did those observed in S and IS neurons. This slow component was mediated by KARs and was characterized by its resistance to either 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466, 100 microM) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[lsqb]f[rsqb]quinoxaline-7-sulfonamide (NBQX, 1 microM), relatively slow decay kinetics, and sensitivity to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM). KAR-mediated EPSCs in pyramidal-like NS neurons contributed significantly more to the combined non-NMDA EPSC than did those from S and IS neurons. Layer III neurons of the mEC are selectively susceptible to degeneration in human temporal lobe epilepsy (TLE) and animal models of TLE such as kainate-induced status epilepticus. Characterizing differences in the complement of postsynaptic receptors expressed in injury prone versus injury resistant mEC neurons represents an important step toward understanding the vulnerability of layer III neurons seen in TLE.

Characterisation of in vitro-selected mutants of Ureaplasma parvum resistant to macrolides and related antibiotics.

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.

Transdermal absorption of the herbicide 2,4-dichlorophenoxyacetic acid is enhanced by both ethanol consumption and sunscreen application.

Xenobiotics absorption is a health concern and skin is a major exposure site for many of these chemicals. Both alcohol consumption and topical sunscreen application act as transdermal penetration enhancers for model xenobiotics. The effect of combining these two treatments on transdermal absorption of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was therefore examined. Skin from rats ingesting low (1.5 g/kg) medium (4.3 g/kg) or high (6 g/kg) ethanol doses or saline control was treated with a commercially available sunscreen containing titanium dioxide and octyl methoxycinnimate and transdermal absorption of 2,4-D was monitored. Ethanol increased penetration by a factor of 1.9, 2.0 and 2.5 for animals treated with 1.5, 4.3 and 6 g/kg respectively, demonstrating an ethanol-induced dose response. Sunscreen application to skin from ethanol gavaged rats caused 2,4-D absorption above that induced by ethanol alone by an additional factor of 1.3, 2.1 and 2.9 for 1.5, 4.3 and 6 g/kg respectively. Comparing 2,4-D transdermal absorption after exposure to both ethanol and sunscreen with a theoretical value (sum of penetration after ethanol or sunscreen treatment) demonstrates that these two treatments enhance additively at the higher doses tested. Results of this study emphasize the importance of limiting excessive alcohol consumption in individuals with potential herbicide exposure rather than discouraging the use of sunscreens, since the consequences of UV-induced skin cancer are far more series than the risks that would be associated with observed increases in chemical exposure.

Emergence of a 23S rRNA mutation in Mycoplasma hominis associated with a loss of the intrinsic resistance to erythromycin and azithromycin.

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.

In vitro development of resistance to six and four fluoroquinolones in Mycoplasma pneumoniae and Mycoplasma hominis, respectively.

Selection of resistant mutants in sequential subcultures with increasing concentrations of six and four different fluoroquinolones was studied for one reference strain each of Mycoplasma pneumoniae and Mycoplasma hominis, respectively. All fluoroquinolones tested selected for resistance, with alterations affecting the quinolone resistance-determining regions of the four target topoisomerase genes.

Increased expression of two multidrug transporter-like genes is associated with ethidium bromide and ciprofloxacin resistance in Mycoplasma hominis.

Two genes, md1 and md2, coding for multidrug resistance ATP-binding cassette transporters were identified in Mycoplasma hominis PG21. Expression of these two genes, quantified by quantitative competitive reverse transcription-PCR, was significantly increased in ethidium bromide-resistant strains of M. hominis compared to that in M. hominis PG21.

In vitro selection and characterization of resistance to macrolides and related antibiotics in Mycoplasma pneumoniae.

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.

Effects of chronic alcohol consumption on dermal penetration of pesticides in rats.

Topically applied ethanol is a well-known dermal penetration enhancer. The purpose of this work was to determine if ethanol consumption might also increase transdermal penetration. Male rats were fed either an ethanol containing or control diet for 6-8 wk. After the feeding regime was completed, skin was removed and placed in an in vitro diffusion system. The transdermal absorption of four very commonly used herbicides was determined. Penetration through skin from ethanol-fed rats was enhanced when compared to control by a factor of 5.3 for paraquat, 2.4 for atrazine, and 2.2 for 2,4-dichlorophenoxyacetic acid (2,4-D), and reduced by a factor 0.6 for trifluralin. Comparison of physical factors of the herbicides to the penetration enhancement revealed an inverse linear correlation with lipophilicity, as defined by log octanol/water partition coefficient (log Kow) with r2 =.98. These changes were at least partially reversible after 1 wk of abstinence from ethanol. These experiments demonstrate that regular ethanol consumption can alter the properties of the dermal barrier, leading to increased absorption of some chemicals through rat skin. If ethanol consumption has the same effect on human skin it could potentially have adverse health effects on people regularly exposed to agricultural, environmental, and industrial chemicals.

DNA gyrase and topoisomerase IV mutations in clinical isolates of Ureaplasma spp. and Mycoplasma hominis resistant to fluoroquinolones.

Twelve clinical isolates of Ureaplasma spp. and one isolate of Mycoplasma hominis were examined for resistance to fluoroquinolones. Previously described mutations at positions 83 and 95 in GyrA (Escherichia coli numbering) and positions 80 and 87 in ParC were found. Unusual alterations were described at positions ParC 123 and 134.

Mutations in 23S rRNA account for intrinsic resistance to macrolides in Mycoplasma hominis and Mycoplasma fermentans and for acquired resistance to macrolides in M. hominis.

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.

Evidence of active efflux in resistance to ciprofloxacin and to ethidium bromide by Mycoplasma hominis.

The uptake of fluoroquinolones was characterized for the fluoroquinolone-susceptible strain PG21 of Mycoplasma hominis. Accumulation of fluoroquinolones appeared to occur by passive diffusion. Addition of arginine as the energizer significantly reduced the uptake of fluoroquinolones, suggesting the presence of an energy-dependent efflux process. Reserpine and orthovanadate, two multidrug pump inhibitors, increased significantly the ciprofloxacin (CIP) uptake. In contrast, such a strong effect was not observed for moxifloxacin and pefloxacin uptakes. Two ethidium bromide (EtBr)-resistant strains, selected in vitro, showed a resistance profile compatible with a multidrug-resistant phenotype, with increased MICs for the hydrophilic fluoroquinolones, CIP and norfloxacin, EtBr, and acriflavine. Taking the EtBr-resistant strain RB1La as a model, a significant decrease of the CIP and EtBr uptakes was observed compared to the reference strain PG21. In the presence of reserpine and orthovanadate, both inhibitors of ATP-dependent efflux pumps, the CIP uptake increased significantly, reaching approximately the same level as that of the susceptible strain. Similar results were obtained with EtBr uptake and efflux experiments. Our data suggest the presence of an active efflux system, possibly an ABC-type efflux pump, implicated in the resistance to CIP and unrelated compounds like EtBr in the human mycoplasma M. hominis.

[Local cutaneous side effects of interferons]. / Effets secondaires cutanés locaux des interférons.

UNDERESTIMATED SIDE EFFECTS: Skin reactions to interferon (INF) treatment are uncommon in the larger series in the literature and are usually considered to be minor. They account for 5 to 12% of adverse effects to IFN and are encountered increasingly in patients with active chronic hepatitis C. Reactions may be local, occurring exclusively at the site of injection, or general. REPORTED EFFECTS AND TREATMENTS: We reviewed the literature on local skin reactions at the site of injection of the different interferons to study the underlying pathophysiological mechanisms and management schemes used. TWO TYPES OF REACTIONS: Local skin reactions can be divided into two types depending on the potential gravity and management. Minor reactions (transient erythema, eczema, depilation) have few clinical or therapeutic implications. More serious reactions, necrosis, vasculitis or injection can be potentially severe and require definitive interruption of treatment. PREVENTION: Preventive measures include careful education concerning self-injections using proper asepsia, variation of injection sites, and self-assessment of persistent skin reactions.