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1.
J Infect Dis ; 2023 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-38041851

RESUMO

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

2.
Front Microbiol ; 13: 705479, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35464992

RESUMO

Biofilm on the skin surface of chronic wounds is an important factor in the pathology, inhibiting wound healing. The polymicrobial nature of these infected wounds and bacterial interactions inside this pathogenic biofilm are the keys for understanding chronic infection. The aim of our work was to develop an innovative in vitro medium that closely mimics the chronic wound emphasizing the microbiological, cellular, and inflammatory environment of chronic wounds but also focusing on the pH found at the wound level. This new medium, called chronic wound medium (CWM), will thus facilitate the study of pathogenic biofilm organization. Clinical Staphylococcus aureus and Pseudomonas aeruginosa strains coisolated from diabetic foot infection were collected and cultivated in this new medium for 24 h in monoculture and coculture. Bacterial growth (growth curves), presence of small colony variant (SCV), biofilm formation (BioFilm Ring Test® assay, biofilm biomass quantification), and virulence (survival curve in a Caenorhabditis elegans model) were evaluated. After 24 h in the in vitro conditions, we observed that P. aeruginosa growth was not affected, compared with a control bacterial medium, whereas for S. aureus, the stationary phase was reduced by two logs. Interestingly, S. aureus growth increased when cocultured with P. aeruginosa in CWM. In coculture with P. aeruginosa, SCV forms of S. aureus were detected. Biofilm studies showed that bacteria, alone and in combination, formed biofilm faster (as soon as 3 h) than the bacteria exposed in a control medium (as soon as 5 h). The virulence of all strains decreased in the nematode model when cultivated in our new in vitro medium. Taken together, our data confirmed the impact of the chronic wound environment on biofilm formation and bacteria virulence. They indicated that P. aeruginosa and S. aureus cooperated in coinfected wounds. Therefore, this in vitro model provides a new tool for bacterial cooperation investigation and polymicrobial biofilm formation.

3.
Drug Discov Today ; 26(9): 2182-2189, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34119667

RESUMO

Antimicrobial susceptibility tests (AST) are based on the minimal inhibitory concentration (MIC), the method used worldwide to guide antimicrobial therapy. Despite its relevance in correctly predicting clinical outcome for most acute infections, this approach is misleading for multiple clinical cases in which pathogens do not grow rapidly, uniformly or with physical protection. This behaviour, named 'metabolic evasion' (ME), enables bacteria to survive antimicrobials. ME can result from different, and sometimes combined, bacterial mechanisms such as biofilms, intracellular growth, persisters or dormancy. We discuss how ME can influence the MIC-based probability of target attainment. We identify clinical cases in which this approach is undermined by ME and propose a new approach that takes ME into account in order to improve patient management and the evaluation of innovative drugs.


Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Bactérias/metabolismo , Árvores de Decisões , Humanos , Resultado do Tratamento
4.
Pediatr Res ; 90(6): 1215-1220, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33627818

RESUMO

BACKGROUND: In neonatal intensive care units (NICUs), hygiene and disinfection measures are pivotal to protect neonates from nosocomial infections. This study aimed to evaluate the efficacy of the classical incubators disinfection procedure and to follow-up neonates housed in the incubators for the development of late-onset sepsis (LOS). METHODS: In a tertiary NICU, 20 incubators were monitored for bacterial contamination at three times: before disinfection, after disinfection, and 24 h after turning on and housing a new neonate. Clinical data of neonates housed in these incubators were retrieved from the medical records. RESULTS: All 20 incubators were contaminated at the 3 times of the study, mainly on mattresses and balances. Coagulase-negative Staphylococci, Enterococcus, and Bacillus-resisted disinfection while enterobacteria and Staphylococcus aureus were eradicated. After 24 h, the bacterial colonisation was similar to the one observed before disinfection. The bacteria isolated on incubators were also found on the caregivers' hands. During the study, two preterm neonates developed a LOS involving a bacterial species that has been previously isolated in their incubator. CONCLUSION: Pathogenic contaminants persist on incubators despite disinfection and represent a risk for subsequent infection in preterm neonates. Improvements are needed concerning both the disinfection process and incubator design. IMPACT: Procedures of disinfection that are usually recommended in NICUs do not allow for totally eradicating bacteria from incubators. Preterm neonates are housed in incubators colonised with potentially pathogenic bacteria. The control of nosocomial infections in NICUs requires further researches concerning mechanisms of bacterial persistence and ways to fight against environmental colonisation.


Assuntos
Desinfecção , Incubadoras para Lactentes/microbiologia , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Sepse/microbiologia
5.
Pathogens ; 9(12)2020 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-33352641

RESUMO

Biofilms are characterized by high tolerance to antimicrobials. However, conventional antibiograms are performed on planktonic microorganisms. Through the clinical Biofilm Ring Test® (cBRT), initially aimed to measure the adhesion propensity of bacteria, we discerned a variable distribution of biofilm-producer strains among P. aeruginosa samples isolated from expectorations of cystic fibrosis (CF) patients. Despite a majority of spontaneous adherent isolates, few strains remained planktonic after 5 h of incubation. Their analysis by an adapted protocol of the cBRT revealed an induction of the biofilm early formation by sub-inhibitory doses of ß-lactams. Microscopic observations of bacterial cultures stained with Syto 9/Propidium Iodide (PI) confirmed the ability of antimicrobials to increase either the bacterial biomass or the biovolume occupied by induced sessile cells. Finally, the cBRT and its derivatives enabled to highlight in a few hours the potential inducer property of antibiotics on bacterial adhesion. This phenomenon should be considered carefully in the context of CF since patients are constantly under fluctuating antimicrobial treatments. To conclude, assays derived from the Biofilm Ring Test® (BRT) device, not only define efficient doses preventing biofilm formation, but could be useful for the antimicrobial selection in CF, to avoid inducer molecules of the early biofilm initiation.

6.
Int J Mol Sci ; 21(17)2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872360

RESUMO

Staphylococcus argenteus is an emerging species responsible for infections comparable to those induced by Staphylococcus aureus. It has been involved in few chronic or persistent infections so far. In this study, we described a case of a persistent prosthetic-joint infection (PJI) affecting a young woman. We investigated in vitro the virulence traits of the incriminated S. argenteus strain (bone cell invasion, biofilm formation and induction of inflammation) and analyzed its genome, in comparison with two other strains of S. argenteus and two S. aureus isolates. It appeared that this S. argenteus PJI strain combined biofilm formation, osteoblast invasion and intracellular persistence abilities together with genes potentially involved in the escape of the host immune defenses, which might explain the chronicization of the infection.


Assuntos
Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus/patogenicidade , Fatores de Virulência/genética , Sequenciamento Completo do Genoma/métodos , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Feminino , Humanos , Evasão da Resposta Imune , Osteoblastos/citologia , Osteoblastos/microbiologia , Infecções Relacionadas à Prótese/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus/imunologia , Staphylococcus/isolamento & purificação , Adulto Jovem
7.
J Antimicrob Chemother ; 75(6): 1466-1473, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32125419

RESUMO

BACKGROUND: Targeting biofilm-embedded and intraosteoblastic Staphylococcus aureus, rifampicin gained a pivotal role in bone and joint infection (BJI) treatment. Two other rifamycins, rifabutin and rifapentine, may represent better-tolerated alternatives, but their activity against bacterial reservoirs associated with BJI chronicity has never been evaluated. OBJECTIVES: To evaluate the activities of rifampicin, rifabutin and rifapentine in osteoblast infection models. METHODS: Using three S. aureus isolates, rifamycins were compared regarding: (i) their intracellular activity in 'acute' (24 h) and 'chronic' (7 days) osteoblast infection models at 0.1× MIC, 1× MIC, 10× MIC and 100× MIC, while impacting infection-induced cytotoxicity (MTT assay), intracellular phenol-soluble modulin (PSM) secretion (RT-PCR), resistance selection and small colony variant (SCV) emergence; and (ii) their minimal biofilm eradication concentration (MBEC) and their MIC to prevent biofilm formation (bMIC). RESULTS: At 0.1× MIC, only rifabutin significantly reduced intracellular inoculum and PSM secretion. All rifamycins allowed a 50% reduction of intraosteoblastic inoculum at higher concentrations, with no difference between acute and chronic infection models, while reducing infection-induced cytotoxicity and PSM secretion. Dose-dependent emergence of intracellular SCVs was observed for all molecules. No intracellular emergence of resistance was detected. bMICs were equivalent for all molecules, but MBEC90s of rifapentine and rifabutin were 10- to 100-fold lower than those of rifampicin, respectively. CONCLUSIONS: All rifamycins are efficient in reducing the S. aureus intraosteoblastic reservoir while limiting infection-induced cytotoxicity, with a higher activity of rifabutin at low concentrations. All molecules prevent biofilm formation, but only rifapentine and rifabutin consistently reduce formed biofilm-embedded bacteria for all isolates. The activity of rifabutin at lower doses highlights its therapeutic potential.


Assuntos
Rifamicinas , Infecções Estafilocócicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Humanos , Testes de Sensibilidade Microbiana , Rifabutina/farmacologia , Rifamicinas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus
8.
Front Med (Lausanne) ; 7: 539501, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33585497

RESUMO

Introduction: Corynebacteria represent often-neglected etiological agents of post-traumatic and/or post-operative bone and joint infection (BJI). We describe here clinical characteristics and bacteriological determinants of this condition. Methods: A retrospective cohort study described characteristics, outcome and determinants of treatment failure of all patients with proven Corynebacterium spp. BJI (i.e., ≥2 culture-positive gold-standard samples). Available strains were further characterized regarding their antibiotic susceptibilies, abilities to form early (BioFilm Ring Test®) and mature (crystal violet staining method) biofilms and to invade osteoblasts (gentamicin protection assay). Results: The 51 included BJI were mostly chronic (88.2%), orthopedic device-related (74.5%) and polymicrobial (78.4%). After a follow-up of 60.7 weeks (IQR, 30.1-115.1), 20 (39.2%) treatment failures were observed, including 4 Corynebacterium-documented relapses, mostly associated with non-optimal surgical management (OR 7.291; p = 0.039). Internalization rate within MG63 human osteoblasts was higher for strains isolated from delayed (>3 months) BJI (p < 0.001). Infection of murine osteoblasts deleted for the ß1-integrin resulted in a drastic reduction in the internalization rate. No difference was observed regarding biofilm formation. Conclusions: Surgical management plays a crucial role in outcome of BJI involving corynebacteria, as often chronic and device-associated infections. Sanctuarisation within osteoblasts, implicating the ß1 cellular integrin, may represent a pivotal virulence factor associated with BJI chronicity.

9.
Rheumatology (Oxford) ; 59(1): 77-83, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31834404

RESUMO

OBJECTIVE: To identify the role of Staphylococcus aureus (SA) or its intracellular small-colony variant phenotype (SCV) with co-trimoxazole (CTX) or ANCA-associated vasculitis (AAV) activity. METHODS: All consecutive AAV patients with granulomatosis with polyangiitis (GPA), eosinophilic GPA or microscopic polyangiitis, followed at the French National Vasculitis Referral Center (09/2012-05/2013), and hospitalized non-AAV controls, exclusively for SA/SCV carriage comparisons, were enrolled. All had bilateral anterior nasal swab cultures. Nasal SA or SCV carriage was determined and associations with relapse(s), BVAS, ANCA-positivity, anti-staphylococcal and immunosuppressant use, were analysed ⩾4 years post-inclusion. RESULTS: Nasal SA carriage rates did not differ among AAVs (P = 0.53): GPA (24/80; 30%), EGPA (7/28; 25%) and microscopic polyangiitis (3/11; 27.3%); and the rate was less frequent in controls than in GPA patients not taking CTX (P = 0.04). AAV patients taking CTX prophylaxis had less nasal SA carriage (8.7% vs 36.2%; P = 0.02). Nasal SA carriage or CTX use did not modify relapse rates, BVAS or ANCA-positivity at inclusion or during follow-up. Nasal SCV carriage, found in 15/207 (7.2%) patients, was similar for GPA (10/24; 41.7%), microscopic polyangiitis (2/7; 28.6%) and eosinophilic GPA (2/3; 66.7%), but higher (P = 0.02) than controls (1/14; 7.1%). SCV carriage by AAV groups did not modify relapse rates or ANCA positivity at inclusion or during follow-up; a trend towards higher BVAS was observed only for anti-PR3 ANCA patients. CONCLUSION: Nasal SA or SCV carriage was comparable among AAVs but more frequent than in controls. Nasal SA or SCV carriage and CTX use did not modify AAV relapse rates.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/microbiologia , Antibioticoprofilaxia/métodos , Prevenção Secundária/métodos , Staphylococcus aureus/crescimento & desenvolvimento , Combinação Trimetoprima e Sulfametoxazol/administração & dosagem , Adulto , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Síndrome de Churg-Strauss/tratamento farmacológico , Síndrome de Churg-Strauss/microbiologia , Feminino , França , Granulomatose com Poliangiite/tratamento farmacológico , Granulomatose com Poliangiite/microbiologia , Humanos , Masculino , Poliangiite Microscópica/tratamento farmacológico , Poliangiite Microscópica/microbiologia , Pessoa de Meia-Idade , Cavidade Nasal/microbiologia , Fenótipo , Estudos Prospectivos , Recidiva , Infecções Estafilocócicas/prevenção & controle , Resultado do Tratamento
10.
Front Microbiol ; 10: 47, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30761099

RESUMO

Background: Methicillin resistance in Staphylococcus aureus (MRSA) is classically conferred by the acquisition of the mecA gene encoding an additional penicillin binding protein with low affinity for beta-lactams. A mecA variant, named mecC, was described in 2011. MRSA isolates harboring mecC of both animal and human origin have since been collected in different European countries. In France, animal cases were reported in 4 dairy farms between 2008 and 2013 in the Meurthe-et-Moselle county, all located in a 30 km perimeter, suggesting a possible dissemination of mecC-positive MRSA strains. We performed a prospective study to evaluate the local epidemiology of such strains in terms of (i) dissemination among animals, humans and in the environment, and (ii) persistence in Meurthe-et-Moselle dairy cattle farms. Methods: The 4 French dairy farms with previous reports of mecC-positive MRSA strains and 14 farms in the same perimeter were included in this study. In each farm, nasal swabs, rectal swabs and milk samples were collected from 10 randomly selected cows, as well as nasal samples from family pets, volunteer farmers and veterinarians. One farm (E0), in which mecC-MRSA isolates were detected, was selected to study more deeply the dissemination of mecC-positive strains within the farm. After pre-enrichment of swabs and milk, they were subcultured on MSSA/MRSA chromogenic selective agar plates. S. aureus colonies were tested with a multiplex PCR to detect the mecA and mecC genes. The mecC-positive strains were characterized using DNA microarray. Results: mecC-positive strains were recovered in four farms, corresponding to the ones with previous reports of mecC-positive MRSA strains, and originated only from dairy cow samples. The screening in the E0 farm showed that 22% of the dairy cows carried mecC-positive MRSA. Three strains were also isolated from the environmental samples. All mecC-positive strains belonged to the clonal complex CC130 and harbored the same spa-type t1736. Conclusion: This study found that mecC-positive MRSA isolates are able to persist within the same farms for several years after being introduced in this setting and are able to widely disseminate but only among dairy cows suggesting that milking machines might be a key player.

11.
J Antimicrob Chemother ; 74(3): 625-632, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30517641

RESUMO

OBJECTIVES: Prolonged use of linezolid for bone and joint infection (BJI) is limited by its long-term toxicity. The better safety profile of tedizolid, a recently developed oxazolidinone, could offer an alternative. However, its efficacy against biofilm-embedded and intracellular Staphylococcus aureus, the two main bacterial reservoirs associated with BJI chronicity, is unknown. METHODS: Using three S. aureus strains (6850 and two clinical BJI isolates), linezolid and tedizolid were compared regarding their ability: (i) to target the S. aureus intracellular reservoir in an in vitro model of osteoblast infection, using three concentrations increasing from the bone concentration reached with standard therapeutic doses (Cbone = 2.5 × MIC; Cplasm = 10 × MIC; Cmax = 40 × MIC); (ii) to eradicate mature biofilm [minimal biofilm eradication concentration (MBEC)]; and (iii) to prevent biofilm formation [biofilm MIC (bMIC) and confocal microscopy]. RESULTS: Linezolid and tedizolid weakly reduced the intracellular inoculum of S. aureus in a strain-dependent manner despite the similar MICs for the tested strains, but improved cell viability even in the absence of an intracellular bactericidal effect. Conversely, linezolid and tedizolid were ineffective in eradicating mature biofilm formed in vitro, with MBEC >2000 and >675 mg/L, respectively. bMICs of tedizolid were 4-fold lower than those of linezolid for all strains. CONCLUSIONS: Linezolid and tedizolid alone are not optimal candidates to target bacterial phenotypes associated with chronic forms of BJI. Despite weak intracellular activity, they both reduce infection-related cytotoxicity, suggesting a role in modulating intracellular expression of staphylococcal virulence factors. Although inactive against biofilm-embedded S. aureus, both-but particularly tedizolid-are able to prevent biofilm formation.


Assuntos
Antibacterianos/farmacologia , Células Precursoras de Granulócitos/microbiologia , Linezolida/farmacologia , Osteoartrite/microbiologia , Oxazolidinonas/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Tetrazóis/farmacologia , Biofilmes/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Modelos Teóricos
12.
PLoS One ; 13(8): e0200064, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30161132

RESUMO

Biofilm formation is a critical virulence factor responsible for treatment failure and chronicity in orthopaedic device-related infections (ODIs) caused by Staphylococcus aureus. Clonal lineages differ in terms of their biofilm forming capacities. The aim of this study was to investigate the correlation between the clonal complex (CC) affiliation and biofilm phenotype of 30 clinical S. aureus isolates responsible of ODI based on i) early biofilm formation using BioFilm Ring Test® and mature biofilm formation using crystal violet assays, ii) biofilm composition using DNase and proteinase K activity, and iii) prevention of biofilm formation by cloxacillin, teicoplanin and vancomycin using Antibiofilmogram® (biofilm minimal inhibitory concentration-bMIC). In terms of early biofilm formation, the CC30 strains were significantly slower than the CC5, CC15 and CC45 strains. CC45 strains produced significantly more mature biofilm than other group of strains did. The formation of biofilms was highly dependent on the presence of extracellular DNA in the CC5, CC15 and CC30 strains whereas it was mostly dependent on the presence of proteins in CC45. Finally, the CC30 group highlighted higher proportion of susceptible (bMIC < breakpoints of EUCAST guidelines) for cloxacillin, teicoplanin and vancomycin compared to the other CCs. These results demonstrate that the biofilm phenotype of clinical S. aureus isolates from ODIs is strongly related to their respective CC affiliation.


Assuntos
Artrite Infecciosa/microbiologia , Biofilmes , Doenças Ósseas Infecciosas/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Adulto , Artrite Infecciosa/terapia , Proteínas de Bactérias/genética , Doenças Ósseas Infecciosas/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Especificidade da Espécie , Infecções Estafilocócicas/terapia , Staphylococcus aureus/isolamento & purificação , Infecção da Ferida Cirúrgica/microbiologia , Infecção da Ferida Cirúrgica/terapia , Falha de Tratamento , Fatores de Virulência/genética
13.
Sci Rep ; 8(1): 13040, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158585

RESUMO

Biofilm has become a major topic of interest in medical, food, industrial, and environmental bacteriology. To be relevant, investigation of biofilm behavior requires effective and reliable techniques. We present herein a simple and robust method, adapted from the microplate technique, in which steam is used as a soft washing method to preserve biofilm integrity and to improve reproducibility of biofilm quantification. The kinetics of steam washing indicated that the method is adapted to remove both planktonic bacteria and excess crystal violet (CV) staining for S. aureus, S. epidermidis, S. carnosus, P. aeruginosa, and E. coli biofilm. Confocal laser scanning microscopy confirmed that steam washing preserved the integrity of the biofilm better than pipette-based washing. We also investigated the measurement of the turbidity of biofilm resuspended in phosphate-buffered saline (PBS) as an alternative to staining with CV. This approach allows the discrimination of biofilm producer strains from non-biofilm producer strains in a way similar to CV staining, and subsequently permits quantification of viable bacteria present in biofilm by culture enumeration from the same well. Biofilm quantification using steam washing and PBS turbidity reduced the technical time needed, and data were highly reproducible.


Assuntos
Bactérias/crescimento & desenvolvimento , Técnicas Bacteriológicas/métodos , Biofilmes/crescimento & desenvolvimento , Manejo de Espécimes/métodos , Vapor , Microscopia Confocal , Coloração e Rotulagem
14.
Diagn Microbiol Infect Dis ; 86(3): 262-264, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27608539

RESUMO

We evaluated the performance of an immunochromatographic assay (PBP2a Culture Colony Test - Alere™), detecting protein-binding penicillin 2a on staphylococci primary isolates in only 6minutes. The assay is highly sensitive for the direct detection of MRSA on various culture media whereas it requires cefoxitin induction for methicillin-resistant coagulase-negative staphylococci.


Assuntos
Cromatografia de Afinidade/métodos , Resistência a Meticilina , Proteínas de Ligação às Penicilinas/análise , Staphylococcus/química , Antibacterianos/metabolismo , Cefoxitina/metabolismo , Sensibilidade e Especificidade , Staphylococcus/efeitos dos fármacos , Fatores de Tempo , Ativação Transcricional
15.
Int J Antimicrob Agents ; 48(4): 459-62, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27575729

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is involved in community-acquired and nosocomial diseases. The means of MRSA transmission and dissemination in the community remain uncertain. Studies have shown that public transport systems could be a source of MRSA and may serve as a potential source for community-acquired MRSA infections. This study aimed to investigate MRSA contamination on Lyon's metropolitan network (Métro) in France. Hand-touched surfaces were sampled with sterile swabs (Transystem(®)) during a 1-day transversal study by collecting 50 samples in seven hub stations and two trains for each of the four Métro lines. Then, during a longitudinal study, one sample was collected twice daily for 30 consecutive days in the busiest and most congested hub station. All swabs were incubated in enrichment medium for 24 h and then each suspension was plated onto a chromogenic selective medium for MRSA. After 24 h at 36 °C, all presumptive MRSA colonies were tested using VITEK(®) MS to confirm identification as S. aureus as well as by Alere™ PBP2a Culture Colony Test and mecA/mecC PCR to check methicillin resistance. Of the 110 swabs tested, 24 presumptive MRSA colonies were isolated, of which 2 were confirmed as S. aureus by VITEK(®) MS. These two isolates were tested negative using the PBP2a Culture Colony Test and PCR. Unlike other foreign cities such as Lisbon, the current data suggest a low level of MRSA contamination of hand-touched surfaces on Lyon's Métro. This should be put in perspective with the low level of MRSA colonisation in the French community.


Assuntos
Microbiologia Ambiental , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Instalações de Transporte , Técnicas Bacteriológicas , França , Humanos , Estudos Longitudinais , Reação em Cadeia da Polimerase , Manejo de Espécimes
16.
Pathog Dis ; 74(6)2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27316688

RESUMO

Biofilms are complex communities of microorganisms embedded in an extracellular matrix and adherent to a surface. The development was described as a four-stage process leading to the formation of a mature biofilm which was resistant to immune system and antibiotic actions. In bone and joint infections (BJIs), the formation of biofilms is a leading cause of treatment failure. Here we study the capacity of 11 antibiotics commonly used in the treatment of BJIs to inhibit the biofilm formation on 29 clinical Staphylococcus aureus isolates by a new test called Antibiofilmogram(®) The minimal inhibitory concentration (MIC) and biofilm MIC (bMIC) were determined in vitro and showed similar values for clindamycin, fusidic acid, linezolid and rifampin. Reversely, daptomycin, fosfomycin, gentamicin and ofloxacin showed a bMIC distribution different from MIC with bMIC above breakpoint. Finally, cloxacillin, teicoplanin and vancomycin revealed an intermediate bMIC distribution with a strain-dependent pattern. A murine in vivo model of catheter-associated S. aureus infection was made and showed a significant reduction, but not total prevention, of catheter colonization with cloxacillin at bMIC, and no or limited reduction with cloxacillin at MIC. Antibiofilmogram(®) could be of great interest after surgical operations on contaminated prostheses and after bacteremia in order to prevent the colonization of the device.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções Relacionadas à Prótese/microbiologia , Animais , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/microbiologia , Doenças Ósseas Infecciosas/diagnóstico , Doenças Ósseas Infecciosas/microbiologia , Infecções Relacionadas a Cateter/microbiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos
17.
Diagn Microbiol Infect Dis ; 85(4): 419-21, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27216499

RESUMO

Using 30 clinical isolates of Staphylococcus aureus representative of the most prevalent clones circulating in France, the performance of the Alere™ PBP2a Culture Colony Test (CCT) and the Slidex(®) MRSA detection kit (SMD) were compared in 5 different labs. CCT demonstrated better performance and was easier to conduct in routine.


Assuntos
Testes de Aglutinação/métodos , Técnicas Bacteriológicas/métodos , Cromatografia de Afinidade/métodos , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificação , França , Humanos , Sensibilidade e Especificidade
18.
Cell Microbiol ; 18(10): 1405-14, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26918656

RESUMO

Staphylococcus aureus bone and joint infection (BJI) is associated with significant rates of chronicity and relapse. In this study, we investigated how S. aureus is able to adapt to the human environment by comparing isolates from single patients with persisting or relapsing BJIs that were recovered during the initial and recurrent BJI episodes. In vitro and in vivo assays and whole-genome sequencing analyses revealed that the recurrent isolates induced a reduced inflammatory response, formed more biofilms, persisted longer in the intracellular compartments of host bone cells, were less cytotoxic and induced less mortality in a mouse infection model compared with the initial isolates despite the lack of significant changes at the genomic level. These findings suggest that S. aureus BJI chronicization is associated with an in vivo bacterial phenotypical adaptation that leads to decreased virulence and host immune escape, which is linked to increased intraosteoblastic persistence and biofilm formation.


Assuntos
Artrite Infecciosa/microbiologia , Biofilmes , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Adaptação Fisiológica , Adulto , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Células Cultivadas , Doença Crônica , Progressão da Doença , Feminino , Proteínas Hemolisinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Masculino , Osteoblastos/imunologia , Osteoblastos/microbiologia
19.
J Med Microbiol ; 64(9): 1021-1026, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26297246

RESUMO

Treatment of orthopaedic infections remains challenging owing to the inability of antibiotics to eradicate biofilms and prevent their regrowth. The present study characterized the effects of 12 antibiotics on in vitro biofilm formed by a representative strain of meticillin-susceptible Staphylococcus aureus (MSSA) isolated from a bone infection. Determination of the minimum biofilm eradication concentrations indicated that in vitro eradication of 24 h-old biofilms required concentrations up to 51,200 times higher than MICs. The influence of the same panel of antibiotics was also investigated on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the suspensions (individual cells) and the biofilm biomass. Except for fusidic acid, the presence of antibiotics during the initial steps of biofilm formation resulted in significant decreases in the number of sessile viable bacteria at the highest concentrations tested. Ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin were the most effective drugs. Confocal microscopy analysis indicated that daptomycin was more efficient at bacteria lysis than gentamicin and vancomycin. However, viable individual cells were still detectable in the assays performed with ceftarolin, fosfomycin, ofloxacin, rifampicin and vancomycin at concentrations for which no sessile cells were detected. Although none of the molecules tested was effective at classical therapeutic concentrations against 24 h-old MSSA biofilms, all except fusidic acid were able to impair biofilm formation at concentrations near the breakpoints. However, presence of viable individual unattached cells could imply a significant risk of microbial dissemination and increased risk of infections.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Artropatias/microbiologia , Osteomielite/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Humanos , Artropatias/tratamento farmacológico , Testes de Sensibilidade Microbiana , Osteomielite/tratamento farmacológico , Staphylococcus aureus/fisiologia
20.
Antimicrob Agents Chemother ; 59(4): 2029-36, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25605365

RESUMO

Although Staphylococcus aureus persistence in osteoblasts, partly as small-colony variants (SCVs), can contribute to bone and joint infection (BJI) relapses, the intracellular activity of antimicrobials is not currently considered in the choice of treatment strategies for BJI. Here, antistaphylococcal antimicrobials were evaluated for their intraosteoblastic activity and their impact on the intracellular emergence of SCVs in an ex vivo osteoblast infection model. Osteoblastic MG63 cells were infected for 2 h with HG001 S. aureus. After killing the remaining extracellular bacteria with lysostaphin, infected cells were incubated for 24 h with antimicrobials at the intraosseous concentrations reached with standard therapeutic doses. Intracellular bacteria and SCVs were then quantified by plating cell lysates. A bactericidal effect was observed with fosfomycin, linezolid, tigecycline, oxacillin, rifampin, ofloxacin, and clindamycin, with reductions in the intracellular inocula of -2.5, -3.1, -3.9, -4.2, -4.9, -4.9, and -5.2 log10 CFU/100,000 cells, respectively (P < 10(-4)). Conversely, a bacteriostatic effect was observed with ceftaroline and teicoplanin, whereas vancomycin and daptomycin had no significant impact on intracellular bacterial growth. Ofloxacin, daptomycin, and vancomycin significantly limited intracellular SCV emergence. Overall, ofloxacin was the only molecule to combine an excellent intracellular activity while limiting the emergence of SCVs. These data provide a basis for refining the choice of antibiotics to prioritise in the management of BJI, justifying the combination of a fluoroquinolone for its intracellular activity with an anti-biofilm molecule, such as rifampin.


Assuntos
Antibacterianos/farmacologia , Osteoblastos/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Doenças Ósseas Infecciosas/tratamento farmacológico , Doenças Ósseas Infecciosas/microbiologia , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
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