Importance of antibiotic penetration in the antimicrobial resistance of biofilm formed by non-pigmented rapidly growing mycobacteria against amikacin, ciprofloxacin and clarithromycin.

OBJECTIVES: To study the resistance of biofilms developed by non-pigmented rapidly growing mycobacteria (NPRGM) against amikacin, ciprofloxacin and clarithromycin in an in vitro model using clinical strains of different species. DESIGN: Antimicrobial susceptibilities of different clinical strains of Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium mucogenicum and Mycobacterium mageritense using conventional techniques were measured. Biofilm resistance was measured by using the sandwich technique developed by Anderl et al. using a concentration of antibiotic of 50mg/L. Penetration of antibiotics through biofilm was measured using the same technique with minimal modifications. RESULTS: NPRGM biofilms showed drug resistance (percentages of viable bacteria >1% of those of controls) against antibiotics that are commonly used for the treatment of infections caused by these organisms, although there are intraspecies differences between strains. We have detected differences in antibiotic penetration through biofilms with an important permeability barrier for ciprofloxacin. However, other mechanisms must be probably more important to explain the antimicrobial resistance of NPRGM biofilm. CONCLUSIONS: Biofilms formed by NPRGM are resistant to amikacin, ciprofloxacin and clarithromycin. As no resistance differences between the tested antibiotics have been observed, it is likely that biofilm permeability of antibiotics is of low importance for antimicrobial resistance of biofilms.

Importance of antibiotic penetration in the antimicrobial resistance of biofilm formed by non-pigmented rapidly growing mycobacteria against amikacin, ciprofloxacin and clarithromycin

Objectives To study the resistance of biofilms developed by non-pigmented rapidly growing mycobacteria (NPRGM) against amikacin, ciprofloxacin and clarithromycin in an in vitro model using clinical strains of different species. Design Antimicrobial susceptibilities of different clinical strains of Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium mucogenicum and Mycobacterium mageritense using conventional techniques were measured. Biofilm resistance was measured by using the sandwich technique developed by Anderl et al. using a concentration of antibiotic of 50mg/L. Penetration of antibiotics through biofilm was measured using the same technique with minimal modifications. Results NPRGM biofilms showed drug resistance (percentages of viable bacteria >1% of those of controls) against antibiotics that are commonly used for the treatment of infections caused by these organisms, although there are intraspecies differences between strains. We have detected differences in antibiotic penetration through biofilms with an important permeability barrier for ciprofloxacin. However, other mechanisms must be probably more important to explain the antimicrobial resistance of NPRGM biofilm. Conclusions Biofilms formed by NPRGM are resistant to amikacin, ciprofloxacin and clarithromycin. As no resistance differences between the tested antibiotics have been observed, it is likely that biofilm permeability of antibiotics is of low importance for antimicrobial resistance of biofilms (AU)

Objetivos Estudiar la resistencia de biopelículas formadas por micobacterias no pigmentadas de crecimiento rápido (MNPCR) frente amicacina, ciprofloxacino y claritromicina en in modelo in vitro empleando aislamientos clínicos de diferentes especies. Material y MétodosSe estudiaron las sensibilidades de las diferentes cepas clínicas de Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium mucogenicum y Mycobacterium mageritense mediante técnicas convencionales. La resistencia de dichas bacterias en la biopelícula fue estudiada mediante la técnica de sándwich descrita por Anderl et al. utilizando una concentración de antibiótico de 50mg/L. La penetración de los antibióticos a través de la biopelícula fue estudiada mediante la misma técnica con mínimas modificaciones. Resultados Las biopelículas de MNPCR presentaron resistencia (porcentajes de bacterias viables > 1% de los recuentos obtenidos en los controles) frente a todos los antibióticos que son empleados habitualmente en las infecciones causadas por estos organismos, si bien se detectaron diferencias dentro de la misma especie entre las diferentes cepas. Hemos detectado diferencias en la penetración de antibióticos a través de la biopelícula, especialmente con una importante disminución de la permeabilidad frente a ciprofloxacino. Sin embargo, otros mecanismos son, probablemente, más importantes para explicar la resistencia antimicrobiana de las biopelículas de MNPCR. Conclusiones Las biopelículas formadas por MNPCR son resistentes frente a amicacina, ciprofloxacino y claritromicina. Como no se demostraron diferencias importantes entre los distintos antibióticos, es probable que la permeabilidad de la biopelícula frente a los antibióticos tenga poca importancia en la resistencia antimicrobiana de las biopelículas (AU)

High frequency of macrolide resistance mechanisms in clinical isolates of Corynebacterium species.

The genus Corynebacterium includes a high number of species that are usually isolated from human skin as saprophytes. However, these microorganisms have also been reported as infectious agents in a broad group of patients and have showed broad-spectrum resistance. We studied the susceptibility profiles against macrolides, clindamycin, and streptogramins of 254 clinical strains belonging to the species Corynebacterium urealyticum (120), Corynebacterium amycolatum (66), Corynebacterium jeikeium (17), Corynebacterium striatum (20), Corynebacterium coyleae (12), Corynebacterium aurimucosum (11), and Corynebacterium afermentans subsp. afermentans (8). The MLS(B) phenotype was detected in 186 strains and was associated with the presence of methylase enzymes codified by the erm(X) gene in 171 strains. The erm(B) gene was only detected in two C. urealyticum strains. Fourteen strains showed macrolide resistance, but they did not carry erm genes. mef genes were not detected despite eight C. amycolatum strains showed the M phenotype. Also, the presence of hydrolytic enzymes codified by ere(B) was evaluated, but all results were negative. Resistance to macrolide in Corynebacterium sp. is mainly due to the presence of erm(X) methylase, although other resistance mechanisms could be involved.

Influence of surface porosity and pH on bacterial adherence to hydroxyapatite and biphasic calcium phosphate bioceramics.

Hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramic materials are widely employed as bone substitutes due to their porous and osteoconductive structure. Their porosity and the lowering of surrounding pH as a result of surgical trauma may, however, predispose these materials to bacterial infections. For this reason, the influence of porosity and pH on the adherence of common Gram-positive bacteria to the surfaces of these materials requires investigation. Mercury intrusion porosimetry measurements revealed that the pore size distribution of both bioceramics had, on a logarithmic scale, a sinusoidal frequency distribution ranging from 50 to 300 nm, with a mean pore diameter of 200 nm. Moreover, total porosity was 20 % for HA and 50 % for BCP. Adherence of Staphylococcus aureus and Staphylococcus epidermidis was studied at a physiological pH of 7.4 and at a pH simulating bone infection of 6.8. Moreover, the effect of pH on the zeta potential of HA, BCP and of both staphylococci was evaluated. Results showed that when pH decreased from 7.4 to 6.8, the adherence of both staphylococci to HA and BCP surfaces decreased significantly, although at the same time the negative zeta-potential values of the ceramic surfaces and both bacteria diminished. At both pH values, the number of S. aureus adhered to the HA surface appeared to be lower than that for BCP. A decrease in pH to 6.8 reduced the adherence of both bacterial species (mean 57 %). This study provides evidence that HA and BCP ceramics do not have pores sufficiently large to allow the internalization of staphylococci. Their anti-adherent properties seemed to improve when pH value decreased, suggesting that HA and BCP bioceramics are not compromised upon orthopaedic use.

Biofilm development by potentially pathogenic non-pigmented rapidly growing mycobacteria.

BACKGROUND: A study to evaluate the biofilm-development ability in three different media (Middlebrook 7H9, sterile tap water and PBS-5% glucose) was performed with 19 collection strains from 15 different species on non-pigmented rapidly growing mycobacteria (NPRGM). A microtiter plate assay was developed to evaluate the percentage of covered surface of the microtiter plate wells in different days from day 1 to day 69. RESULTS: All strains were able to develop biofilm in all the tested media. Middlebrook 7H9 showed the fastest growth, followed by sterile tap water and PBS-5% glucose. A sigmoid growth curve was detected in all the strains both in Middlebrook 7H9 and in sterile tap water. A difference could be detected for Mycobacterium abscessus in tap water, where it showed faster growth than all the other strains. CONCLUSION: Biofilm development seems to be a property of all the species of NPRGM and it depends on the nutrients present in the medium. The microtiter plate assay described here is a useful tool to evaluate differences in biofilm development among the different species of rapidly growing mycobacteria.

Evaluation of quantitative analysis of cultures from sonicated retrieved orthopedic implants in diagnosis of orthopedic infection.

To improve the microbiological diagnosis of device-related osteoarticular infections, we have developed a protocol based on the sonication of device samples, followed by concentration and inoculation of the sonicate in a broad variety of media in a quantitative manner. Sixty-six samples from 31 patients were included in the study (17 of them with clinical diagnosis of infection). The sonication procedure had a sensitivity of 94.1%, which is better than that of conventional cultures (88.2%). One case of contamination and six cases of unexpected positive cultures were detected (specificity of 42.8%): two of these were considered to represent true infection, while the other four were considered to be nonsignificant (corrected specificity of 50%), although the clinical importance of these isolates is questionable. When we analyzed the number of CFU, no breakpoint between significant and nonsignificant isolates could be established. Based on our results, the procedure of sonication of retrieved implants is better than conventional cultures for the diagnosis of device-related infections. The significance of some isolates in patients without clinical infection remains uncertain. However, they may become pathogens and cannot be routinely considered to be contamination.