Effect of sub-inhibitory concentrations of antibiotics on biofilm formation and expression of virulence genes in penicillin-resistant, ampicillin-susceptible Enterococcus faecalis.

Biofilm formation is a key factor in the pathogenesis of enterococcal infections. Thus, the biofilm-forming ability and frequency of biofilm-related genes in penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) compared to penicillin- and ampicillin-susceptible E. faecalis (PSASEF) were assessed in the present study. In addition, the effect of sub-inhibitory concentrations (sub-MICs) of antibiotics on biofilm formation and expression of virulence genes was evaluated. Twenty PRASEF and 21 PSASEF clinical isolates were used to determine the effect of sub-MICs of antibiotics (ampicillin, penicillin, and gentamicin) on biofilm formation, and ten selected isolates were subjected to RT-qPCR to detect the transcript levels of virulence genes (efaA, asa1, esp, and ace). Antibiotic susceptibility was evaluated by the microdilution broth method. Biofilm formation assay was performed using the microtiter plate method. All PSASEF and PRASEF isolates produced biofilms in vitro. Most isolates had three or four virulence genes. Sub-MICs of ampicillin significantly decreased biofilm production and expression of ace and asa1 genes, although the transcript levels were significantly lower (-350% and -606.2%, respectively) among the PSASEF isolates only. Sub-MICs of gentamicin did not have any significant effect on biofilm formation, but slightly increased the transcript levels of efaA. In conclusion, this study showed that the biofilm-forming ability and frequency of the evaluated virulence genes were similar among the PRASEF and PSASEF isolates. Further, in vitro antibiotic sub-MICs were confirmed to interfere with the expression pattern of virulence genes and biofilm formation by E. faecalis. However, further studies are required to clarify the role of sublethal doses of antibiotics on enterococcal biofilms.

High Prevalence of the aac(6')-Ie-aph(2'')-Ia Gene in Hospital Isolates of Enterococcus faecalis Co-Resistant to Gentamicin and Penicillin.

Objectives: This study aimed to characterize the molecular mechanism of resistance to gentamicin among penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates by investigating the presence of the aac(6')-Ie-aph(2'')-Ia gene. The co-resistance to antimicrobials of other classes was also evaluated. Results: Among the 151 isolates evaluated, 70 were PRASEF and 81 were penicillin-susceptible and ampicillin-susceptible E. faecalis (PSASEF). No ß-lactamase producing isolate was detected. Eighty-three (55.0%) and 35 (23.2%) out of the 151 E. faecalis isolates showed high-level gentamicin resistance (HLGR) and high-level streptomycin resistance (HLSR) phenotypes. However, a significantly higher rate of PRASEF (88.6%) showed HLGR phenotype in comparison with PSASEF (23.5%) (p < 0.01). Conversely, a significantly lower rate of PRASEF (14.3%) showing HLSR was observed in comparison with PSASEF (30.9%) (p = 0.02). The prevalence of isolates displaying multidrug resistance (MDR) phenotype was significantly higher (p < 0.01) in the group of PRASEF (81.4%) than in PSASEF (18.6%). The majority of PSASEF (61.9%) and PRASEF (90.3%) isolates showing HLGR phenotype was harboring the aac(6')-Ie-aph(2'')-Ia gene, which encodes a bifunctional enzyme that inactivates all aminoglycosides except streptomycin. Conclusion: The aac(6')-Ie-aph(2'')-Ia gene was prevalent among the Brazilian PRASEF isolates that usually exhibit co-resistance to gentamicin and to multiple other drugs.

Carnosic acid acts synergistically with gentamicin in killing methicillin-resistant Staphylococcus aureus clinical isolates.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to different commonly used antibiotics, stressing the need for further strategies to treat this human pathogen with worldwide prevalence. The use of phytochemicals within the current pharmacology is a promising approach to enhance the antimicrobial activity of common antibiotics in the battle against these bacteria. PURPOSE: The purpose of this study was to determine the antimicrobial effectiveness of carnosic acid, the major constituent of Rosmarinus officinalis L. leaves, in combination with gentamicin against multi-drug resistant MRSA clinical isolates obtained from pediatric patients with bacteremia. MATERIALS AND METHODS: Anti-MRSA activity was studied using the broth microdilution assay and time-kill method. Combinations of subinhibitory concentrations of carnosic acid and gentamicin were examined using the checkerboard method. RESULTS: Carnosic acid exhibited a good antibacterial activity against all multidrug-resistant MRSA clinical isolates tested, which are resistant to four up to nine antibiotics. In addition, the combination of carnosic acid with gentamicin not only decreased the minimal inhibitory concentration (MIC) of both by 4- to 5-fold, but also improved the bactericidal potency of the common antibiotic by 32- to 40-fold against both gentamicin-susceptible and gentamicin-resistant MRSA clinical isolates. A clear bactericidal synergistic activity between carnosic acid and gentamicin in killing multidrug-resistant MRSA clinical isolates with a fractional bactericidal concentration index (FBCI) of 0.28-0.35 was demonstrated. CONCLUSIONS: Our findings show the potential use of carnosic acid in combination with gentamicin as a promising alternative for the control of healthcare-associated infections caused by multidrug-resistant MRSA.

Clinical, epidemiological, and microbiological characteristics of bacteremia caused by high-level gentamicin-resistant Enterococcus faecalis

Enterococcus spp bacteremia is associated with high mortality and the appearance of high-level gentamicin resistance (HLGR) created additional challenges for the treatment of these infections. We evaluated the epidemiological and clinical characteristics of patients with bacteremias caused by HLGR and non_HLGR Enterococcus faecalis isolates at a teaching hospital in the State of São Paulo, Brazil. Patients with bacteremia due to E. faecalis diagnosed between January 1999 and December 2003 were included in the study. We collected clinical, epidemiological, and microbiological data from medical records. Banked isolates were typed using pulsed-field gel electrophoresis. We identified 145 cases of E. faecalis bacteremia: 66 (45.5 percent) were caused by HLGR isolates and 79 (54.5 percent) by non_HLGR. In the univariate analysis, patients with HLGR infection were older, had higher rates of bladder catheterization, and more often had treatment with cephalosporin, quinolone, and/or carbapenem compared with patients with non_HLGR infection (P < 0.05). Multivariate analysis indicated that older age, hematological malignancy, and previous use of vancomycin were independently associated with HLGR (P < 0.05). Mortality rates were not significantly different among patients with HLGR (50 percent) and non_HLGR (43 percent) infections (P = 0.40). Of the 32 genotyped isolates, 16 were distributed into 6 main electrophoresis patterns and 16 others had distinct patterns. E. faecalis bacteremia is associated with high mortality and is frequently caused by HLGR isolates at this teaching hospital. The variability among genotyped isolates suggests that endogenous infections, rather than patient-to-patient transmission of E. faecalis, are more common at this institution.