Effect of subinhibitory concentrations of tigecycline and ciprofloxacin on the expression of biofilm-associated genes and biofilm structure of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of foreign body-associated infections. This is related to the bacterium's ability to form biofilms on synthetic materials. Bacteria within a biofilm may be exposed to subinhibitory concentrations (sub-MICs) of antibiotics because of an agent's limited penetration into the biofilm core. Here, we investigated the effect of sub-MICs of tigecycline and ciprofloxacin on the expression of biofilm-associated genes, i.e. icaA, altE and sigB, and the biofilm structure of five clinical isolates of S. epidermidis. For most tested isolates, the expression of these genes increased after exposure to 0.25 MIC and 0.5 MIC tigecycline. A slight decrease in icaAmRNA levels was observed only in two isolates in the presence of 0.25 MIC tigecycline. The effect of ciprofloxacin exposure was isolate-dependent. At 0.5 MIC, ciprofloxacin induced an increase of sigB and icaAmRNA levels in three of the five tested isolates. At the same time, expression of the altE gene increased in all isolates (from 1.3-fold to 42-fold, depending on the strain). Confocal laser scanning microscopy analysis indicated that sub-MIC ciprofloxacin decreased biofilm formation, whereas tigecycline stimulated this process. Our data suggest that sub-MIC tigecycline may have bearing on the outcome of infections.

Coagulase-negative staphylococci: pathogenesis, occurrence of antibiotic resistance genes and in vitro effects of antimicrobial agents on biofilm-growing bacteria.

Coagulase-negative staphylococci (CoNS) are opportunistic pathogens that particularly cause infections in patients with implanted medical devices. The present research was performed to study the virulence potential of 53 clinical isolates of Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus cohnii and Staphylococcus caprae. All clinical strains were clonally unrelated. Isolates carried genes encoding resistance to ß-lactam (mecA) (15 %), aminoglycoside [aac(6')/aph(2″)(11 %), aph (3')-IIIa (15 %), ant(4')-Ia (19 %)] and macrolide, lincosamide and streptogramin B (MLSB) [erm(A) (4 %), erm(B) (13 %), erm(C) (41 %), msr(A) (11 %)] antibiotics. CoNS isolates (64 %) were able to form biofilms. Confocal laser scanning microscopy revealed that these biofilms formed a three-dimensional structure composed mainly of living cells. All biofilm-positive strains carried the ica operon. In vitro studies demonstrated that a combination treatment with tigecycline and rifampicin was more effective against biofilms than one with ciprofloxacin and rifampicin. The minimum biofilm eradication concentration values were 0.062-0.5 µg ml-1 for tigecycline/rifampicin and 0.250-2 µg ml-1 for ciprofloxacin/rifampicin. All CoNS strains adhered to the human epithelial cell line HeLa, and more than half of the isolates were able to invade the HeLa cells, although most invaded relatively poorly. The virulence of CoNS is also attributed to their cytotoxic effects on HeLa cells. Incubation of HeLa cells with culture supernatant of the CoNS isolates resulted in cell death. The results indicate that the pathogenicity of S. capitis, S. auricularis, S. lugdunensis, S. cohnii and S. caprae is multi-factorial, involving the ability of these bacteria to adhere to human epithelial cells, form biofilms and invade and destroy human cells.

Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water.

The presence and persistence of antibiotic resistant bacteria in the environment is thought to be a growing threat to public health. The route of the spread of multiresistant bacteria from human communities to aquatic environment may lead through wastewater treatment plants that release treated wastewater to a water reservoir. In this study we used multiplex PCR assay to determine the frequency of integron presence in Escherichia coli isolates cultured from wastewater treatment plant (WWTP) (integrons were detected in 11% of E. coli isolates), river water upstream (6%) and downstream (14%) the discharge of WWTP, and clinical specimens (56%). Antimicrobial resistance of the integron-positive isolates, determined by disk diffusion method, varied between E. coli of different origin. Isolates from the downstream river, compared to those cultured from upstream river, were more frequently resistant to kanamycin, cephalotin, co-trimoxazole, trimethoprim, and fluoroquinolones. Moreover, they displayed broader resistance ranges, expressed as the number of classes of antimicrobials to which they were resistant. The results may suggest that WWTP effluent contributes to increased frequency of integron-positive E. coli isolates in the river downstream the WWTP and to their elevated resistance level.

Phylogenetic groups, virulence genes and quinolone resistance of integron-bearing Escherichia coli strains isolated from a wastewater treatment plant.

We investigated phylogenetic affiliation, occurrence of virulence genes and quinolone resistance in 109 integron-containing strains of Escherichia coli isolated from a wastewater treatment plant. Selection for integron-bearing strains caused a shift toward phylogroup D, which was most numerous, followed by A, B1 and B2. Phylogroups D and B2, both of which are reported to include virulent extraintestinal pathotypes, made up 50.5% of all isolates and were present in every stage of wastewater treatment, including final effluent. Diarrheagenic pathotypes made up 21% of the strains. The average virulence factor genes score was low (1.40) and the range was from 0 to 5. Quinolone and fluoroquinolone resistance was observed in 56.0% and 50.4% of the strains, respectively; however, it was not associated with virulence factor score. Although the average virulence factor score was low, 17.4% of strains had three and more virulence genes. They were isolated mostly from raw sewage, but 30% of them were cultured from final effluent. Release of multiresistant integron-bearing E. coli strains with virulence traits into the environment may create potential threat and be of public health concern.