Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Differential pulse polarographic determination of poly(8-hydroxyquinoline) in the presence and absence of an insulating poly(vinyl alcohol) matrix.

The electrochemical activity of poly(8-hydroxyquinoline) (PHQ) in acid and alkaline media has been investigated by use of differential pulse polarography (DPP). The reduction peak height (I(p)) of PHQ in universal buffer solutions is not useful as an analytical signal, because it is highly affected by hydrogen evolution in acid media and appears as a small peak located at more negative potential values in alkaline media. A new and highly sensitive reduction peak (E(p)=-0.45, pH 9.25) appears, however, after addition of trace amounts of PHQ to Cu(II), or vice versa. This reduction peak is a result of the reduction of Cu(II) chelates in the PHQ-Cu(II) complex and is highly promising for the trace determination of PHQ at nanomolar and submicromolar levels. The response current (I(p)/mu A) for the reduction peak of Cu(II) chelates in a PHQ-Cu(II) matrix results in sensitivity to the concentration of PHQ at least three orders of magnitude higher than that for the reduction peak of PHQ alone under the same conditions. The limit of detection is as low as 5.264 ppb (microg L(-1)). The effect of a variety of anions and cations and of an insulating poly(vinyl alcohol) (PVA) matrix has been investigated. Electroactive PHQ-Cu(II) at a level of 0.685% could induce a current of approximately 240 nA in an insulating PVA matrix, suggesting possible application for the preparation of a PHQ-Cu(II)-PVA electroactive composite.

Interaction between biofilms formed by Staphylococcus epidermidis and quinolones.

The interaction between pefloxacin, ciprofloxacin, norfloxacin, and ofloxacin and biofilms formed by Staphylococcus epidermidis (20 clinical isolates) was studied. In the presence of 1/2-MIC and 1/8-MIC of quinolones, the optical density of the biofilms was reduced to 22-24% and 65-74% of the controls, respectively. Treatment of preformed biofilms with quinolones in concentrations ranging from 12.5 microg/ml to 400 microg/mL caused reduction in the optical density of the adherent biofilms to 45-77% of the control. In an in vitro model of vascular catheter colonization, subinhibitory concentrations (12, 14 and 1/8 MIC) of fluoroquinolones reduced the number of adherent bacteria to 24-28%, 48-55% and 58-76% of the controls, respectively. The vascular catheter segments precolonized with Staphylococcus epidermidis for 24 h and exposed to the fluoroquinolones in 8-16 times MIC (100 microg/mL) for 2 h showed no growth of adherent cells. The activity of pefloxacin in reducing the bacterial adhesion and eradicating the preformed biofilms was demonstrated by scanning electron microscope. These data show that subinhibitory concentrations of ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin inhibit the adhesion of Staphylococcus epidermidis to plastic surfaces and vascular catheters. Higher concentrations of fluoroquinolones were able to eradicate the preformed biofilms on vascular catheters.

Effectiveness of immersion treatments with acids, trisodium phosphate, and herb decoctions in reducing populations of Yarrowia lipolytica and naturally occurring aerobic microorganisms on raw chicken.

Yarrowia lipolytica, one of the predominant yeasts in raw poultry, is believed to play a role in spoilage. This study was undertaken to investigate treatments to control the growth of Y. lipolytica on raw chicken stored at refrigeration temperature. Raw chicken wings inoculated with a mixture of five strains of Y. lipolytica isolated from raw poultry were dipped in solutions containing 2, 5, or 8% lactic acid, 2% lactic acid containing 0.2, 0.4, or 0.8% potassium sorbate or sodium benzoate, and 4, 8, or 12% trisodium phosphate solution. Populations of the yeast and total aerobic microorganisms were determined before and after treatment. Immersion of wings in 2% lactic acid (with or without 0.2% potassium sorbate or sodium benzoate) or 4% trisodium phosphate caused a significant (alpha = 0.05) reduction in numbers of Y. lipolytica and aerobic microorganisms. Treatment with 2% lactic acid containing 0.4 or 0.8% preservative did not result in additional significant reductions. Treatment of chicken wings with 2% lactic acid or 8% trisodium phosphate significantly reduced numbers of Y. lipolytica by 1.47 and 0.65 log10 cfu/g, respectively, and aerobic microorganisms by 2.60 and 1.21 log10 cfu/g, respectively, compared to controls. Growth of Y. lipolytica on wings stored at 5 degrees C for up to 9 days, however, was not affected by these treatments. Significant reductions in the population of Y. lipolytica occurred when the yeast was inoculated into 100% basil, marjoram, sage, and thyme decoctions, but not in 100% oregano or rosemary decoctions, held at 5 degrees C for 24 h. Treatment of chicken wings with 100% sage or thyme decoctions significantly reduced populations of Y. lipolytica but did not control its growth during storage at 5 degrees C for up to 9 days. The small, temporary decreases in numbers of Y. lipolytica and aerobic microorganisms resulting from immersion treatment of chicken wings with sage and thyme decoctions render these treatments of questionable value as preservation interventions.

Presence and changes in populations of yeasts on raw and processed poultry products stored at refrigeration temperature.

A study was undertaken to determine populations and profiles of yeast species on fresh and processed poultry products upon purchase from retail supermarkets and after storage at 5 degrees C until shelf life expiration, and to assess the potential role of these yeasts in product spoilage. Fifty samples representing 15 commercial raw, marinated, smoked, or roasted chicken and turkey products were analyzed. Yeast populations were determined by plating on dichloran rose bengal chloramphenicol (DRBC) agar and tryptone glucose yeast extract (TGY) agar. Proteolytic activity was determined using caseinate and gelatin agars and lipolytic activity was determined on plate count agar supplemented with tributyrin. Populations of aerobic microorganisms were also determined. Initial populations of yeasts (log10 cfu/g) ranged from less than 1 (detection limit) to 2.89, and increased by the expiration date to 0.37-5.06, indicating the presence of psychrotrophic species. Highest initial populations were detected in raw chicken breast, wings, and ground chicken, as well as in turkey necks and legs, whereas roasted chicken and turkey products contained less than 1 log10 cfu/g. During storage, yeast populations increased significantly (P < or = 0.05) in whole chicken, ground chicken, liver, heart and gizzard, and in ground turkey and turkey sausage. Isolates (152 strains) of yeasts from poultry products consisted of 12 species. Yarrowia lipolytica and Candida zeylanoides were predominant, making up 39 and 26% of the isolates, respectively. Six different species of basidiomycetous yeasts representing 24% of the isolates were identified. Most Y. lipolytica strains showed strong proteolytic and lipolytic activities, whereas C. zeylanoides was weakly lipolytic. Results suggest that yeasts, particularly Y. lipolytica, may play a more prominent role than previously recognized in the spoilage of fresh and processed poultry stored at 5 degrees C.

Modulation of biofilms of Pseudomonas aeruginosa by quinolones.

The interaction between four fluoroquinolones (ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin) and biofilms of Pseudomonas aeruginosa in wells of microtiter plates and on segments of vascular catheters were studied in an in vitro model of vascular catheter colonization. Subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of the fluoroquinolones reduced the adherence of P. aeruginosa to 30 to 33, 44 to 47, and 61 to 67% of that of controls, respectively. The addition of high concentrations of the fluoroquinolones (12.5 and 400 micrograms/ml) to preformed biofilms (grown for 48 h at 37 degrees C) decreased the adherence of P. aeruginosa to 69 to 77 and 39 to 60% of that of controls, respectively. In an in vitro model of vascular catheter colonization, subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of fluoroquinolones reduced the number of adherent bacteria to 21 to 23, 40 to 46, and 55 to 70% of that of the controls, respectively. Scanning electron microscopy demonstrated a significant reduction in glycocalyx formation and adherent bacteria in the presence of pefloxacin at one-half to one-eight of the MIC. Vascular catheter segments precolonized with P. aeruginosa for 24 h and exposed to the fluoroquinolones at 4 to 25 times the MIC (50 micrograms/ml) for 2 h showed <5% growth of adherent cells compared with controls. No adherent organisms were cultured in the presence of 8 to 50 times the MIC (100 micrograms/ml). Scanning electron microscopy studies of preformed biofilms exposed to pefloxacin verified the results obtained by culture. These data show that subinhibitory concentrations of ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin inhibit the adherence of P. aeruginosa to plastic surfaces and vascular catheters. Clinically achievable concentrations of fluoroquinolones (50 to 100 micrograms/ml) were able to eradicate preformed biofilms on vascular catheters.

Biofilms in device-related infections.

The use of various medical devices including indwelling vascular catheters, cardiac pacemakers, prosthetic heart valves, chronic ambulatory peritoneal dialysis catheters and prosthetic joints has greatly facilitated the management of serious medical and surgical illness. However, the successful development of synthetic materials and introduction of these artificial devices into various body systems has been accompanied by the ability of microorganism to adhere to these devices in the environment of biofilms that protect them from the activity of antimicrobial agents and from host defense mechanisms. A number of host, biomaterial and microbial factors are unique to the initiation, persistence and treatment failures of device-related infections. Intravascular catheters are the most common devices used in clinical practice and interactions associated with these devices are the leading cause of nosocomial bacteremias. The infections associated with these devices include insertion site infection, septic thrombophlebitis, septicemia, endocarditis and metastatic abscesses. Other important device-related infections include infections of vascular prostheses, intracardiac prostheses, total artificial hearts, indwelling urinary catheters, orthopedic prostheses, endotracheal tubes and extended wear lenses. The diagnosis and management of biofilm-associated infections remain difficult but critical issues. Appropriate antimicrobial therapy is often not effective in eradicating these infections and the removal of the device becomes necessary. Several improved diagnostic and therapeutic modalities have been reported in recent experimental studies. The clinical usefulness of these strategies remains to be determined.

Tolerance of Staphylococcus epidermidis grown from indwelling vascular catheters to antimicrobial agents.

During a prospective study of indwelling vascular catheter-related infections, 134 isolates of Staphylococcus epidermidis were grown from 700 catheter tips. In vitro antimicrobial susceptibility testing of these isolates to oxacillin, vancomycin and ofloxacin was performed using the standard broth microdilution technique. These results were compared to those for the same organisms grown in biofilm before the addition of antimicrobial agents. In 96-well flat bottom microtiter plates, 10(4)-10(5) colony forming units of S. epidermidis in 0.1 ml broth were grown for 18 h at 37 degrees C, at which time a biofilm was observed for all isolates. Different concentrations of antimicrobial agents (0.1 ml) were then added to the plates. The plates were incubated for 18 h at 37 degrees C. Since MICs could not be estimated in these plates, all the wells were subcultured after mixing the biofilm with the broth. Minimum bactericidal concentrations (MBCs) were defined as 99.9% reduction in colony forming units. For organisms grown in suspension, 100% of the isolates were susceptible to vancomycin, 81% to ofloxacin and 40% to oxacillin. MBCs of susceptible isolates were within four-fold differences for vancomycin (53%), oxacillin (50%), and ofloxacin (51%). When grown as a biofilm, 78%, 93% and 71% of isolates had MBCs of > or = 2048 micrograms ml-1 of oxacillin, vancomycin and ofloxacin respectively. These data demonstrate the reduced bactericidal activity of antimicrobial agents against S. epidermidis in a biofilm and a simple method for its detection in the microbiology laboratory.