Significant Reduction In Bacterial Counts Research Articles

A novel cell wall-anchored peptidoglycan hydrolase (autolysin), IspC, essential for Listeria monocytogenes virulence: genetic and proteomic analysis

We have recently concluded that a Listeria monocytogenes 86 kDa immunogenic surface protein, IspC, is a cell wall-anchored peptidoglycan hydrolase (autolysin), capable of degrading the cell wall peptidoglycan of the bacterium itself. To determine if this enzyme has any biological functions and/or plays a role in virulence, we in-frame-deleted the ispC gene from the L. monocytogenes chromosome. This DeltaispC mutant exhibited complete abrogation of expression of IspC and displayed no defects in in vitro growth, colony and microscopic morphologies, or biochemical characteristics. Lack of IspC led to attenuated virulence in mice, evidenced by a significant reduction in bacterial counts in livers and brains and no mortality compared with the wild-type. Furthermore, the data from assays using various eukaryotic cells for adhesion, invasion, actin tail formation, plaque formation and intracellular growth indicated that the mutant was severely attenuated in virulence in a cell culture model in a cell type-dependent manner. The findings that (i) the mutant was impaired for adhesion to certain eukaryotic cells, and (ii) both purified IspC and its C-terminal cell wall-binding domain were capable of binding sheep choroid plexus (SCP) epithelial cells and Vero cells, supported the role of IspC as an adhesin in virulence. The DeltaispC mutant exhibited a marked defect in adhesion to and invasion of SCP cells but not human brain microvascular endothelial cells (HBMEC), suggesting that IspC is necessary for crossing the blood-cerebrospinal fluid barrier. Proteomic and immunological analysis showed a reduced surface expression of some known or putative virulence factors (e.g. ActA, InlC2 and a flagellin homologue, FlaA) due to IspC deficiency. Altogether, this study demonstrates that IspC, expressed as a minor autolysin in vitro, is not important for cell division or separation but is essential for full virulence of L. monocytogenes in vivo.

Triphala Promotes Healing of Infected Full-Thickness Dermal Wound

Infection is a major problem in the management of wounds. Even though the development of synthetic antimicrobial agents persists, drug resistance and toxicity hinder their way. Many plants with multi-potent pharmaceutical activities may offer better treatment options, and Triphala (dried fruits of Terminalia chebula, Terminalia bellirica, and Phyllanthus emblica) are potential formulations evaluated for healing activity on infected wound as it possesses numerous activities. Alcoholic extract of Triphala has shown in vitro antimicrobial activity against wound pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes. An ointment was prepared from the Triphala extract (10% w/w) and assessed for in vivo wound healing on infected rat model by rate of healing, bacterial count, biochemical analysis, and expression of matrix metalloproteinases. The treated group has shown significantly improved wound closure. Assessment of granulation tissue on every fourth day showed significant reduction in bacterial count with significant level of collagen, hexosamine, uronic acid, and superoxide dismutase in the treated group (P < 0.01). Reduction of matrix metalloproteinase expression observed in the treated group by gelatin zymography and immunoblotting confirms our in vivo assessment. The above results showed the antibacterial, wound healing, and antioxidant activities of Triphala ointment, necessary for the management of infected wounds. Active principles of the Triphala may be further evaluated and used as an excellent therapeutic formulation for infected wounds.

Efficacy of colistin/rifampin combination in experimental rat models of sepsis due to a multiresistant Pseudomonas aeruginosa strain*

To investigate the efficacy of rifampin and colistin in three experimental rat models of Pseudomonas aeruginosa sepsis. Prospective, randomized, controlled animal study. Research laboratory in a university hospital. Adult male Wistar rats. Adult male Wistar rats were given a) an intraperitoneal injection of 1 mg of P. aeruginosa 10 lipopolysaccharide; b) 2 x 10(10) colony-forming units of P. aeruginosa ATCC 27853; and c) 2 x 10(10) colony-forming units of one clinically multiresistant strain of P. aeruginosa. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 10 mg/kg rifampin, 1 mg/kg colistin, and 10 mg/kg rifampin plus 1 mg/kg colistin. Lethality, bacterial growth in blood and peritoneum, and endotoxin and tumor necrosis factor-alpha concentrations in plasma were measured. Colistin exerted a strong antimicrobial activity and achieved a significant reduction of plasma endotoxin and tumor necrosis factor-alpha concentration compared with control and rifampin-treated groups. Rifampin exhibited no antimicrobial activity with no substantial impact on endotoxin and tumor necrosis factor-alpha plasma concentrations. The combination of colistin and rifampin resulted in a significant reduction in bacterial count compared with colistin monotherapy, whereas no significant difference was found in positive hem cultures and mortality rates between the two groups. Colistin and rifampin might have a role in the therapy of multiresistant P. aeruginosa infection.

Effects of Chemomechanical Preparation With 2.5% Sodium Hypochlorite and Intracanal Medication With Calcium Hydroxide on Cultivable Bacteria in Infected Root Canals

This clinical study was conducted to assess the bacterial reduction after chemomechanical preparation with 2.5% NaOCl as an irrigant and the additive antibacterial effect of intracanal dressing with calcium hydroxide. According to stringent inclusion criteria, 11 teeth with primary intraradicular infections and chronic apical periodontitis were selected and monitored in the study. Bacterial samples were taken at the baseline (before treatment) (S1), after chemomechanical preparation with 2.5% NaOCl as an irrigant (S2), and after a 7-day dressing with a calcium hydroxide paste in glycerin (S3). Cultivable bacteria recovered from infected root canals at the 3 stages were counted and identified by means of 16S rRNA gene sequencing analysis. At S1, all canals were positive for bacteria, with the mean number of 2.8 taxa per canal (range, 1-6). At S2, 5 cases (45.5%) still harbored cultivable bacteria, with 1 or 2 species per canal. At S3, bacteria were cultured from 2 cases (18.2%), with 1 species per positive case. There was no indication that any specific bacterial taxon was more resistant to treatment. A significant reduction in bacterial counts was observed between S1 and S2, and S1 and S3. However, no statistically significant difference was observed for comparisons involving S2 and S3 samples with regard to the number of cases yielding negative cultures (P = .18) or quantitative bacterial reduction (P = .19). It was concluded that the whole antibacterial protocol used in this study significantly reduced the number of bacteria in the canal and rendered most canals free of cultivable bacteria.

Biological evaluation of pyrazinamide liposomes for treatment of Mycobacterium tuberculosis

Pyrazinamide liposomes were prepared employing the phospholipid molar ratios; dipalmitoyl phosphatidyl choline (7):cholesterol (2) neutral and dipalmitoyl phosphatidyl choline (7):cholesterol (2):dicetyl phosphate (1) negatively charged. Swelling at 52 °C led to higher trapping efficiencies. An optimum sterilizing dose of 25 kGy was exhibited by gamma (γ)-irradiation. Neutral pyrazinamide liposomes (7:2), swollen for 24 h, were employed in biological evaluation for treatment of mice infected by Mycobacterium tuberculosis. Liposomal pyrazinamide could effect highly significant reduction in bacterial counts (colony forming units/g lung), 10, 20 and 30 days after the last treatment dose. Histopathological examination of mice lungs showed highest severity of infection in drug-free liposomes (control) group > pyrazinamide liposomes > free pyrazinamide 6 days/week. The results indicate high therapeutic efficacy of pyrazinamide liposomes, injected twice weekly, in treatment of M. tuberculosis in mice.

Efficacy of linezolid plus rifampin in an experimental model of methicillin-susceptible Staphylococcus aureus endocarditis.

The efficacy of linezolid, alone or in combination with rifampin, against methicillin-susceptible Staphylococcus aureus in rabbits with experimental endocarditis was investigated. Linezolid (50 or 75 mg/kg of body weight), rifampin, and linezolid (25, 50, or 75 mg/kg) plus rifampin produced statistically significant reductions in bacterial counts compared with those in untreated controls. Plasma or valvular vegetation levels of linezolid in the groups treated with the linezolid-rifampin combination were similar to those in the respective linezolid-only treatment groups. At therapeutic levels of linezolid, rifampin resistance was not observed. The results from this experimental model of endocarditis suggest that while rifampin did not provide synergy to the linezolid dosing, it did not antagonize the efficacy of linezolid.

Effects of sulfamethizole and amdinocillin against Escherichia coli strains (with various susceptibilities) in an ascending urinary tract infection mouse model.

Resistance to antibiotics used for the treatment of urinary tract infections (UTIs) is increasing worldwide. The impact of in vitro resistance on clinical outcome in UTIs requires further study, since most studies of both humans and animals have evaluated only the efficacy of antibiotics toward bacteria susceptible in vitro. We were interested in evaluating the relationship between the in vitro antibacterial effect and the in vivo efficacy after antibiotic treatment. We simulated a natural ascending UTI by use of the ascending UTI mouse model and used Escherichia coli strains with various susceptibilities to amdinocillin (mecillinam) and sulfamethizole. Mice were treated for 3 days with antibiotic doses approximating human urinary tract concentrations after a standard oral dose. For a susceptible strain (MIC, 0.5 micro g/ml) and a resistant strain (MIC, 128 micro g/ml), respectively, there were significant reductions in bacterial counts in the urine, bladder, and kidneys after treatment with amdinocillin, whereas for a strain for which the MIC was 16 micro g/ml, there was a significant reduction in bacterial counts in the kidneys only (P < 0.05). Treatment with sulfamethizole resulted in a significant reduction in bacterial counts in all samples from a susceptible strain (MIC, 128 micro g/ml) and a resistant strain (MIC, 512 micro g/ml). Infection with a sulII gene-positive strain (MIC, >2,048 micro g/ml) could not be treated with sulfamethizole, as no effect could be demonstrated in the urine, bladder, or kidneys. For amdinocillin, there was no clear-cut relationship between the in vitro susceptibility and the in vivo outcome, while for sulfamethizole, we found a relationship between the MIC for the strain and the effect in the urinary tract.

Routine disinfection of patients’ environmental surfaces. Myth or reality?

We have evaluated the need for daily disinfection of environmental surfaces not contaminated by biological fluids, in patient areas of a medical unit with two wings [North (N) and South (S)] at the University Hospitals of Geneva, Switzerland. Weekly bacteriological monitoring of surfaces was carried out at random (N = 1356 samples). In the S wing (control), we used detergent/disinfectant for daily cleaning of the floors and furniture. In the N wing we began by using a detergent for floors and furniture; after four weeks the results suggested changing to a rotation of detergent, dust attracting disposable dry mops and disinfectant. During this period the furniture was cleaned with an active oxygen-based compound. The average differences in contamination before and after cleaning floors were (mean reduction in bacterial counts and 95% confidence intervals; CI95): disposable mops: 92·7 cfu/24 cm2(CI95; 74–112), active oxygen based compound 111·1 (90–133), and quaternary ammonium compound –0·6 (–27–26). Use of detergent alone was associated with a significant increase in bacterial colony counts: on average by 103·6 cfu (CI9573–134). The quaternary ammonium compound was inadequate for disinfecting bathrooms and toilets but the active oxygen based compound was satisfactory. For furniture, there was a significant reduction in bacterial counts with both the methods using disinfectants. As the detergent was contaminated, by using it alone for cleaning, we were actually seeding surfaces with bacteria.A total of 1117 patients was studied and we observed no change in the incidence of nosocomial infections during the four months of the trial. In conclusion, uncontrolled routine disinfection of environmental surfaces does not necessarily make it safe for the patient and could seed the environment with potential pathogens.

The effects of low-frequency ultrasound on Staphylococcus epidermidis.

Low frequency ultrasound (LFUS) significantly enhances skin permeability to a variety of drugs; however, its bacterial effects have not been well studied. Staphylococcus epidermidis organisms were grown and standardized to 10(5) cfu/ml 24 h prior to investigation and suspended in normal saline. LFUS was applied with two probes immersed in the bacterial suspensions over a range of suspension volumes, intensities, and exposure times. The suspension temperature was measured, and a sample was removed, streaked onto blood agar plates, and incubated at 37 degrees C for 24 h. Quantitative bacterial counts were then obtained. LFUS resulted in significant reductions in bacterial counts that correlated with fluid temperature. Probe size and ultrasound intensity appeared to affect bacterial counts, but were also correlated with temperature. Bacterial growth was minimal with temperatures exceeding 45 degrees C. While LFUS can reduce bacterial counts, these conditions have the potential to cause burns in humans.

The Possible Role of Interleukin (IL)-12 and Interferon-γ-Inducing Factor/IL-18 in Protection against ExperimentalMycobacterium lepraeInfection in Mice

Cell-mediated immunity participates in host defense against mycobacterial infection. Both interleukin 12 (IL-12) and interferon-γ-inducing factor (IGIF/IL-18), produced mainly by macrophages, play a critical role in expression of cell-mediated immunity. To investigate the role of IL-12 and IGIF/IL-18in vivo,we examined cytokine profile, bacterial growth, and the potential benefit of cytokine therapy in susceptible and resistant mice infected withMycobacterium leprae.The early expression of IL-12 p40 and IGIF/IL-18 at the site of inoculation was found in resistant mice 3–72 h after the infection, but not in susceptible mice. Both strains of mice did not show expression of IFN-γ and IL-4. IL-12 administration resulted in a significant reduction of bacterial counts in mice with establishedM. lepraeinfection. The results imply that susceptible mice exhibit decreased expression of type 1 helper T (Th1) response without reciprocal increased Th2 response and show responsiveness to exogenous IL-12. IL-12 therapy may be a possible rationale for treatment ofM. lepraeinfection.

Enhancement of the in vitro and in vivo activities of clarithromycin against Haemophilus influenzae by 14-hydroxy-clarithromycin, its major metabolite in humans

MICs of clarithromycin and its major human metabolite, 14-hydroxy-clarithromycin, for Haemophilus influenzae in combination were reduced two- to fourfold compared with the MICs of each compound alone. Serum reduced the MICs of the parent compound and metabolite two- to fourfold compared with the MICs in medium without serum. In serum spiked with clinically relevant concentrations of clarithromycin and 14-hydroxy-clarithromycin at a fixed ratio of 4:1, 15 of 16 strains (94%) were inhibited and killed by combinations containing 1.2 and 0.3 micrograms/ml, respectively. In time kill experiments, the combination of parent compound and metabolite at one-fourth and one-half of their individual MICs, respectively, reduced bacterial counts by greater than 5 log CFU. The postantibiotic effect of clarithromycin combined with 14-hydroxy-clarithromycin was twice that of clarithromycin when tested alone. When orally administered to gerbils with H. influenzae otitis media, the 14-hydroxy metabolite was significantly more active than clarithromycin in reducing bacterial counts from the middle ear. The in vivo activity of the two compounds in combination was synergistic or additive, depending on the level of H. influenzae present at the time treatment was initiated. Significant reductions in bacterial counts and increases in cure rates were observed when clarithromycin at 50 or 100 mg/kg of body weight was combined with 14-hydroxy-clarithromycin at 12 mg/kg or higher. Results from in vitro and in vivo combinations suggest that routine susceptibility tests and animal efficacy studies with clarithromycin alone may underestimate its potential efficacy against H. influenzae.

Oral temafloxacin versus vancomycin for therapy of experimental endocarditis caused by methicillin-resistant Staphylococcus aureus

We compared oral temafloxacin, a new fluoroquinolone agent, with vancomycin, each with and without rifampin, in the therapy of rats with aortic valve endocarditis caused by a clinical isolate of methicillin-resistant Staphylococcus aureus. The temafloxacin, vancomycin, and rifampin MICs and MBCs were 0.78 and 1.56, 1.56 and 3.13, and less than 0.024 and 0.78 microgram/ml, respectively. The animals were classified into the following six treatment groups: vancomycin (60 mg/kg) +/- rifampin (6 mg/kg) each intramuscularly every 12 h for 5 days; temafloxacin (100 mg/kg) orally +/- rifampin (6 mg/kg) intramuscularly every 12 h for 5 days; rifampin (6 mg/kg) intramuscularly every 12 h for 5 days; and untreated controls. All regimens with either vancomycin or temafloxacin resulted in improved survival over controls, but only temafloxacin regimens resulted in a significant reduction in bacterial counts in vegetations. These data support further investigation of the efficacy of temafloxacin in treating serious infections caused by methicillin-resistant S. aureus.

The Therapeutic Response of Cephalosporin-Treated E. coli Pyelonephritis of the Rat, in Relation to Variations of the Infection Model

In the E. coli pyelonephritis, induced in female Wistar rats by retrograde infection (high pressure reflux), we investigated the influence of 1) the time of commencement of therapy, 2) the renal bacterial counts, i.e. the inflammatory activity of the pyelonephritis after endovesical instillation of cultures with different bacterial concentrations, and 3) the level of infection resistance of the experimental animal strain on the therapeutic response of the model infection with single doses of cefoxitin (150 mg/ml) and cefotaxime (5 mg/ml). 1. Early commencement of therapy post inoculation was therapeutically advantageous provided the intrarenal multiplication of the infective organisms was not delayed or the initial bacterial concentrations were not too high. 2. The mild form of pyelonephritis with lower renal bacterial concentrations and poor inflammatory activity after endovesical instillation of a low inoculum (10 4 cfu/ml) was less amenable to treatment than the inflammatorily active pyelonephritis with high renal bacterial counts, using a high inoculum (10 7 cfu/ml). High renal bacterial counts after retrograde inoculation of an E. coli culture of 10 8 cfu/ml resulted in significant reduction of bacterial counts 48, 72 and 96 h post infectionem, with i.m. application of cefoxitin 12 h prior. 3. For Wistar rat strain Bor:WIST, which showed a stronger infection resistance with lower renal bacterial concentrations and a stronger tendancy to spontaneous healing, application of a single dose of cefotaxime (5 mg/ml) was therapeutically ineffective, whereas, in contrast, with Han: WIST rats the acute phase of E. coli pyelonephritis could be treated effectively.