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Occurrence of macrolide-lincosamide-streptogramin resistances among staphylococcal clinical isolates at a university medical center. Is false susceptibility to new macrolides and clindamycin a contemporary clinical and in vitro testing problem?

A total of 2189 staphylococcal strains at the University of Iowa Hospitals and Clinics (Iowa City, IA) were initially screened to determine the incidence of constitutive (29.8%) and potential inducible macrolide-lincosamide-streptogramin (MLS) resistance (11.3%). Staphylococcus haemolyticus and S. epidermidis (62.5% and 55.3%) showed the highest incidence of constitutive resistance. Staphylococcus hominis had the highest incidence of inducible resistance (40.6%), while S. aureus had the lowest rate for both resistance types. The overall ratio of constitutive-inducible MLS resistance was 4:1. Among strains initially speciated using the Vitek System GPI card, there was only a 69% species identification reproducibility, and 78% accuracy versus a reference identification method. A random sample of 105 Staphylococcus spp. isolates with discordant macrolide (erythromycin resistant) and lincosamide (clindamycin susceptible) susceptibility patterns were tested against 16 antimicrobial agents by using a reference broth microdilution method. All erythromycin-resistant Staphylococcus spp. were also resistant to other 14-member macrolides and azithromycin, while all organisms remained susceptible to clindamycin, rifampin, vancomycin, and the streptogramin compounds (RP59500 and virginiamycin). Resistance to teicoplanin was identified among some oxacillin-resistant S. haemolyticus strains. Of 105 isolates, 65 (62%) showed inducible MLS resistance, 28 (27%) were noninducible, and 12 (11%) were either fully susceptible or resistant to the MLS drugs (Vitek System interpretation errors). MLS disk induction tests revealed two inducible resistance phenotypes: ML and MLS. Staphylococcus aureus showed the highest inducible resistance rate at 95% with an MLS-predominant pattern. In contrast, endemic S. haemolyticus isolates did not demonstrate inducible resistance that is, efflux-mediated erythromycin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗

Staphylococcal and micrococcal adherence to canine and feline corneocytes: quantification using a simple adhesion assay.

In this paper a simple adhesion assay suitable for the assessment of bacterial adhesion to both canine and feline corneocytes is described. Using this assay Staphylococcus intermedius, Staphylococcus aureus and Staphylococcus chromogenes were shown to adhere well to both canine and feline corneocytes. The numbers of adherent bacteria were, however, generally lower for feline corneocytes. Both Staphylococcus hominis and a Micrococcus species adhered poorly to canine and feline corneocytes. This is the first report documenting bacterial adhesion to feline corneocytes.

Animals↗

Recognition of Staphylococcus saprophyticus in urine cultures by screening colonies for production of phosphatase.

Phenolphthalein diphosphate was incorporated into a primary blood agar medium for use in performing quantitative urine cultures. Phosphatase-negative staphylococci, such as Staphylococcus saprophyticus, were differentiated from phosphatase-positive species, such as Staphylococcus epidermidis, by spot testing colonies on filter paper saturated with 1 N NaOH. Phosphatase-positive colonies turned pink within seconds, and phosphatase-negative colonies showed no color. None of 55 S. saprophyticus isolates showed production of phosphatase on this medium. Of 193 consecutive coagulase-negative staphylococci isolated from the urine of 190 adolescent female patients, 84% were phosphatase positive, non-S. saprophyticus species; 16% were phosphatase-negative and indicated S. saprophyticus (22), Staphylococcus haemolyticus (4), Staphylococcus simulans (2), Staphylococcus warneri (1), and Staphylococcus hominis (1). Phosphatase activity was variable in the other flora encountered in the urine cultures. Mixtures of phosphatase-positive and -negative organisms did not cause false-positive reactions.

Adolescent↗

Teat skin normal flora and colonization with mastitis pathogen inhibitors.

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial flora became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow teats with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.

Animals↗

A clinical and bacteriological evaluation of the effect of sulphamethoxazole-trimethoprim in acne vulgaris, resistant to prior therapy with tetracyclines.

42 patients with acne vulgaris, clinically resistant to prior therapy with tetracyclines, were evaluated after therapy with sulphamethoxazole-trimethoprim (400 + 80 mg) twice daily. Initially and after 6, 12 and 18 weeks of treatment in each patient the different acne lesions were counted and pus specimens from unhealed pustules were taken for bacteriological analysis. Complete remission or excellent results were obtained in 33 patients (79%) at the end of treatment despite a relative increase of Staphylococcus hominis and Propionibacterium granulosum. These species were more resistant in agar dilution test to the combination sulphamethoxazole-trimethoprim (20:1) than other isolated species.

Acne Vulgaris↗

Hub colonization as the initial step in an outbreak of catheter-related sepsis due to coagulase negative staphylococci during parenteral nutrition.

A prospective study was carried out to determine the pathogenesis of coagulase negative staphylococci catheter-related sepsis during parenteral nutrition. Forty-three catheters were cultured by semiquantitative and quantitative methods. The skin around the puncture site was cultured at the time of catheter removal and three segments of the catheter were cultured apart: the hub, the proximal subcutaneous segment, and the tip. Skin cultures were negative (89%) or yielded different coagulase negative staphylococci from those recovered in catheter and/or blood. Seventeen catheters were the source of sepsis. In 15 cases an infected hub was associated with an infected tip. In two cases the hub was negative (one sepsis due to mixture contamination and the other due to hematogenous seeding of the catheter tip). Sixteen cases of sepsis were due to coagulase negative staphylococci. Staphylococcus epidermidis has been the species most commonly isolated, followed by Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus hominis. In our patients most catheter sepsis have their origin in an infected hub and are not due to migration of skin bacteria along catheter subcutaneous tunnel.

Cross Infection↗

Wound healing accelerated by Staphylococcus aureus.

While comparing the effects on wound healing of a heated scalpel with those of the cold scalpel, we discovered that inoculation of rat skin incisions with a strain of Staphylococcus aureus dramatically accelerated the gain in wound strength. The accelerating effect was evident four days postoperatively, was maximal at seven to ten days, and was still present at 28 days. The accelerating effect was correlated with the number of S aureus organisms introduced into the wound, and was found in conventional rats and rats germ free up to the time of monocontamination with S aureus. There was no evidence of infection on gross examination; on histologic examination an occasional microabscess was seen in some rats. There may be both local and systemic mechanisms underlying the S aureus accelerating effect. Seven strains of S aureus with varying characteristics demonstrated the wound-healing accelerating effect. In sharp contrast, Staphylococcus epidermidis (three strains), Staphylococcus hominis (one strain), and Pseudomonas aeruginosa (two strains) did not show this effect. The increases in wound healing due to S aureus were substantially greater than reported previously for any nutritional supplement, drug, or other chemical or physical agent.

Animals↗

In vivo binding of the salivary glycoprotein EP-GP (identical to GCDFP-15) to oral and non-oral bacteria detection and identification of EP-GP binding species.

Extra Parotid Glycoprotein (EP-GP) is a glycoprotein isolated from human saliva, having homologues in several other body fluids. The biological role of EP-GP and its homologues is unknown. Recently, EP-GP was shown to bind in vitro to the bacterium Streptococcus salivarius HB. In contrast, no binding to a number of other oral microorganisms could be demonstrated. In the present study we have determined whether binding of EP-GP to bacteria occurs in vivo in saliva and in other EP-GP containing body fluids. Therefore the presence of EP-GP on bacteria in vivo was determined by analyzing oral, skin and ear floras by confocal fluoresence microscopy using specific antibodies. About 12% of the in vivo oral flora had EP-GP present on their surface, while approximately 5% of the bacteria from ear canal or skin was positive for EP-GP. IgA was detected on approximately 65% of the salivary bacteria, whereas the high-molecular weight mucin (MG1) and cystatin C were not detectable on any oral bacterium. Using a replica-plate assay, a number of EP-GP binding strains in saliva were isolated and identified as Gemella haemolysans, Gemella morbillorium, Streptococcus acidominimus, Streptococcus oralis, Streptococcus salivarius and Streptococcus parasanguis. Bacteria from the ear canal and skin bacteria were identified as Staphylococcus hominis. It is concluded that EP-GP is selectively bound in vivo to several oral and non-oral bacterial species.

Body Fluids↗

Study of the antiseptic efficacy of a quick drying rubbing type povidone-iodine alcoholic disinfectant solution by the glove juice method.

The antiseptic efficacy of HAD Hand Wash (83% ethanol containing 0.5% povidone-iodine) was investigated by the glove-juice method in 29 cases amongst staff members of our university. The number of viable bacteria on the hands and fingers was counted three times, and the mean of these counts was used as the baseline value for each subject. The number of viable bacteria on both hands was the same prior to disinfection. After the hands were disinfected by rubbing with 3 ml of HAD Hand Wash until it dried, the subjects put on sterile gloves. The number of viable bacteria was counted on the right hand of all the subjects immediately after disinfection. The number of viable bacteria on the left hand was counted in 8 subjects 30 minutes after disinfection, and in 7 subjects each 1, 2 and 4 hours after disinfection. The bacteria were identified by subculture. The ratio of bacteria sterilized (bacterial reduction rate) was 75.4% immediately after disinfection, and 69.0%, 71.9%, 35.8% and 27.6% at 30 minutes, 1 hour, 2 hours and 4 hours, respectively. All of the bacteria detected were Gram-positive bacteria that were native to the sebaceous glands, sweat glands or skin surface; i.e. Staphylococcus, Micrococcus, Bacillus, and Streptococcus. The frequency of detection of Staphylococcus warneri was the highest before disinfection, followed by Staphylococcus epidermidis and Staphylococcus hominis, in that order. After disinfection, all these species showed a marked decrease in the number of bacteria. Micrococcus remained in half the subjects, while other species remained in less than one third of the subjects.

Antisepsis↗

Evaluation of the Staph-Zym system with staphylococci isolated from bovine intramammary infections.

A total of 148 staphylococci isolated from bovine intramammary infections were used to evaluate the Staph-Zym system (ROSCO, Taastrup, Denmark). The overall accuracy of the system was 91.9%. The system correctly identified all strains of Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus xylosus and 95% of Staphylococcus intermedius strains. Of 33 Staphylococcus hyicus strains, 31 (93.9%) were classified correctly by the Staph-Zym system, as well as 8 (80%) of 10 Staphylococcus chromogenes strains. All 11 Staphylococcus epidermidis strains and the 1 Staphylococcus haemolyticus strain included in the study were identified, but the Staph-Zym system had difficulty distinguishing strains of Staphylococcus warneri and Staphylococcus hominis from other species in the S. epidermidis group. The Staph-Zym system correctly identified all six S. xylosus strains and two of three Staphyloccus sciuri strains. The Staph-Zym system was considered an acceptable alternative to conventional methods for identification of bovine mammary gland isolates.

Animals↗

Alcohol increases hemolysis by staphylococci.

It was recently found that alcohols can confer hemolytic properties on certain species of yeast. Here, it is reported that alcohol can promote hemolysis by various species of staphylococci, including strains of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus hominis. In order to study this novel phenomenon in S. aureus and S. epidermidis, strains that exhibit this phenomenon (e.g. S. aureus 8325-4, COL, SH1000, S. epidermidis), as compared with strains that exhibited little alcohol-enhanced hemolysis (e.g. S. aureus 8325-4 DeltaTRAP, RN6911) were examined. Both ethanol and n-butanol caused upregulation of the virulence regulator-RNAIII, with a concomitant increase in the production of alpha, beta and gamma-hemolysins in strain 8325-4. In S. aureus COL and SH1000, there was an increase in RNAIII but no change in transcription levels of alpha, beta and gamma hemolysins. Staphylococcus epidermidis stain sofi exhibited increased RNAIII and beta hemolysin production. Staphylococcus aureus mutant strains (8325-4 DeltaTRAP and RN6911) showed no change in the transcription level of the RNAIII regulator and the above hemolysins. Increased hemolysis in S. aureus COL, SH1000 and mutant strains may be caused by other hemolysins (not regulated by RNAIII) or through other mechanisms such as hyperoxidation or cytotoxic lipids.

1-Butanol↗

Application of molecular typing methods to characterize nosocomial coagulase-negative staphylococci collected in a Greek hospital during a three-year period (1998-2000).

A total of 143 methicillin-resistant coagulase-negative staphylococci (MR-CNS) collected between 1998 and 2000 at the University Hospital of Patras, Greece, were characterized by antibiogram and genomic typing to define the clonal types endemic in this hospital and their evolution during the 3-year period. These isolates corresponded to 93 methicillin-resistant Staphylococcus epidermidis (MRSE) and 50 other MR-CNS, which were isolated from patients in different wards, exclusively from blood and catheter tips cultures. Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with mecA and murE DNA probes were performed. The application of these methodologies demonstrated the existence, persistence and spread of MRSE, MR-Staphylococcus haemolyticus, and MR-Staphylococcus hominis clones in this hospital, whereas the SmaI/murE hybridization pattern was shown to be a valuable tool for the MRSE identification.

Bacterial Typing Techniques↗

Production of experimental endocarditis by coagulase-negative staphylococci: variability in species virulence.

The abilities of 11 coagulase-negative staphylococcal isolates of human origin to establish infective endocarditis in a rat model were compared. Seven of the strains were Staphylococcus epidermidis, the species most commonly causing endocarditis in humans. The other four strains were Staphylococcus hominis, an infrequent cause of endocarditis. Four of the seven strains of S. epidermidis and two of the four strains of S. hominis elaborated surface slime. With an inoculum of 10(7) colony-forming units, endocarditis was established in all of 77 rats injected with S. epidermidis but in only five (12.5%) of 40 animals injected with S. hominis (P less than .001). Strains of S. epidermidis were significantly more resistant to phagocytic killing in vitro than were strains of S. hominis. These data indicate that there are major differences in the virulence of various species of coagulase-negative staphylococci, both in vivo and in vitro, and that these differences are reflected in the observed incidence of disease caused in humans by the respective species.

Animals↗

Characteristics of coagulase-negative staphylococci that help differentiate these species and other members of the family Micrococcaceae.

One hundred reference strains and 1,240 clinical isolates representing 26 species of the family Micrococcaceae were used to evaluate the potential of tests for synergistic hemolysis, adherence to glass, pyroglutamyl-beta-naphthylamide hydrolysis, and susceptibility to a set of five antimicrobial agents for differentiating these species and strains within the species. Sixty-eight percent of the clinical isolates exhibited synergistic hemolysis; 69% of the clinical staphylococci but none of the micrococci or stomatococci were adherence positive, and 92% of the strong positive adherence reactions were produced by strains of Staphylococcus epidermidis. Strains from 15 of the species were pyroglutamyl-beta-naphthylamide positive, but this test separated Staphylococcus xylosus from other novobiocin-resistant staphylococci and Staphylococcus intermedius from other coagulase-positive species. A polymyxin B disk helped differentiate S. epidermidis from most other coagulase-negative staphylococci, and a bacitracin disk (10 U) helped differentiate Staphylococcus haemolyticus from most other novobiocin-susceptible staphylococci. All strains that were susceptible to furazolidone and resistant to Taxo A disks (bacitracin, 0.04 U; BBL Microbiology Systems, Cockeysville, Md.) were staphylococci. We observed a 91% correlation between species identification obtained with the Staph-Ident system (Analytab Products, Plainview, N.Y.) and conventional methods; but the micrococci and stomatococci were incorrectly identified as staphylococci with Staph-Ident, and several isolates of S. epidermidis were misidentified as Staphylococcus hominis because they were alkaline phosphatase negative. Both these problems can be prevented by adding the simple tests we describe to those already recommended when the Staph-Ident system is used to identify isolates of gram-positive, catalase-positive cocci.

Bacitracin↗

Potentiation of antibiotic activity by EDTA-tromethamine against three clinically isolated gram-positive resistant bacteria. An in vitro investigation.

The in vitro synergistic effects of combinations of EDTA-tromethamine and five antimicrobial agents (ampicillin, cephalexin, oxytetracycline, streptomycin and sulphadimethoxine) on three clinically isolated Gram-positive bacteria (Staphylococcus aureus, Staphylococcus hominis and Streptococcus faecium) were investigated. The bacteria had been isolated from three cases of canine otitis resistant to beta-lactam antibiotic therapy. The antimicrobial activity was evaluated by measuring the minimal inhibitory concentration for the antibiotics alone or in combination with EDTA-tromethamine. EDTA-tromethamine potentiated the activity of cefalexin against S. aureus and S. hominis, of oxytetracycline against S. aureus and S. faecium and of streptomycin against S. faecium. No significant effects were noted on the activity of oxytetracycline against S. hominis. The remaining combinations gave a slight synergistic effect. As previously shown for Gram-negative resistant bacteria, these data suggest that the association of EDTA-tromethamine and appropriate antibiotic therapy may be useful to overcome persistent infections of soft tissues in domestic animals.

Animals↗

Epidemiology and susceptibilities to mercury preservatives of staphylococci isolated from used eye-drops preserved with thiomersal.

Minimum inhibitory concentrations (MICs) of seven independent isolates of Staphylococcus hominis isolated in the same week from used eye-drops, preserved with thiomersal and collected from wards and clinics in the same hospital, ranged between 1 and 0.03 mg L-1 for thiomersal, 1 and 0.01 mg L-1 for phenyl mercuric nitrate and 10 and 3 mg L-1 for mercuric chloride. Although MIC values determined on solid nutrient medium indicated a 100-fold variation in susceptibility to the bacteriostatic effect of phenyl mercuric nitrate, after 5 h in an aqueous solution containing the bactericidal concentration of 10 mg L-1 phenyl mercuric nitrate, the survival levels of the six S. hominis isolates were similar, with a mean of 13.4% (s.d. 11.0), compared with 100 and 0.8%, respectively, for the most resistant and most sensitive control staphylococcal strains tested. Antibiotic susceptibilities and plasmid profiles of the S. hominis isolates indicated they were the same strain. It is concluded that laboratory indicators of preservative efficacy, such as MIC determination or susceptibility to bactericidal concentrations of preservatives, do not necessarily correlate with the epidemiology of contaminating bacterial strains or their survival in preserved pharmaceuticals.

Anti-Infective Agents, Local↗

Species dependent variability in the susceptibility of coagulase-negative staphylococci to various antimicrobial agents.

The in-vitro activity of 20 beta-lactam antibiotics and vancomycin was determined against three different species of coagulase-negative-staphylococci. All isolates were susceptible to vancomycin (MIC90 3.12-6.25 mg/l) although occasional tolerance was seen. Most isolates (85%) of Staphylococcus hominis were susceptible to methicillin and other beta-lactams including cephalothin, cefapirin, cefamandole, imipenem and BMY28142, while 90% of Staph. haemolyticus isolates were resistant to these agents. Staph. epidermidis was intermediate in susceptibility with 50% of isolates being methicillin resistant (MR). These MR isolates exhibited cross-resistance or tolerance to the other beta-lactam agents except cefamandole.

Anti-Bacterial Agents↗

D-value determinations are an inappropriate measure of disinfecting activity of common contact lens disinfecting solutions.

Determination of a D value for specific test organisms is a component of the efficacy evaluation of new contact lens disinfecting solutions. This parameter is commonly defined as the time required for the number of surviving microorganisms to decrease 1 logarithmic unit. The assumption made in establishing a D value is that the rate of kill exhibits first-order kinetics under the specified conditions. Such exponential kill rates are seen with thermal contact lens disinfection system. A comparison of the death rate kinetics for a variety of chemical contact lens disinfecting solutions was undertaken to ascertain the suitability of D-value determination for these chemical disinfectants. The active agents of these different solutions included hydrogen peroxide, thimerosal, chlorhexidine, tris(2-hydroxyethyl)tallow ammonium chloride, thimerosal, polyaminopropyl biguanide, and polyquaternium-1. The solutions were challenged with 10(6) CFU of either Pseudomonas aeruginosa, Serratia marcescens, or Staphylococcus hominis per ml, and survival rate was determined. This study clearly demonstrates the nonlinear nature of the inactivation curves for most contact lens chemical disinfecting solutions for the challenge organisms. D-value determination is, therefore, an inappropriate method of reporting the biocidal activity of these solutions.

Contact Lenses↗