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PubMed2003

PCR rapidly detects methicillin-resistant staphylococci periprosthetic infection.

Optimal treatment of methicillin-resistant staphylococcal periprosthetic infections is predicated on rapid and reliable detection of these organisms. Culture has served as the gold standard for identification of these organisms despite shortcomings with sensitivity and processing time. The objective of the current study was to investigate a polymerase chain reaction assay aimed at rapid genomic detection of methicillin-resistance in staphylococci (mecA gene). The feasibility of the molecular approach first was validated using a septic arthritis model consisting of 73 synovial fluid samples inoculated with methicillin-resistant staphylococci and four negative controls. MecA polymerase chain reaction then was done on 35 clinical samples from 18 patients obtained at the time of revision arthroplasty. Results of the polymerase chain reaction were compared with culture. MecA polymerase chain reaction successfully predicted the presence of methicillin-resistant staphylococci in the septic arthritis model. In the clinical samples studied, the polymerase chain reaction results were concordant with culture results in 34 of the 35 samples tested. The one discordant result represented a false-positive culture result. The molecular assay was processed in less than 5 hours compared with 2 to 3 days for culture. Detection of methicillin-resistant staphylococci involved in periprosthetic infections by the polymerase chain reaction is a rapid and reliable approach.

Arthritis, Infectious↗
PubMed1982

Intraphagocytic protection of staphylococci from extracellular penicillin.

In a system in which unphagocytosed bacteria were removed by differential centrifugation after a 30-min phagocytosis period, staphylococci associated with rabbit polymorphonulcear (PMN) leukocytes were completely protected from the effects of benzyl penicillin 1 microgram/ml, but not completely protected from the effects of 5 micrograms/ml. When unphagocytosed bacteria were lysed with lysostaphin, effective protection could be observed over a range of penicillin concentrations from 0.25 to 200 micrograms/ml. 14C-benzyl penicillin failed to accumulate in rabbit PMN leukocytes, whether or not they had previously phagocytosed staphylococci, in conditions in which mouse peritoneal macrophages readily accumulated penicillin. Mixed granule extracts prepared from the PMN leukocytes interacted synergically with penicillin against staphylococci at physiological pH (7.2) but failed to show synergy at an intraphagolysosomal pH of 5.0 unless the bacteria were first sublethally treated with penicillin. Experiments in which the pH value of culture media was changed either from 7.2 to 5.0 or from 5.0 to 7.2 indicated that the partial nature of the protective effect of the intraphagolysosomal environment could be attributed to the growth-limiting effects of the low phagolysosomal pH, which prevents full expression of the synergic potential of granule contents and penicillin. The concentration-dependent nature of the protection and its incompleteness are explained by supposing that a proportion of the staphylococci not ingested during the 30-min phagocytosis period are modified by penicillin in a way that opsonises them and potentiates the intrinsic bactericidal mechanisms of the PMN leukocytes when the bacteria are subsequently ingested.

Animals↗
PubMed1991

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci.

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. mitior produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1-12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher (P less than 0.001, chi 2 test and P less than 0.05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus (P less than 0.001 and P less than 0.01) and of oral Staph. epidermidis over oral Staph. aureus (P less than 0.01 and P less than 0.05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological viewpoints are discussed.

Adult↗
PubMed1990

Subgingival occurrence of enteric rods, yeasts and staphylococci after systemic doxycycline therapy.

The occurrence of subgingival enteric rods, yeasts and staphylococci (pre-treatment and post-treatment) was examined in 21 adult periodontitis patients receiving mechanical periodontal therapy and systemic doxycycline (200 mg on first day, then 100 mg/day for 20 days). Subgingival samples obtained prior to, and 2 weeks and 4 weeks after completion of the antibiotic therapy were transported in VMGA III and plated onto TSBV (for enteric rods and yeasts) and Staphylococcus 110 media. At baseline, small numbers of enteric rods (3 patients), yeasts (5 patients) and staphylococci (11 patients) were detected subgingivally. After the antibiotic therapy, more than 10-fold increases were seen in subgingival numbers of Enterobacter aerogenes (2 patients), Escherichia coli (1 patient), Candida albicans (2 patients), and staphylococci (11 patients). These findings demonstrating that systemic doxycycline therapy may result in at least temporary subgingival overgrowths of enteric rods, yeasts and staphylococci, may indicate the risk of frequent use of tetracycline in periodontal therapy.

Adult↗
PubMed2006

SCCmec in staphylococci: genes on the move.

Staphylococcal cassette chromosome (SCC) elements are, so far, the only vectors described for the mecA gene encoding methicillin resistance in staphylococci. SCCmec elements are classified according to the type of recombinase they carry and their general genetic composition. SCCmec types I-V have been described, and SCC elements lacking mecA have also been reported. In this review, we summarize the current knowledge about SCC structure and distribution, including genetic variants and rudiments of the elements. Its origin is still unknown, but one assumes that staphylococcal cassette chromosome is transferred between staphylococci, and mecA-positive coagulase-negative staphylococci may be a potential reservoir for these elements. Staphylococcal genomes seem to change continuously as genetic elements move in and out, but no mechanism of transfer has been found responsible for moving SCC elements between different staphylococcal species. Observations suggesting de novo production of methicillin-resistant staphylococci and horizontal gene transfer of SCCmec will be discussed.

Chromosomes, Bacterial↗
PubMed1985

Studies on coagulase-negative staphylococci in patients undergoing cardiac surgery. Different hypotheses for the origin of multiply-resistant isolates.

Hypotheses for the origin of multiply-resistant coagulase-negative staphylococci from 146 patients undergoing cardiac surgery were tested. All received cephalothin per-operatively. Antibiotic susceptibility testing, phage-typing, bio-typing, and test for Tween-80-splitting enzyme were used to characterize 132 isolates from nose swabs. Seventy-five percent of the pre-operative susceptible isolates were of biotype 1, while biotypes 3 and 4 made up 59% of the post-operative, multiply-resistant isolates. Fifty-three percent of the isolates were typable by phage-typing. Typability of isolates of biotype 1 was high (56%) while almost 75% of biotype 4 were untypable. Susceptible isolates were more often typable than multiply-resistant ones. Of the 146 patients, 105 (72%) carried coagulase-negative staphylococci pre-operatively, only two of them carried multiply-resistant strains. Fifty-nine patients (41%) were colonized with multiply-resistant coagulase-negative staphylococci post-operatively. By combining the results of bio-typing, phage-typing, and test for Tween-splitting enzyme the study made it probable that a maximum of ten patients (6.8%) already carried multiply-resistant strains on admission to the hospital or were carriers of initially susceptible strains which developed multiple-resistance during administration of antibiotics. It therefore seemed likely that most of the patients were deprived of their natural bacterial flora by antibiotic treatment and subsequently colonized post-operatively with multiply-resistant coagulase-negative staphylococci from the environment.

Cardiac Surgical Procedures↗
PubMed1993

Induction of oxidative burst response in human neutrophils by adherent staphylococci. Comparison between Staphylococcus epidermidis and Staphylococcus aureus.

The ability of staphylococci adherent to silicone surfaces to induce superoxide anion (O2-) production by polymorphonuclear leukocytes (PMNs) was investigated and compared with the same activity induced by planktonic bacteria. The responses to Staphylococcus aureus strain E 2371 and Staphylococcus epidermidis strain ATCC 14990 were compared. The staphylococci were allowed to adhere to silicone catheters for 2 h at 37 degrees C. After opsonization of adherent bacteria in 30% human AB-positive serum, the induction of superoxide anion production by PMNs was measured in a cytochrome C reduction assay. Both bacterial strains, when adhered to the surfaces, were able to induce superoxide anion production by PMNs to about the same extent. Comparing adherent and planktonic bacteria with these two bacterial strains, it was found that planktonic S. epidermidis induced one to three times higher superoxide anion production than the adherent bacteria, whereas planktonic S. aureus induced four to seven times higher superoxide anion production than the adherent bacteria. Interstrain variation between the response to adherent and planktonic staphylococci was found. The lower phagocytic response to adherent staphylococci as compared to the response to planktonic organisms may interfere with the killing process and thereby contribute to poor clearance of these bacteria when adherent to foreign bodies such as catheters.

Humans↗
PubMed1995

Comparison of adherent and non-adherent staphylococci in the induction of polymorphonuclear leukocyte activation in vitro.

The ability to consume complement and activate neutrophils was investigated for staphylococci adherent to silicone surfaces and non-adherent staphylococci. Staphylococcus epidermidis strain ATCC 14990 and Staphylococcus aureus strain E 2371 were used in this study. The bacteria were allowed to adhere to silicone catheter segments for 2 h at 37 degrees C. Complement consumption was measured by reduction in serum haemolytic activity against sheep red blood cells. The induction of chemiluminescence was measured after opsonization of the staphylococci in 20% AB-positive human serum for 60 min at 37 degrees C. The bacteria consumed complement to approximately the same extent when adherent to the catheter segments, but more slowly in comparison with planktonic bacteria. When planktonic bacteria were compared, complement was consumed more quickly by S. epidermidis than by S. aureus. Measuring the induction of chemiluminescence by planktonic bacteria, S. epidermidis induced a lower response than S. aureus, while when adherent to the catheter segments the bacteria induced similar responses. These responses were only 15 to 20% of those induced by planktonic bacteria and only slightly higher than the spontaneous chemiluminescence by the neutrophils. Inter-strain variation was found, but all strains induced about the same low chemiluminescence when adherent to the catheter segments. The reduction in inflammatory response caused by adherence of staphylococci to catheter segments may interfere with phagocytosis and elimination of S. epidermidis during the early establishment of a foreign body infection.

Bacterial Adhesion↗
PubMed1987

Prevalence of macrolides-lincosamides-streptogramin B resistance and erm gene classes among clinical strains of staphylococci and streptococci.

A total of 332 staphylococcal and 263 streptococcal isolates from three hospital microbiology laboratories were tested with erythromycin, clindamycin, and vernamycin B alpha to determine the prevalence of macrolides-lincosamides-streptogramin B resistance. Constitutive resistance was detected in 28 Staphylococcus aureus isolates (15.5%), 53 coagulase-negative staphylococci (35.1%), and 20 streptococci (7.6%). Inducible resistance was observed in 13 S. aureus isolates (7.2%), 25 coagulase-negative staphylococci (16.6%), and 2 streptococci (0.8%). Eleven coagulase-negative staphylococci (7.3%) exhibited a novel phenotype, namely inducible resistance to erythromycin and vernamycin B alpha but not clindamycin. Among the staphylococci, two variants of the inducible phenotype detected with the agar diffusion assay correlated with the presence of classical ermA or ermC genes, respectively, by dot-blot analysis. The prevalence of the staphylococcal phenotypes were different in the hospitals surveyed, and there was an apparent inverse correlation between the resistance observed and the use of erythromycin in each hospital.

Anti-Bacterial Agents↗
PubMed1991

Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction.

A rapid method for the detection of methicillin resistance in staphylococci was developed. The method was based on the polymerase chain reaction (PCR) and primers that targeted the internal region of the coding frame of the mec gene. The amplification reaction was carried out with crude cell lysates as a source of target DNA and provided data in less than 5 h. Seventy-four isolates of coagulase-negative staphylococci were tested by PCR, DNA hybridization with a probe derived from the mec gene, and an agar dilution susceptibility assay. PCR results showed a 100% correlation with the susceptibility assay carried out with high inocula (10(8) CFU) and incubation at 32 degrees C for 48 h. PCR was more sensitive and specific than DNA hybridization in detecting methicillin resistance in coagulase-negative staphylococci. The former technique identified the mec gene in all the strains which were phenotypically resistant but which did not hybridize with the probe. Identification of methicillin-resistant strains by PCR offers a very specific, sensitive, and rapid alternative to traditional susceptibility tests and DNA hybridization as a guide for the treatment of infections caused by staphylococci.

Coagulase↗
PubMed2004

In vitro activity of the new quinolone WCK 771 against staphylococci.

The activity of WCK 771, an experimental quinolone developed to overcome quinolone resistance in staphylococci and other bacteria, was determined against quinolone-susceptible and -resistant Staphylococcus aureus and S. epidermidis. WCK 771 MICs for 50 and 90% of the strains tested (MIC(50) and MIC(90), respectively) were 0.008 and 0.015 microg/ml for S. aureus (n = 43) and 0.015 and 0.03 microg/ml for S. epidermidis (n = 44) for quinolone-susceptible isolates, compared to ciprofloxacin values of 0.12 and 0.25 microg/ml and 0.25 and 0.5 microg/ml, respectively. Values for levofloxacin were 0.12 and 0.25 microg/ml and 0.12 and 0.25 microg/ml, those for clinafloxacin were 0.015 and 0.03 microg/ml and 0.015 and 0.03 microg/ml, those for moxifloxacin were 0.03 and 0.06 microg/ml and 0.06 and 0.12 microg/ml, and those for gatifloxacin were 0.06 and 0.12 microg/ml and 0.12 and 0.25 microg/ml, respectively. The WCK 771 MIC(50) and MIC(90), respectively, were 0.5 and 1 microg/ml for both species of staphylococci (n = 73 for S. aureus, n = 70 for S. epidermidis) for isolates highly resistant to ciprofloxacin (MIC(50) and MIC(90), >32 and >32 microg/ml, respectively). Values for levofloxacin were 8 and 32 microg/ml and 8 and 32 microg/ml, those for clinafloxacin were 1 and 2 microg/ml and 0.5 and 2 microg/ml, those for moxifloxacin 4 and >4 microg/ml and 4 and >4 microg/ml, and those for gatifloxacin were 4 and >4 microg/ml and 2 and >4 microg/ml, respectively. WCK 771 and clinafloxacin demonstrated MICs of 1 microg/ml against three vancomycin-intermediate strains. WCK 771 showed concentration-independent killing for up to 24 h at 2, 4, and 8 times the MICs against quinolone-resistant staphylococci and was also bactericidal after 8 h for high-density inocula (10(8) CFU/ml) of quinolone-resistant strains at 5 microg/ml, whereas moxifloxacin at 7.5 microg/ml was bacteriostatic. WCK 771 was not a substrate of the NorA efflux pump as evident from the similar MICs against both an efflux-positive and an efflux-negative strain. Overall, WCK 771 was the most potent quinolone tested against the staphylococci tested, regardless of quinolone susceptibility.

Anti-Bacterial Agents↗
PubMed1964

STAPHYLOCOCCI IN COMPETITION. III. INFLUENCE OF PH AND SALT ON STAPHYLOCOCCAL GROWTH IN MIXED POPULATIONS.

Previous results showed definite repressive effects on the growth of staphylococci in mixed cultures due to the competitive growth of psychrophilic saprophytes. This study was continued, and the influence of other environmental factors, pH and salt, on the competition between staphylococci and saprophytes was investigated. Initial pH values varied from 5 to 9. At the extremes of the pH range, staphylococci failed to grow, while the saprophytes grew under all of the conditions tested. At pH 5, the growth curves for the saprophytes were markedly altered from those obtained at neutral pH. The lag phases were greatly lengthened at and below 20 C, but normal numbers of saprophytes were reached in the stationary phase. At pH 6 and 8, staphylococcal growth showed the same inhibition observed at pH 7, at and below 20 C; normal multiplication was observed above this temperature, but with accelerated death phases. Thus, pH did not primarily effect staphylococcal growth through its influence on saprophyte growth and competition, but rather directly affected the growth of Staphylococcus cultures. Salt concentrations from 3.5 to 9.5% were investigated for influence on staphylococcal growth in mixed populations. Above 3.5% salt, staphylococcal inhibition at and above 20 C was not as marked as in the controls, although normal numbers were never reached. The saprophytes were increasingly inhibited, and their lag phases materially lengthened as salt concentration was increased. Salt acted directly on the Staphylococcus population and also, by repressing saprophyte growth, decreased competition, which allowed the staphylococci to grow.

Cold Temperature↗
PubMed1964

STAPHYLOCOCCI IN COMPETITION. IV. EFFECT OF STARCH AND KIND AND CONCENTRATION OF SUGAR ON STAPHYLOCOCCAL GROWTH IN MIXED POPULATIONS.

Foods containing large amounts of carbohydrate have frequently been involved in staphylococcal food poisoning. Custard has been considered to be a highly favorable culture medium for staphylococci; however, it may be a selective medium rather than an ideal one. The influence of dextrose, lactose, and sucrose in varying amounts from 0.25 to 18%, and of starch, on the growth of staphylococci in mixed populations with saprophytes was determined. The inhibitory effect of the sugars was much greater on the saprophyte population than on the staphylococci. Of the three sugars, sucrose was most inhibitory to the saprophytes. It greatly decreased their lag periods as the concentration of sugar increased. Dextrose was the least inhibitory; in fact, 0.5% dextrose gave considerable stimulus to saprophyte growth. This sharply repressed staphylococcal growth. Lactose occupied an intermediate position. Rapid onset of the death phase of the staphylococci was observed in all increased sugar concentrations and seemed to be a pH effect rather than a result of competition. Sucrose exerted an inhibitory effect on the growth of saprophytes at and above room temperature. In the presence of 2.5% corn starch, staphylococcal growth in mixed cultures was slightly inhibited, while the death phase was sharply accelerated. Thus, carbohydrates exert their influence on staphylococcal growth in mixed cultures through their effect on the saprophytes by decreasing or increasing competition.

Carbohydrates↗
PubMed1965

Bacteriophage types and antibiotic sensitivity of staphylococci from bovine milk and human nares.

The number, phage types, and antibiotic sensitivity of coagulase-positive staphylococci from grade A raw milk samples produced on 40 farms in the Athens, Ga., milkshed were determined. Counts of mannitol-positive staphylococci in milk ranged from 100 to 3,580 per milliliter, with an arithmetic mean of 1,047. Examination of the nares of 48 dairymen on 34 of the farms also revealed that 13 (27%) were carriers of coagulase-positive staphylococci. Isolates from milk (412) and from nares (39) were tested against the Coles, Seto-Wilson, and International phage sets and 87, 68, and 56%, respectively, proved typable. Nine isolates were not typable. Each of the 33 phages used lysed one or more of the isolates. Staphylococcal phage types per milk sample ranged from 0 to 5, 0 to 7, and 0 to 8, with arithmetic means of 1.9, 2.3, and 2.3, respectively. Of the 13 narial carriers, 7 harbored staphylococci of one or more of the same phage types as those isolated from the milk at the respective farms. Randomly selected isolates were tested against high and low concentrations of 12 common antibiotics. All were either moderately sensitive or resistant to polymixin B. Over 30% were moderately sensitive or resistant to dihydrostreptomycin and penicillin individually. With but few exceptions, all isolates were sensitive to chlortetracycline, bacitracin, carbomycin, chloramphenicol, erythromycin, neomycin, novobiocin, oxytetracycline, and tetracycline individually.

Animals↗
PubMed1968

Lysozyme production as an aid for identification of potentially pathogenic strains of staphylococci.

Lysozyme production is a frequent property of potentially pathogenic staphylococci. In the present study, 1,186 strains of human origin, 85 strains of animal origin, and 156 strains of Staphylococcus albus (epidermidis) were tested. Of 1,114 coagulase-positive strains of human and animal origin, 1,098 were lysozyme-positive (98.5%). On the other hand, of 157 coagulase-negative strains which, based on further investigations, belong to the potentially pathogenic staphylococci, all were lysozyme-positive. All of the 156 strains (100%) belonging to the species S. albus (epidermidis) were lysozyme-negative. We conclude that lysozyme production is a better index of potentially pathogenic staphylococci than the measurement of free coagulase, especially in cases of strains of animal origin. It is possible that lysozyme production allows a differentiation between pathogenic and nonpathogenic coagulase-negative staphylococci.

Animal Population Groups↗
PubMed1972

Use of shake cultures in a semisolid thioglycolate medium for differentiating staphylococci from micrococci.

The standard diagnostic test for differentiating staphylococci from micrococci is based on the ability of the former to produce acid anaerobically in a glucose-containing growth medium. This test has been modified to provide greater convenience, easier interpretation of results, and better correlation with deoxyribonucleic acid (DNA) base composition. In the modified test, shake cultures in Brewer's fluid thioglycolate medium with 0.3% agar added are observed for growth in the anaerobic zone of the tubes. This test was applied to 125 strains of staphylococci and micrococci, and all except two strains gave results that were consistent with other criteria. Of particular interest were eight strains of Micrococcus saprophyticus and three strains of M. lactis that have a DNA composition of 30 to 37% guanine plus cytosine (GC). All 11 of these cultures produced anaerobic growth and thus would be classified as staphylococci. Strains of M. lactis that have a high GC content in their DNA grew only aerobically. Some cultures of staphylococci produced characteristic band patterns of anaerobic growth and other cultures produced only a few anaerobic colonies from an inoculum of 10(6) to 10(7) cells. These observations suggest some interesting genetic and metabolic capabilities in such cultures.

Aerobiosis↗
PubMed1974

Slide agglutination method for the serological identification of Neisseria gonorrhoeae with anti-gonococcal antibodies adsorbed to protein A-containing staphylococci.

A rapid slide agglutination test has been developed for the identification of Neisseria gonorrhoeae that are primarily detected as oxidase-positive colonies in gonococcal cultures. The technique is based on the specific nonimmune reactivity between the Fc portion of immunoglobulin (Ig)G and staphylococcal protein A. IgG molecules adsorbed to stabilized staphylococci will thereby become oriented with their antigen-reactive sites that are directed outwards. Protein A-containing staphylococci with unabsorbed anti-gonococcal antibodies gave positive co-agglutination reactions with gonococci but also with meningococci, some Moraxella, Haemophilus, and Pseudomonas strains. These crossreactions were eliminated by absorption of the anti-gonococcal antiserum with meningococcal and Moraxella organisms prior to the coating of reagent staphylococci. In the routine culture diagnosis of N. gonorrhoeae the use of specific gonococcal reagent staphylococci gave concordant results with fermentation procedures and immunofluorescent techniques.

Adsorption↗
PubMed1988

Methicillin-resistant staphylococci.

Strains of staphylococci resistant to methicillin were identified immediately after introduction of this drug. Methicillin-resistant strains have unusual properties, the most notable of which is extreme variability in expression of the resistance trait. The conditions associated with this heterogeneous expression of resistance are described. Methicillin resistance is associated with production of a unique penicillin-binding protein (PBP), 2a, which is bound and inactivated only at high concentrations of beta-lactam antibiotics. PBP2a appears to be encoded by the mec determinant, which also is unique to methicillin-resistant strains. The relationships between PBP2a and expression of resistance and implications for the mechanism of resistance are discussed. The heterogeneous expression of methicillin resistance by staphylococci poses problems in the detection of resistant strains. Experience with several susceptibility test methods is reviewed and guidelines for performance of these tests are given. Treatment of infections caused by methicillin-resistant staphylococci is discussed. Vancomycin is the treatment of choice. Alternatives have been few because methicillin-resistant strains often are resistant to multiple antibiotics in addition to beta-lactam antibiotics. New agents which are active against methicillin-resistant staphylococci are becoming available, and their potential role in treatment is discussed.

Bacteriological Techniques↗