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Evaluation of different disk diffusion/media combinations for detection of methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci.

In order to find a disk diffusion method with both high sensitivity and specificity for determination of methicillin resistance primarily for S. aureus but also for coagulase-negative staphylococci we screened several methodological variants using a material of 66 S. aureus comprising of 11 methicillin-susceptible, 18 borderline-resistant, and 37 methicillin-resistant strains. Only four of the combinations studied performed with both high sensitivity and specificity. Two of these, the Columbia agar +4.5% NaCl and Mueller Hinton agar +2% NaCl combined with a 5 microg oxacillin disk, confluent inoculum and 24 h incubation at 35 degrees C were further evaluated using 105 MRSA and 91 mecA-negative S. aureus and 193 clinical isolates of coagulase-negative staphylococci. The Columbia agar +4.5% NaCl performed excellently for both S. aureus and coagulase-negative staphylococci. For Columbia agar +4.5% NaCl using a 5 microg oxacillin disk we suggest an interpretive zone diameter of R < or =15 mm and S > or =16 mm for S. aureus and R < or =24 mm and S >or =26 mm for coagulase-negative staphylococci. The Mueller Hinton agar +2% NaCl performed well for coagulase-negative staphylococci but for S. aureus at least three (3%) very major errors were found, making this method less attractive.

Coagulase↗

Rapid detection of the methicillin-resistance gene, mecA, in coagulase-negative Staphylococci.

Phenotypical methods are routinely used to detect methicillin resistance in Staphylococci. These methods are time-consuming and there are difficulties in detecting all resistant strains carrying the mecA gene. We detected methicillin-resistant Staphylococci in biological samples by PCR amplification of mecA, without the time-consuming step of identifying a bacterial isolate. Methicillin-resistant Staphylococci isolates were also detected by screening on agar supplemented with oxacillin. The biological samples were collected from the hands of 17 healthcare workers at the Department of Paediatrics at the University Hospital of Tromsø. mecA was amplified in 12 of the 17 samples. The gene was verified by DNA sequencing of the PCR amplicon. Using the phenotypical method, methicillin-resistant Staphylococci were isolated from 6 of the samples. In all 6 of these samples, mecA was amplified by PCR. We conclude that PCR is a sensitive and specific method for detecting methicillin resistance in Staphylococci. The PCR detection of mecA is rapid, fairly simple and can easily be assimilated into the routines of a clinical microbiological laboratory.

Bacterial Proteins↗

New insights in the pathogenesis of foreign body infections with coagulase negative staphylococci.

Foreign body infections by coagulase negative Staphylococci are an important and growing problem in our hospitals. Only recently have we started to get some data on the specific virulence factors that permit the otherwise non-pathogenic Coagulase Negative Staphylococci (CNS) to be so successful in causing foreign body infections. Adherence of the Coagulase Negative Staphylococci to the foreign body is a first and crucial step. Several genes and gene-products have been identified that enhance staphylococcal adherence to biomaterials. Adherence is followed by accumulation; in this phase the Coagulase negative Staphylococci organise themselves into a complex multilayer of cells covered with polysaccharide. This we call the biofilm. Finally coagulase negative Staphylococci undergo complex and as yet non-defined metabolic changes that in combination with biofilm formation allow them to persist on the foreign body and become less susceptible to antibiotics. Few data are available on the factors involved in the accumulation and persistence phase.

Bacterial Adhesion↗

Fate of staphylococci within human leukocytes.

Direct observations by phase microscopy have demonstrated that small numbers of pathogenic staphylococci survive prolonged periods of time within living human polymorphonuclear leukocytes. Non-pathogenic microorganisms are rapidly destroyed in similar preparations. Leukocytes in which staphylococci remained viable often appeared less vigorous after ingesting microorganisms, but intracellular survival could not be correlated with obvious leukocyte damage with any consistency. Both pathogenic and non-pathogenic cocci were seen to divide within living granulocytes during the first few minutes after ingestion. Occasionally pathogenic staphylococci multiplied in dying cells after long periods of intracellular residence. Phagocytosis of more than one pair of staphylococci by a single leukocyte appeared to act as a stimulus to bacterial destruction. Multiple ingestions of pathogenic staphylococci reduced the incidence of survival of the total microbiol population contained within the cell.

Granulocytes↗

Bacteremia caused by staphylococci with inducible vancomycin heteroresistance.

The clinical significance of bacteremia due to vancomycin-heteroresistant staphylococci and a rapid laboratory screening method were examined; 203 strains of staphylococci isolated from patients with clinically significant bacteremia were screened by the disk-agar method with use of vancomycin-salt agar to demonstrate satellitism around an aztreonam disk as well as by conventional population screening. Eighteen isolates (three Staphylococcus aureus and 15 coagulase-negative staphylococci) were shown to be heteroresistant to vancomycin. A case-control clinical study showed that the interval between admission and bacteremia, admission to the intensive care unit, prior use of vancomycin and/or beta-lactams, and isolation of methicillin-resistant staphylococci were significantly more common among patients with bacteremia due to staphylococci with heteroresistance to vancomycin; these patients had an overall mortality of 44.4%. The use of vancomycin and admission to the intensive care unit were independently significant risk factors on multivariate analysis. Vancomycin heteroresistance is inducible by salt and beta-lactams. Indiscriminate sequential use of beta-lactams and glycopeptides may facilitate the emergence of glycopeptide resistance.

Adolescent↗

Patterns of multidrug resistance among methicillin-resistant hospital isolates of coagulase-positive and coagulase-negative staphylococci collected in the international multicenter study RESIST in 1997 and 1998.

The primary purpose of the multicenter international study "RESIST" was to obtain an update on the degree of multidrug resistance among methicillin-resistant staphylococci collected from a geographically diverse sample. A total of 3,307 staphylococcal isolates were recovered from single patients and primarily from clinical specimens that were collected at 20 collaborating regional health centers located in several countries in Europe, Asia, and Latin America during a 3- to 4-month period each in 1997 and 1998. All strains were deposited at the Laboratory of Molecular Genetics at ITQB/UNL in Oeiras, Portugal, for quality control and for testing by microbiological and molecular typing techniques; the Laboratory of Microbiology at The Rockefeller University serving as organizational center. The majority of strains, 3,100, were methicillin-resistant, of which 1,749 were coagulase positive (methicillin-resistant Staphylococcus aureus, MRSA), and 1,351 were coagulase negative (methicillin-resistant coagulase negative staphylococci, MRCNS). The overall frequency of drug resistance traits among the 1,749 MRSA strains was high (over 70% and up to and over 90% of the strains) to ciprofloxacin, erythromycin, clindamycin, gentamicin, and tetracycline, and was somewhat less frequent to sulfamethoxazole-trimethoprim (45%), chloramphenicol (30%), and rifampin (38%). None of the 3,307 staphylococcal isolates showed reduced susceptibility to vancomycin except for a single methicillin-resistant coagulase-negative isolate. The great majority of staphylococci were also susceptible to the new antimicrobial Synercid. In contrast, resistance to teicoplanin was significant among methicillin-resistant strains of coagulase-negative staphylococci, particularly among Staphylococcus haemolyticus. MRSA isolates showed marked geographic variation in their patterns of multiresistance, most likely reflecting the properties of unique multiresistant MRSA clones dominant in the hospitals that provided the MRSA isolates from the various geographic areas. The multiresistance patterns of MRSA strains and strains of methicillin-resistant coagulase-negative staphylococci originating at the same country source also showed striking differences, suggesting that resistance to antimicrobial agents emerged under different antibiotic pressures in these bacterial species.

Anti-Bacterial Agents↗

Long-term studies of the effect of rifampin on nasal carriage of coagulase-positive staphylococci.

Eighty healthy men with nasal carriage of coagulase-positive staphylococci were given cloxacillin, rifampin, cloxacillin plus rifampin, or no drug. Treatment with rifampin alone or with rifampin plus cloxacillin eradicated the original strain from 80% of the men for 12 weeks after treatment; 15%-20% of the 80 subjects acquired a strain of a different phage type. Treatment with cloxacillin alone did not significantly alter the rate of carriage. Only one of 40 men receiving rifampin (either alone or with cloxacillin) acquired rifampin-resistant, coagulase-positive staphylococci; seven of the 40 acquired rifampin-resistant, coagulase-negative staphylococci. Rifampin-resistant organisms did not persist in repeat nasal cultures five months after treatment. Of the 40 subjects given rifampin alone or rifampin plus cloxacillin, 60% had repeat nasal cultures done one year later. Only 50% were found to be carriers of coagulase-positive staphylococci. In another study, coagulase-positive staphylococci were eradicated from 70% of 20 carriers undergoing renal dialysis who received rifampin plus cloxacillin.

Carrier State↗

Resistance of staphylococci in The Netherlands: surveillance by an electronic network during 1989-1995.

An electronic surveillance network for monitoring antibiotic resistance in The Netherlands has been in operation since 1989. Seven public health laboratories participate and the system covers about 25% of all bacteriological determinations in The Netherlands. This paper reports the results of staphylococci isolated in the period 1989-1995. About 0.3% of the Staphylococcus aureus isolates in the study period were resistant to methicillin. This low percentage may be due to the restrictive use of antibiotics and to strict isolation measures aimed at eradicating methicillin-resistant S. aureus. Low frequencies of resistance among methicillin-resistant S. aureus were found for vancomycin (0%), chloramphenicol (11%), cotrimoxazole (11%), mupirocin (3% low-level resistance) and fusidic acid (7%). Twenty-one percent of the coagulase-negative staphylococci were resistant to methicillin. Low frequencies of resistance among these methicillin-resistant coagulase-negative staphylococci were those to vancomycin (0.4%), nitrofurantoin (2%), doxycycline (20%) and amikacin (20%). Coagulase-negative staphylococci from cerebrospinal fluid, blood and skin were less often resistant to quinolones than isolates from respiratory tract, faeces and urine. A significant increase in resistance of coagulase-negative staphylococci to methicillin, erythromycin, gentamicin and ciprofloxacin was observed in the investigated period but the resistance to doxycycline and co-trimoxazole decreased in the last few years. To confirm the determination of methicillin resistance and coagulase production, a PCR method was developed which detects both the mecA and the coagulase gene. The results of the PCR method correlated well with the methicillin MIC as determined by an agar-dilution method.

Anti-Bacterial Agents↗

Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. The ESPRIT Trial.

Multiresistant staphylococci (82 Staphylococcus aureus and 114 coagulase-negative staphylococci) were characterized by testing with rapid multiplex polymerase chain reaction (PCR) assays for species identification and detection of associated antibiotic resistance genes. These 196 staphylococci were isolated from 149 adult patients who developed wound infection after elective coronary artery bypass grafts and/or valve surgery. The multiplex PCR assays allowed identification of the most common staphylococcal species with S. aureus- and Staphylococcus epidermidis-specific primers as well as the detection of the erythromycin resistance genes ermA, ermB, ermC and msrA, the aminoglycoside resistance gene aac(6')-aph(2"), the oxacillin resistance gene mecA and the penicillin resistance gene blaZ. There was a very good correlation between the genotypic analysis by PCR and the phenotype determined by standard methods of susceptibility testing and identification of staphylococcal species: 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin resistance and 100% for S. aureus and S. epidermidis species identification. This study suggests that the incidence and distribution of the tested clinically relevant antibiotic resistance genes in staphylococci associated with infections after cardiac surgery do not differ from those in strains from other infections. These multiplex PCR assays may be used as diagnostic tools to replace or complement standard methods of susceptibility testing and identification of staphylococci.

Adult↗

The antibacterial activity of honey against coagulase-negative staphylococci.

OBJECTIVES: Development of antibiotic-resistant strains of coagulase-negative staphylococci has complicated the management of infections associated with the use of invasive medical devices, and innovative treatment and prophylactic options are needed. Honey is increasingly being used to treat infected wounds, but little is known about its effectiveness against coagulase-negative staphylococci. The aim of this study was to determine the minimum active dilution of two standardized, representative honeys for 18 clinical isolates of coagulase-negative staphylococci. METHODS: An agar incorporation technique was used to determine the minimum active dilution, with dilution steps of 1% (v/v) honey [or steps of 5% (v/v) of a sugar syrup matching the osmotic effect of honey]. The plates were inoculated with 10 microL spots of cultures of the isolates. RESULTS: The honeys were inhibitory at dilutions down to 3.6 +/- 0.7% (v/v) for the pasture honey, 3.4 +/- 0.5% (v/v) for the manuka honey and 29.9 +/- 1.9% (v/v) for the sugar syrup. CONCLUSIONS: Typical honeys are about eight times more potent against coagulase-negative staphylococci than if bacterial inhibition were due to their osmolarity alone. Therefore, honey applied to skin at the insertion points of medical devices may have a role in the treatment or prevention of infections by coagulase-negative staphylococci.

Anti-Bacterial Agents↗

Toxic shock syndrome associated with TSST-1 producing coagulase-negative staphylococci.

Coagulase-positive staphylococci have been isolated from more than 95% of cases of toxic shock syndrome (TSS). Toxic shock syndrome toxin-1 (TSST-1) is produced in more than 90% of cases. A case is described of recurrent TSS in which a coagulase-negative staphylococcus was isolated from the vagina and produced TSST-1. No coagulase-positive staphylococci were isolated. Coagulase-negative staphylococci should be considered as causative agents in cases of TSS. Assay for TSST-1 should be performed on coagulase-negative staphylococci obtained from the appropriate clinical sites if no coagulase-positive staphylococci are isolated.

Adult↗

Application of current methods for isolation and identification of staphylococci in raw bovine milk.

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.

Animals↗

Activity of cephalosporins against methicillin-susceptible and methicillin-resistant, coagulase-negative staphylococci: minimal effect of beta-lactamase.

Eight cephalosporins were tested for their activity against methicillin-susceptible and methicillin-resistant, coagulase-negative staphylococci and for their resistance to beta-lactamase from methicillin-resistant, coagulase-negative staphylococci. Susceptibility testing by the agar plate method was evaluated for the effect of inoculum size and duration of incubation. Methicillin-susceptible, coagulase-negative staphylococci were highly susceptible to the cephalosporins, with cephapirin and cepahlothin showing the greatest activity, followed by cefazolin and cefamandole. Methicillin-resistant, coagulase-negative staphylococci displayed nearly total cross-resistance to the cephalosporins. Resistance increased with increasing inoculum size. Beta-Lactamases produced by methicillin-resistant, coagulase-negative staphylococci had a minimal hydrolytic effect on cepahlothin, cephapirin, cefazolin, and cefamandole and no measurable effect on cefoxitin. There was no correlation between the anti-staphylococcal activity and resistance to beta-lactamases.

Cephalosporins↗

In vitro susceptibilities of four species of coagulase-negative staphylococci.

The in vitro susceptibilities of 260 strains of coagulase-negative staphylococci to penicillin G, oxacillin, nafcillin, methicillin, cephalothin, and seven non-beta-lactam antimicrobial agents were determined and compared with the susceptibilities of 54 strains of Staphylococcus aureus with known patterns of susceptibility. Penicillin G susceptibility for S. aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis was readily determined by using beta-lactamase tests with induced cells and with a standardized microdilution test. MIC criteria for susceptibility used for S. aureus were applicable to the coagulase-negative species. Percentages of organisms susceptible were as follows: S. epidermidis, 7%; S. haemolyticus, 5%; and S. hominis, 47%. Oxacillin susceptibility for these four species was readily determined by using a modification of the microdilution test. MIC criteria for susceptibility used for S. aureus were applicable to S. haemolyticus and S. hominis, but alternate criteria were necessary for S. epidermidis. Percentages of organisms susceptible were as follows: S. epidermidis, 29%; S. haemolyticus, 36%; and S. hominis, 97%. Staphylococcus saprophyticus differed from the other staphylococcal species; all strains were beta-lactamase negative and were penicillin susceptible but had higher penicillin G MICs than did susceptible strains of the other species. There was total cross resistance among the penicillinase-resistant penicillins and cephalothin for the coagulase-negative staphylococci as well as for S. aureus; oxacillin MICs were more reliable than MICs of the other drugs or a standardized disk diffusion test for distinguishing resistant from susceptible strains. Vancomycin, rifampin, and ciprofloxacin were consistently active against all staphylococci. Erythromycin, clindamycin, gentamicin, and trimethoprim-sulfamethoxazole were more active against oxacillin-susceptible staphylococci than against oxacillin-resistant staphylococci.

Anti-Bacterial Agents↗

Detection of methicillin resistance in staphylococci by using a DNA probe.

A DNA probe derived from the PBP 2a gene of the methicillin-resistant Staphylococcus aureus COL was compared with phenotypic microbiologic tests for its ability to identify methicillin-resistant and -susceptible staphylococci. Lysates were applied to nitrocellulose with a dot blot apparatus. Isolates tested were both S. aureus and coagulase-negative staphylococci that had been recovered from a variety of geographic and clinical sources. When compared with a spread plate phenotypic test, the DNA probe gave sensitivity, specificity, and predictive values for both positive and negative tests of 100% for 204 S. aureus isolates (103 positive, 101 negative) and 99, 95, 99, and 95%, respectively, for 249 coagulase-negative staphylococci (210 positive, 39 negative). The probe was more sensitive than broth microdilution and more specific than agar dilution in identifying methicillin-resistant and -susceptible coagulase-negative staphylococci; all tests were equally accurate in identifying the methicillin susceptibility of S. aureus. DNA probe analysis for determining the methicillin susceptibility of staphylococci was rapid, easily interpretable, and equally accurate with radioactive and nonradioactive probes, and it gave results equivalent to the most sensitive microbiologic test for all staphylococcus species studied.

Bacterial Proteins↗

Some effect of subinbilitory concentrations of penicillin on the structure and division of staphylococci.

A strain of Staphylococcus aureus was planted on filter membranes placed on Trypticase soy agar (BBL). After incubation, the membranes with growing staphylococci were transferred to Trypticase soy agar containing a subinhibitory concentration of penicillin (one-third minimal inhibitory concentration) and again incubated. The membranes were then returned to drug-free agar and incubated once more. Counts of the colony-forming units and electron microscopy were carried out at several time intervals. When grown on agar containing penicillin, the staphylococci formed what appeared to be abnormally large cells with multiple and unusually thick septa. Examination of a number of sections showed that these large cells were in reality clusters of staphylococci that had divided but failed to separate. When these large cells were subsequently grown on drug-free agar, smaller cells and normal staphylococci emerged. Subinhibitory concentrations of penicillin do not kill staphylococci; they seem to inhibit lysis of cross walls, preventing the separation of otherwise divided cells.

Cell Division↗

Production of lysozyme by staphylococci and its correlation with three other extracellular substances.

Jay, James M. (Wayne State University, Detroit, Mich.). Production of lysozyme by staphylococci and its correlation with three other extracellular substances. J. Bacteriol. 91:1804-1810. 1966.-Lysozyme production was determined on plates containing 1 mg/ml of Lysozyme Substrate in Heart Infusion Agar with incubation at 37 C for 48 hr. Its production was compared with that of alpha-hemolysin and sheep hemolysin and egg-yolk precipitation, by use of both coagulase-positive and coagulase-negative strains of staphylococci. Of 126 coagulase-positive strains tested, 120 or 95.2% produced lysozyme, 117 or 92.9% produced alpha-hemolysin, 108 or 85.7% precipitated egg yolk, and 102 or 81% produced sheep hemolysin. Of the 49 coagulase-negative strains (which included 22 pathogens), only 4 or 8.1% produced lysozyme, 14 or 28.6% produced alpha-hemolysin, 13 or 26.5% produced sheep hemolysins, and 5 or 10.2% precipitated egg yolk. Only two of the six coagulase-positive strains which failed to produce lysozyme showed any consistent patterns in relation to the four characteristics determined. The four coagulase-negative strains which produced lysozyme were inconsistent for the other characteristics measured. It is suggested that lysozyme production is more a property of coagulase-positive staphylococci, and therefore a better ancillary test of pathogenicity, than either production of alpha-hemolysin or egg-yolk precipitation, because the incidence of lysozyme producers is higher among this group than among those producing the other substances and because fewer coagulase-negative staphylococci produced lysozyme than hemolysins or egg-yolk precipitation. Of 16 other species of bacteria and yeasts tested, all were found negative except Bacillus subtilis. Lysozyme production by staphylococci in heavily contaminated foods was not inhibited on plates containing sodium azide, whereas media containing 7.5% salt and sorbic acid were unsuitable. The possible relationship of lysozyme production to staphylococcal pathogenicity is discussed.

Coagulase↗

Evaluation of the BD Phoenix automated microbiology system for identification and antimicrobial susceptibility testing of staphylococci and enterococci.

We evaluated the Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD) for the identification (ID) and antimicrobial susceptibility testing (AST) of challenge and clinical staphylococci and enterococci recovered from patients in a tertiary-care medical center. In total, 424 isolates were tested: 90 enterococci; 232 Staphylococcus aureus isolates, including 14 vancomycin-intermediate S. aureus isolates; and 102 staphylococci other than S. aureus (non-S. aureus). The Phoenix panels were inoculated according to the manufacturer's instructions. The reference methods for ID comparisons were conventional biochemicals and cell wall fatty acid analysis with the Sherlock microbial identification system (v 3.1; MIDI, Inc. Newark, DE). Agar dilution was the reference AST method. The overall rates of agreement for identification to the genus and the species levels were 99.7% and 99.3%, respectively. All S. aureus isolates and enterococci were correctly identified by the Phoenix panels. For the non-S. aureus staphylococci, there was 98.0% agreement for the ID of 16 different species. The AST results were stratified by organism group. For S. aureus, the categorical agreement (CA) and essential agreement (EA) were 98.2% and 98.8%, respectively. Three of three very major errors (VMEs; 1.7%) were with oxacillin. For non-S. aureus staphylococci, the CA, EA, VME, major errors, and minor error rates were 95.7%, 96.8%, 0.7%, 1.7%, and 2.9%, respectively. The two VMEs were with oxacillin. For the enterococci, there was 100% CA and 99.3% EA. All 36 vancomycin-resistant enterococci were detected by the Phoenix system. The Phoenix system compares favorably to traditional methods for the ID and AST of staphylococci and enterococci.

Anti-Bacterial Agents↗