A rapid latex agglutination assay for the detection of penicillin-binding protein 2'

A simple and rapid slide latex agglutination assay was developed to detect penicillin-binding protein 2' (PBP2') from isolates of staphylococi. PBP2' present in the membranes of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2' could be detected from a single loopful of cells taken from agar plates not containing beta-lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2' positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2' negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.     

Clumping of Staphylococcus aureus by human fibronectin

Clumping of different staphylococci by fibronectin and other purified plasma proteins has been investigated. Purified fibronectin was capable of clumping Staphylococcus aureus strains in concentrations identical with concentrations of fibronectin in human plasma. S. epidermidis and S saprophyticus were not clumped by fibronectin. The binding of fibronectin to S. aureus was not mediated by protein-A, as a strain lacking protein-A clumped in the presence of fibronectin, and the presence of IgG could not inhibit the clumping of S. aureus strains. The fibronectin-binding component on the staphylococcal cell wall seems to be unrelated to the fibrinogen-binding clumping factor.     

Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistant Staphylococcus aureus

Four thousand eighty-eight Staphylococcus aureus isolates obtained from patients hospitalised in a university clinic and four community hospitals over a period of one year were screened for methicillin resistance. A resistance rate of 5% was detected among initial isolates. Distribution of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus showed an increased prevalence of MRSA in clinically significant specimens such as blood, central venous catheter tips, bronchial secretions, and wound secretions. Typing of 110 MRSA strains (initial isolates) by macrorestriction analysis of chromosomal DNA revealed 26 different genotypes that could be divided into five epidemic and 21 sporadic strains. More than 50% of all isolates belonged to one type that was confirmed to be closely related to the "southern-German" epidemic strain. Production of virulence factors such as enterotoxin A-D and toxic shock syndrome-toxin 1 among MRSA strains (initial isolates) occurred in ten of 26 different MRSA types. A strong correlation between genotype and toxin production was demonstrated.     

Typing of Staphylococcus aureus and Staphylococcus epidermidis strains by PCR analysis of inter-IS256 spacer length polymorphisms

IS256 elements are present in multiple copies in the staphylococcal genome, either flanking the transposon Tn4001 or independent of it. PCR-based analysis of inter-IS256 spacer polymorphisms was developed for typing of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis strains. Using SmaI macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE) as the reference method for MRSA typing, excellent reproducibility (100%), discriminatory power (97%), and in vivo stability were observed. Good concordance of the results with those of other molecular typing methods was found for two MRSA collections. Inter-IS256 PCR analysis of a U.S. collection of MRSA strains (n = 36), previously characterized by 15 typing methods, showed more limited discrimination. Agreement was 78% with PFGE analysis and 83% with ribotyping (HindIII). Analysis of a second set of Belgian MRSA strains (n = 17), categorized into two widespread epidemic clones by PFGE analysis, showed 65% agreement. For typing of S. epidermidis strains (n = 26), inter-IS256 PCR showed complete typeability (100%) and good discriminatory power (85%). Inter-IS256 PCR analysis is proposed as an efficient molecular typing assay for epidemiological studies of MRSA or S. epidermidis isolates.     

Rostering: placing the nurse in the picture

The aim of the study was to find out whether methicillin-resistant S. aureus strains (MRSA) are tolerant in a higher degree to disinfectants, and whether a correlation exists between lower sensitivity to these agents and resistance to gentamicin. The study was carried out on 30 strains of MRSA and 20 of MSSA isolated from various clinical materials in various regions of the country. Among the studied MRSA 24 strains were resistant and 6 were sensitive to gentamicin, and in MSSA 3 strains were resistant and 17 sensitive to this antibiotic. The sensitivity to four disinfectants: Manusan, Sterinol, Septyl R and Lysoformin Spezial was determined by measurement of MIC (minimal inhibitory concentration) in agar medium. Most MRSA in Poland showed decreased sensitivity to these disinfectants. Among gentamicin-sensitive and resistant MRSA strains the proportions of strains with higher tolerance of three disinfectants (Manusan, Sterinol and Lysoformin Spezial) were very similar. Reduced sensitivity to disinfectants was found in all gentamicin-resistant MSSA. These data indicate that S. aureus strains possess various mechanisms of tolerance of disinfectants. Nearly half the studied strains (46%) had decreased sensitivity to all three preparations (Manusan, Sterinol and Lysoformin Spezial) belonging to various chemical groups this seems to indicate that increased tolerance to these disinfectants is a non-specific feature of S. aureus strains.     

Induction and measurement of antisperm antibody responses in vitro

We did a statistical study of 294 strains of Staphylococcus aureus (S. aureus) isolated from skin infections during the period from January of 1989 to December of 1991 in the Department of Dermatology, Kansai Medical University Hospital. We especially examined methicillin-resistant S. aureus (MRSA) from the point of view of incidence, variety of skin infections with MRSA, coagulase type, phase type, and resistance against antimicrobial agents. The frequency of isolation of MRSA has been increasing. In 1991, the proportion of MRSA isolates among all S. aureus strains isolated from skin infections was 41.5%. MRSA was isolated most often from infectious decubitus. Coagulase type II and phage group NT (not typable) MRSA were most frequently isolated. The resistance of MRSA to OFLX and IMP/CS had remarkably increased. Notably, the resistance to MINO was low before 1991.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy was studied in a five-month period in 1996, during which all S. aureus isolated were collected. All MRSA isolates (95) and a sample of methicillin-susceptible S. aureus (20) were typed with a variety of phenotypic and genotypic methods. Clonal identities were determined by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests and, for MRSA isolates, by probing ClaI digests with a mecA probe and a Tn554 probe. Overall, MRSA represented 32.3% of all isolates, with very high percentages from the intensive care units (adult and neonatal). PFGE after restriction with SmaI resolved genomic DNA of 95 MRSA strains into 26 major PFGE patterns. The use of southern blot hybridization of ClaI genomic digests with mecA and Tn554 allowed us a significant increase in discrimination, differentiating at least 32 different clones. Two major clones, however, each sharing common ClaI-mecA and Tn554 type and PFGE pattern as well as a common resistance phenotype, represented more than 50% of all MRSA isolates. The recovery of these two clones in the majority of the isolates of adult and neonatal intensive care units, respectively, is indicative of typical nosocomial outbreaks and clonal spread. It is concluded that intensive care units are major areas requiring preventative interventions.     

5-HT2A receptor subtype is involved in the thermal hyperalgesic mechanism of serotonin in the periphery

Quinupristin/dalfopristin (RP59500) is a novel streptogramin and a semisynthetic derivative of pristinamycins IA and IIB. The following properties of RP59500 were investigated: (i) its in-vitro activity against 164 hospital isolates of Staphylococcus aureus, 101 of which were methicillin-resistant (MRSA); (ii) its killing effect against 24 MRSA and seven methicillin-susceptible (MSSA) isolates; (iii) its interactions with rifampicin and ciprofloxacin against 18 MRSA isolates, six susceptible to both rifampicin and ciprofloxacin and 12 resistant to both, at 1 x MIC, 2 x MIC and 4 x MIC. Rifampicin and ciprofloxacin were applied at a concentration equal to their mean serum levels in order to establish the clinical relevance of the results. The MIC50, MIC90, MBC50 and MBC90 of quinupristin/dalfopristin were, respectively, < or = 0.015, 2, 0.12 and 2 mg/L for MRSA isolates and < or = 0.015, 0.06, < or = 0.015 and 0.25 mg/L for MSSA isolates. All isolates were inhibited by quinupristin/dalfopristin. Its killing effect varied with concentration and time, being optimal at 4 x MIC and after 24 h growth. Strains surviving 24 h exposure to this agent had much higher MICs than the parent strain, but only a limited number of them became resistant. Quinupristin/dalfopristin at 2 x MIC and 4 x MIC showed in-vitro synergy with rifampicin against highly resistant isolates mainly at 6 h and 24 h of growth involving 50-83% of MRSA isolates, and showed synergy with ciprofloxacin at 24 h involving 42-75% of isolates. The MIC increase in colonies surviving at 24 h was restricted by the presence of rifampicin or ciprofloxacin. In contrast, the above combinations acted synergically over the total number of MRSA strains susceptible to both rifampicin and ciprofloxacin. The above findings show that quinupristin/dalfopristin is a very potent antistaphylococcal agent, and that its activity against MRSA isolates is enhanced when it is combined with rifampicin or ciprofloxacin.     

Application of an optimized and highly discriminatory method based on arbitrarily primed PCR for epidemiologic analysis of methicillin-resistant Staphylococcus aureus nosocomial infections

The optimization of an arbitrarily primed PCR method for typing 96 methicillin-resistant Staphylococcus aureus (MRSA) isolates was compared with pulsed-field gel electrophoresis. Identical results in the differentiation of MRSA clones and identification of the main cluster that included 82 strains (88% of patients) were obtained by both techniques.     

A contingency-based approach for assessing policies on aging

The analysis of genomic DNA fragment patterns has revealed as a powerful tool for strain discrimination in Staphylococcus aureus; for use as an epidemiological marker, stability during the course of an outbreak is an essential prerequisite. Genomic DNA fragment patterns (SmaI restriction, pulsed-field electrophoresis) of four different epidemic MRSA strains were compared along with intra- and interhospital and country-wide spread over more than 12 months in Germany. Strain I was isolated from infections in 8 hospitals. In one hospital a subclone arised which differed from the original strain by 4 fragments. Strain II was spread among 4 hospitals, isolates from three of these hospitals exhibited a variability of one to three fragments in the 150-200 kb range. Two hospitals in the Hannover-area were affected by strain III; in 17 isolates of this strain a variability up to three fragments was found in the 170-200 kb range. Strain IV was isolated from 19 cases of infections in 3 hospitals in Berlin. The fragment patterns were completely stable. When S. aureus strains are typed by genomic DNA fragment patterns, a variability in a definite range of molecular masses during the course of an epidemic should be taken into consideration.     

Intercontinental spread of a multidrug-resistant methicillin-resistant Staphylococcus aureus clone

Two hundred ten methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered between 1990 and 1997 from three Portuguese hospitals located in Lisbon and Oporto were analyzed by molecular fingerprinting techniques. The hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes combined with pulsed-field gel electrophoresis documented the abrupt appearance and extensive intrahospital spread of the Brazilian epidemic MRSA clone in the 1995 samples of each one of the three hospitals analyzed-suggesting the intercontinental transfer of this strain from Brazil to Portugal. The appearance of this clone may challenge the dominance of another highly epidemic imported clone-the Iberian MRSA, currently the most widely spread MRSA clone in Portuguese hospitals.     

Production of A and C variants of staphylococcal beta-lactamase by methicillin-resistant strains of Staphylococcus aureus

Most methicillin-resistant Staphylococcus aureus (MRSA) strains produce beta-lactamase. To determine whether this enzyme(s) is identical to one or more of the four beta-lactamases produced by methicillin-susceptible strains, the beta-lactamases of 50 MRSA isolates were typed by using substrate profile analysis. Forty type A, no type B, ten type C, and no type D beta-lactamase-producing strains were identified. The beta-lactamase inhibitor sulbactam reduced the MICs of beta-lactamase-labile antibiotics, including ampicillin, penicillin G, and cefazolin, for type A and type C MRSA strains.     

Evaluating compliance with Australia''s first smoke-free public places legislation

The pathogenic methicillin-resistant Staphylococcus aureus (MRSA) has received a voluminous amount of notoriety. The four major reasons are its morbidity, mortality rate, cost of treatment, and constant appearance in intensive care units. Both Staphylococcus aureus and S. epidermidis (MRSE) account for 82% of our gram-positive wound isolates, whereas the gram-negative account for 34% of all isolates. Therefore we compared the morbidity, mortality rate, and cost factors related to MRSA-MRSE and gram-negative infections for a 4-year period, assessing more than 214 documented infections. Morbidity and mortality rates were minor for MRSA. Pseudomonas aeruginosa and Escherichia coli accounted for 57.5% of the total gram-negative isolates. Gram-negative antimicrobial therapy usually requires two therapeutic drugs, which increases morbidity and costs, whereas the staphylococci usually can be treated by one antimicrobial. During this period there were 47 gram-negative infections requiring 10 to 15 additional days of hospital stay, with a daily antibiotic cost of $293.40. Costs for MRSA or MRSE are 28% less. Therefore our preoccupation with MRSA or MRSE infections is unwarranted and unsubstantiated.     

A study of virulence factors produced by MRSA strains isolated from blood samples

Toxic shock syndrome toxin-1 (TSST-1) and enterotoxins are important virulence factors produced by Staphylococcus aureus. It is reported that these toxins are associated with septic shock and toxic shock syndrome. We investigated the toxin production and coagulase types of 701 MRSA strains isolated in Sasebo City General Hospital between 1994 and 1996 TSST-1 or/and enterotoxins were detected in 67% of all MRSA strains, and those were detected in 88% of MRSA strains isolated from blood samples. 45% of all MRSA strains produced both TSST-1 and enterotoxin C, and 70% of MRSA strains obtained from blood produced those toxins. Frequency of TSST-1 or/and enterotoxin production by MRSA strains isolated from blood samples was significantly higher than that by MRSA strains isolated from urine and pharynx (p < 0.05), and frequency of both TSST-1 and enterotoxin C production by MRSA isolates from blood was significantly higher than that by MRSA strains isolated from pharyngeal sample (p < 0.05). This study indicated that investigation of virulence factors produced by MRSA might give the useful information on prevention and treatment of MRSA infection.     

Production of exfoliative toxin A by Staphylococcus aureus isolated from mastitic cow's milk and farm bulk milk

The production of exfoliative toxins A and B (ETA and ETB) by Staphylococcus aureus isolated from mastitic cow's milk and farm bulk milk was examined by the reverse passive latex agglutination method (RPLA). ETA was detected in 2 (1.2%) of 162 isolates from mastitic cow's milk and in 1 (0.6%) of 166 isolates from farm bulk milk. RPLA titers of these isolates were much lower than in human isolates. No ETB was detected in any of the isolates tested. These ETA-positive isolates belonged to bovine ecovar. They were non-typable using the international phage set for human strains. When these ETA-positive isolates were subcutaneously inoculated into neonatal mice, general exfoliation of the epidermis accompanied by the so-called Nikolsky sign was not recognized. By the immunoblotting and PCR methods, however, ETA and eta gene were recognized in the ETA-positive isolates from mastitic cow's milk and farm bulk milk. These data suggest that ETA is also produced by bovine isolates of S. aureus, but in smaller quantities.     

Nonsteroidal antiinflammatory drugs are associated with both upper and lower gastrointestinal bleeding

We retrospectively evaluated antiinfective therapy for methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) endocarditis in 54 patients who had 57 treatment courses for the disease. Three treatments were assessed: 27 nafcillin-treated courses of MSSA endocarditis, 18 vancomycin-treated courses of MSSA endocarditis, and 11 vancomycin-treated courses of MRSA endocarditis. At baseline, patients with MSSA treated with vancomycin had more chronic conditions (p<0.01), a lower frequency of intravenous drug use (p<0.01), a lower hematocrit concentration (p<0.05), and a higher serum creatinine concentration (p<0.05) than the nafcillin group. Vancomycin-treated patients had a higher complication rate during therapy (p<0.05) and a longer duration in an intensive care unit (p<0.01) than the nafcillin group. The trend was for a higher complete response rate in the nafcillin group (74% vs 50%, p=0.12), but no difference in mortality (22% vs 28%, p=0.73). Patients with MRSA infection treated with vancomycin had higher mortality than those with MSSA who received that drug (55% vs 28%, p=0.24). Patients with vancomycin-treated MSSA endocarditis may have a poorer outcome than those who receive nafcillin, but this may be influenced by different or more severe clinical features.     

Nonviolent (empathic) communication for health care providers

As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation. This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test. To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR. Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin. The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.     

Antibacterial activities of combination uses of isepamicin and beta-lactams in vitro against clinically isolated strains. Part 1. Activities against Staphylococcus aureus

In order to evaluate antibacterial activities of combination uses of isepamicin (ISP) and beta-lactams in vitro, minimum inhibitory concentrations (MICs) these drugs were examined singly and in combination against clinically isolated Staphylococcus aureus. The results are summarized as follows; 1. MICs of ISP + cefazolin (CEZ), ISP + cefotiam (CTM) and ISP + flomoxef (FMOX) were low and the activities against methicillin (DMPPC)-susceptible S. aureus (MSSA) were dependent on the concentration of ISP. Combined effects were observed when the concentrations of ISP were at sub-MIC levels (1/2 approximately 1/4 concentrations). 2. MICs of ISP + CEX, ISP + CTM, ISP + FMOX, ISP + imipenem and ISP + panipenem were low and the activities against DMPPC-resistant S. aureus (MRSA) were dependent on the concentration of ISP, and were similar to those against MSSA. Combined effects were observed when the concentrations of ISP were at sub-MIC levels of ISP. Lower MIC50 or MIC90 was observed at ISP concentrations of 4 approximately 16 micrograms/ml. 3. The blood Cmax of ISP exceeded 20 micrograms/ml at one-time administration of ISP 400 mg, and these results suggested that antibacterial activities of combination uses of ISP and beta-lactams was clinically effective against MRSA infections.     

Nasal carriage of staphylococcus aureus and epidemiology of surgical-site infections in a Sudanese university hospital

Surgical site infections (SSI) due to Staphylococcus aureus among 256 male and 158 female patients (mean age, 28 years) undergoing elective surgery at the Soba University Hospital (Khartoum, Sudan) were studied. During an 11-month study period all patients were analyzed for nasal carriage of S. aureus at the time of admission. Follow-up of the development of SSI proceeded until 4 weeks after the operations. In addition, nasal swabs were obtained periodically during the same period from 82 members of the staff. In order to discriminate autoinfection from cross infection, bacterial isolates were typed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments, and restriction fragment length polymorphism analysis of the protein A and coagulase genes. Preoperative cultures revealed the presence of S. aureus in the noses of 98 patients (24%). The overall number of postsurgical wound infections in the entire group was 57 (14%), 24 of which were due to S. aureus. Only 6 of the 98 nasal S. aureus carriers suffered from wound infections by the same species. In these six cases the infecting strain could not be genetically discriminated from the nasal inhabitant, substantiating autoinfection. However, nasal carriage of S. aureus is not a significant risk factor for the development of SSI in this setting (6 of 98 patients with autoinfection versus 18 of 316 patients [414 - 98 patients] with cross infection; P = 0.81), most probably due to the fact that noncarriers are at a significant and relatively large risk for acquiring an independent S. aureus SSI. The other S. aureus strains causing SSI showed a high degree of genetic heterogeneity, demonstrating that it is not an epidemic strain that is causing the SSI. Among the staff personnel screened, 47.4% did not carry S. aureus in the nose at any time during the study period, whereas 13. 2% persistently carried a single strain in the nose. Another 39.5% could be classified as intermittent carriers. When strains derived from staff personnel were genetically typed, it was demonstrated that most of the strains represented genetic variants clearly differing from the isolates causing SSI. On the other hand, possible cross colonization among staff personnel and even cross infection from staff personnel to patients or from patient to patient were demonstrated in some cases, but epidemic spread of a single strain or a few clonally related strains of S. aureus could be excluded.