Capsular serotype of Staphylococcus aureus in the era of community-acquired MRSA

Capsular polysaccharide (CP) plays an important role in the pathogenicity and immunogenicity of Staphylococcus aureus, yet the common serotypes of S. aureus isolated from US pediatric patients have not been reported. We investigated capsular serotype as well as methicillin susceptibility, presence of Panton-Valentine leukocidin (PVL), and clonal relatedness of pediatric S. aureus isolates. Clinical isolates were tested for methicillin susceptibility, presence of mecA, lukS-PV and lukF-PV, cap5 and cap8 genes by PCR, and for capsular or surface polysaccharide expression (CP5, CP8, or 336 polysaccharide) by agglutination. Genetic relatedness was determined by pulsed-field gel electrophoresis. All S. aureus isolates encoded cap5 or cap8. Sixty-nine percent of 2004-2005 isolates were methicillin-susceptible (MSSA) and most expressed a detectable capsule. The majority of MRSA isolates (82%) were unencapsulated, exposing an expressed cell wall techoic acid antigen 336. Pulsed-field type USA300 were MRSA, PVL-positive, unencapsulated strains that were associated with deep skin infections and recurrent disease. Over half (58%) of all isolates from invasive pediatric dermatologic infections were USA300. All pediatric isolates contained either capsule type 5 or capsule type 8 genes, and roughly half of the S. aureus clinical disease isolates from our population were diverse MSSA-encapsulated strains. The majority of the remaining pediatric clinical disease isolates were unencapsulated serotype 336 strains of the PVL(+) USA300 community-associated-MRSA clone.     

Relationship between capsular types 5 and 8 and phage types in Staphylococcus aureus isolates from cow, goat and ewe milk

Abstract In order to check the relationship between capsular polysaccharide (CP) type 5 and 8 and certain phage patterns, previously described in human Staphylococcus aureus clinical isolates, we typed 100 CP types 5 and 8 S. aureus strains isolated from cow, goat and ewe milk, with the human set of phages. The proportion of typable strains was much less than that found with human strains. The association between CP types 5 and 8 and phage patterns reported for human isolates was only partially confirmed and an original correlation between susceptibility to group III phages and CP type 5 was found.     

Staphylococcus cohnii--resident of hospital environment: cell-surface features and resistance to antibiotics

Staphylococcus cohnii strains dominated in the environment of investigated hospitals. We isolated 420 strains of the species mainly from hospitals environments, but also from infants--Intensive Care Units patients, its medical staff and non-hospital environments. S. cohnii subspecies cohnii was seen to dominate (361 strains). Seventy seven percent of these strains expressed cell-surface hydrofobicity, most of them were slime producers (61%) and this feature was correlated with their methicillin resistance. Among S. cohnii ssp. cohnii strains isolated from ICU environment 90% were resistant to methicillin, 43% expressed high-level resistance to mupirocin and high percentages were resistant to many other antibiotics. These strains may constitute a dangerous reservoir of resistance genes in a hospital.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Methicillin-resistant Staphylococcus aureus (MRSA) with high resistance to mupirocin in hospitals of the Gdańsk region

Occurrence of high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from 18 hospitals in Gdańsk area was determined. The study was carried out on 190 MRSA isolated in 1997-2000 from various clinical samples. The strains were tested for high-level mupirocin resistance by 200 micrograms mupirocin disc. The minimum inhibitory concentrations (MIC) for methicillin were estimated by agar dilution. Sensitivity to other antibiotics was determined in disc-diffusion method and to vancomycin in agar dilution method additionally. The strains were typed by set of 10 experimental phages and compared by the method of PCR-RFLP analysis of coagulase gen restriction fragment length polymorphism. There were low frequency of high-level mupirocin resistance in MRSA strains (4.7%) that were found only in 3 hospitals, in 6 patients. All of them were high-resistant also to methicillin and resistant to doxycyclin, gentamycin, erytromycin, klindamycin, ciprofloksacin, rifampicin, resistant or intermediate sensitive to fusidic acid but sensitive to vancomycin, teikoplanin and bacitracin. The origin all of the MRSA strains high-resistant to mupirocin probably was the same, except one strain, because they were belonged to one genetic type and possessed the same phage pattern.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 x 10(9) cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A x 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20 degrees C reduced cell-surface hydrophobicity of all species, while storage at 4 degrees C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4 degrees C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Microbiological external quality assurance program (MEQAP)--evaluation of results obtained in sanitary epidemiological laboratories in 2002

The aim of this study was to evaluate reliability of identification and determination of sensitivity to antibiotics and chemotherapeutics of some Gram positive cocci strains in 34 sanitary-epidemiological stations. All laboratories engaged in this study received 3 strains: S. aureus (S. aureus SC+ CF+, resistant to methicillin., or S. aureus SC - CF+, sensitive to methicillin), coagulase-negative staphylococci (S. epidermidis or S. haemolyticus or S. saprophyticus) and Enterococcus sp. (E. faecalis HLAR-positive or E. faecalis HLAR-negative or E. faecium or E. gallinarum). All these strains previously were identified in Department of Bacteriology of National Institute of Hygiene. Of the 68 staphylococci strains tested, 66 isolates were correctly identified. Among the 34 enterococcal strains studied the greatest difficulty in identification was caused by E. gallinarum strain--(8 out of 13 strains were incorrectly recognised). The determination of the sensitivity of the control strains to antibiotics and chemotherapeutic agents, generally was performed correctly in accordance with to the NCCLS and national recommendations. Some incorrect results of the antibiograms were caused by an erroneous interpretation of the zones of inhibition.     

Serotyping of Dutch Staphylococcus aureus strains from carriage and infection

International epidemiological studies have shown that clinical isolates of Staphylococcus aureus are usually capsulated with either type 5 or 8 capsular polysaccharides (CPs). Because all noncapsulated strains were found to be cross-reactive with polysaccharide 336 (336PS) antibodies, the noncapsulated strains were denoted as type 336PS. The capsular types of 162 Dutch methicillin-susceptible S. aureus strains derived from individuals living in the Rotterdam area were determined. The serotype distribution was 28.4% serotype 5, 53.7% type 8, and 17.9% type 336PS. Serotyping was in agreement with genotyping by amplified fragment length polymorphism (AFLP) and multi locus sequence typing (MLST). Among 49 nasal carriage isolates from healthy children 24.5% belonged to serotype 5, 67.3% were type 8 and 8.2% were type 336PS. For 28 adult patients on chronic ambulatory peritoneal dialysis (CAPD) the serotype incidences among carriage isolates obtained from the nose, catheter exit-site, and abdominal skin were 45.1%, 41.2% and 13.7%, respectively. Among S. aureus strains deriving from blood cultures, the serotype incidences were 17.7% serotype 5, 53.2% type 8, and 29.0% type 336PS. Apparently, type 336PS strains are more prevalent (P=0.017) among bacteraemia isolates as compared with the nasal carriage isolates obtained from healthy children and CAPD patients. In conclusion, all Dutch S. aureus isolates belonged to types 5, 8, or 336PS, which is in agreement with data from other countries. Thus, addition of the 336PS conjugate to a type 5- and type 8-CP protein conjugate vaccine would significantly extend the vaccine coverage.     

Reduced susceptibility to triclosan in methicillin-resistant Staphylococcus aureus

Susceptibility to triclosan in Staphylococcus aureus was determined. The study was carried out on 200 strains, including 100 resistant (MRSA) and 100 susceptibile (MSSA) to methicillin. The examined strains were isolated from varied clinical samples and patients in 18 medical centers, in majority from hospitals in the region of Gdansk. The susceptibility was estimated by the MIC (minimal inhibitory concentration) using dilution test in Mueller-Hinton agar. The antimicrobial resistance patterns were determined, including resistance to methicillin and mupirocin. The most of MRSA strains (62%) demonstrated reduced susceptibility to triclosan (MIC 2mg/L), while 93% of MSSA strains were highly sensitive to this antibacterial agent (MIC 0,031mg/L). The majority (66,1%) of MRSA strains with reduced susceptibility to triclosan demonstrated the same antimicrobial resistance pattern. Reduced susceptibility to triclosan was observed in 8 from 9 high - level mupirocin resistant strains, but the most of MRSA strains with reduced triclosan susceptibility (91,5%) were found among fusidic acid resistant strains.     

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 times 10⁹ cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A × 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20C reduced cell-surface hydrophobicity of all species, while storage at 4C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Toxin profiles, and cell-surface properties of Salmonella strains isolated from Swedish travelers.

Toxin production, cell-surface hydrophobicity and fibronectin-binding properties of 21 Salmonella strains of different species, isolated from Swedish travelers to different parts of the world, were studied. Cell sonicate supernatants from blood agar grown cultures of 80% of the strains induced rabbit skin permeability reaction in the form of induration and/or blueing while 33% of the strains also produced cell necrotizing factor on rabbit skin. Four strains were negative in the rabbit skin permeability test, while only two were negative when tested on CHO cells. When cultured on blood agar, a majority of the strains (17/21) showed low cell-surface hydrophobicity, showing no aggregation even at 1.5 M ammonium sulfate concentration in salt aggregation test (SAT), while only four strains showed high cell-surface hydrophobicity. Furthermore, these strains could be classified as low fibronectin binders due to their poor interaction with fibronectin or its 29 kDa N-terminal fragment.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Extrachromosomal Control of Methicillin Resistance and Toxin Production in Staphylococcus aureus

All of 41 naturally occurring coagulase-positive methicillin-resistant strains of Staphylococcus aureus isolated in various laboratories were resistant to several antibiotics and were lipase-negative. Most strains produced hemolysins, and 38 strains produced enterotoxin B. Acriflavine treatment of four strains resulted in elimination of resistance to methicillin and mercury; in one strain, resistance to cadmium was also lost. Production of enterotoxin B and beta-hemolysin was eliminated in all four strains and penicillinase production was eliminated in one strain. In transduction experiments, methicillin resistance and enterotoxin B production were transferred together at a frequency of 0.2 x 10(-8) to 1.1 x 10(-8) by use of ultraviolet-induced phage lysates from naturally lysogenic methicillin-resistant strains. Cotransductions of resistance to mercury and cadmium, as well as production of penicillinase and beta-hemolysin, were obtained to some extent. The extrachromosomal character of these determinants and their possible genetic association are discussed.     

Nosopharyngeal microflora in ambulatory treated children and adults with upper respiratory tract infections

Upper respiratory tract consists resident and transient bacterial microflora, which in appropriate condition can cause infection. Bacteriological study was performed among 201 patients with upper respiratory tract infections treated in ambulatory. From nasal and pharyngeal swabs Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Streptococci group A, B, C, G were isolated. Antibiotic susceptibility testing of isolated strains was performed using CLSI criteria. All isolated strains of streptococci were susceptible to penicillin; some of them demonstrated resistance to macrolides and lincosamides. Few isolated strains of H. influenzae demonstrated resistance to penicillin and cotrimoxazole. Azitromycin resistant strains were not detected. All isolated strains of M. catarrhalis were beta-lactamase positive and demonstrated resistance to penicillin. Strains of methicillin sensitive S. aureus (MSSA) were isolated most frequently from pharyngeal swabs (35.4%) and S. pneumoniae (33.3)--from nasal swabs.     

Donor activity of methicillin resistant staphylococci in transduction experiments]

Antibiotic resistance gene transmission from methicillin resistant strains of Staphylococcus aureus (MRSA), isolated in a burn care unit, was studied in transduction experiments with type phages 29, 52, 52A and in experiments with prophage induction. The results of the experiments demonstrated high donor activity of MRSA. Recombinants with different antibiotic resistance phenotypes were revealed, but there were no methicillin resistant staphylococci among them. Stability of cloramphenicol resistance gene transmission in the experiments on specific transduction with the prophage induction could be indicative of the prophage localization near the chloramphenicol resistance genes. Variety of the antibiotic resistance combinations in the transductants from the clinical strains of MRSA could prove heterogeneity of the strains even under conditions of one hospital.     

Evaluation of susceptibility to antibiotics of Staphylococcus aureus strains resistant to methicillin]

Methicillin-resistant strains of Staphylococcus aureus (MRSA) constitute a serious diagnostic and therapeutic problem. Over 500 strains of Staphylococcus aureus were tested for susceptibility to methicillin. By application of a screening method, 13.7% of these strains were classified as methicillin-resistant. Over 95% of these strains were isolated from hospital infections. Applying criteria of belonging of these strains to methicillin-resistance classes it was found that 49.3% belonged to class II, 31.2% to class III and 19.5% to class IV. Analysis of susceptibility to antibiotics of MRSA strains demonstrated significant differences between class II and between class III and IV in resistance to imipenem, gentamycin, erythromycin and tetracycline. All tested strains were susceptible to ciprofloxacin, ofloxacin, vancomycin and teicoplanin. The screening method (25 mg methicillin/l of TSA medium) results in obtaining of univocal results of determination of methicillin-resistance in S. aureus.     

Presence of enterotoxin C and toxic shock syndrome toxin--1 (TSST-1) genes in population of Staphylococcus aureus phage type 187

The aim of this study was to examine whether Staphylococcus aureus of phage type 187 possess the genes of enterotoxins and toxic shock syndrom toxin. Sixteen phage type 187 strains were isolated from the hospital patients (12) and the carriers (4) in twelve medical centres in Poland during 1991 and 2005. Biotyping, phage typing, antibiotic susceptibility, detection of the genes of enterotoxins (sea--sed) and toxic shock syndrome toxin (tst) was tested. The results of this study showed that all staphylococci of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Almost all of them (93.8%) had the enterotoxin C gene and TSST-1 gene. This fact allows to consider them the strains of potentially high virulence.