Dynamics of antibiotic resistance of Staphylococcus aureus isolated from chronic osteomyelitis patients

The level of antibiotic resistance of 365 S.aureus strains, isolated from chronic osteomyelitis patients in 1990 and 2000, was determined. As revealed in this study, over 10 years the isolation rate of strains adapted to the antimicrobial action of all tested preparations increased in some degree. The increase of the resistance level to aminoglycosides was most significant. The proportion of polyresistant strains was 9.1% in 1990 and 30.5% in 2000. The isolation rate of methicillin-resistant S. aureus (MRSA) did not increase during these 10 years, but the range of preparations suitable for the treatment of MRSA-infected patients was shown to become essentially narrower. These data confirm the view on the possibility of further decrease in the effectiveness of the conservative treatment of chronic osteomyelitis due to the rapid growth of the antibiotic resistance of S. aureus.     

Cross reactions between Staphylococcus aureus and Neisseria meningitidis

The results of the study of rabbit antisera to meningococci A, B, C in the double diffusion in gel, passive hemagglutination test and enzyme immunoassay with antigenic preparations isolated from S. aureus strains are indicative of the presence of common antigenic determinants of protein and polysaccharide nature in S. aureus and N. meningitidis.     

A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods

The coagulase reaction of Staphylococcus aureus on the PPSA (pork plasma for S. aureus) agar of Devoyod et al. was found to be fibrinogen-deficient. By including bovine fibrinogen (BFG) in the medium, the fibrin halos around S. aureus colonies became more distinct, preparations of pork plasma previously unacceptable for inclusion in the original PPSA agar were performing well, and the amount of pork plasma required in PPSA agar could be reduced by nearly 90%. In the modified medium, designated PPF (pork plasma fibrinogen) agar, the agar base (Baird-Parker agar without egg yolk) was unchanged. After surface plating, the base was covered with 8 mL of a modified overpour agar: 2.5% pork plasma, 0.38% BFG, and 0.0015% soy trypsin inhibitor in 0.7% Bacto agar. Most S. aureus strains could be enumerated after 24 h of incubation at 35 degrees C; the others required 44 h. Without soy trypsin inhibitor, a number of strains showed considerable fibrinolysis between 24 and 44 h of growth; this activity was neutralized by the inhibitor. The S. aureus counts of 27 food samples on PPF agar were essentially the same as the confirmed S. aureus counts obtained by the Baird-Parker method.     

Evaluation of Three Slide Agglutination Tests for Rapid Identification of Staphylococcus aureus

Three slide agglutination tests for identification of Staphylococcus aureus were compared. The agglutination tests used for evaluation were Staphaurex (Wellcome Diagnostics), Staphyslide-Test (BioMerieux), and ANI S. aureus TEST (Ani Biotech Oy). A total of 347 isolates were analyzed, including 288 strains of S. aureus, 49 of S. epidermis, 11 of S. intermedius, 12 strains of other staphylococci and 14 non-staphylococcal strains. One hundred of the S. aureus strains were isolates from cases of food poisoning, 129 from mastitis and 59 from other clinical cases. The sensitivities of the tests were also compared using diluted suspensions of S. aureus strains and with purified Protein A dilutions. The results showed that the sensitivities of the tests were 98.6 %, 97.9 % and 99.0 % for Staphaurex, Staphyslide-test and ANI S. aureus TEST, respectively. The specificities were 100 % for the Staphyslide test and 98.8 % for both the ANI S. aureus TEST and the Staphaurex test. The sensitivities measured with diluted S. aureus strain suspensions and Protein A solutions were equal with the Staphaurex and ANI S. aureus TEST. All the agglutination tests studied proved to be practical, easy to use and accurate for the rapid identification of S. aureus strains from culture isolates.     

Cross-reactions between Staphylococcus epidermidis and 23 other bacterial species

By quantitative immunoelectrophoretic methods, 43 antigens were found in a mixture of sonicated preparations of four Staphylococcus epidermidis strains, using corresponding rabbit antiserum. Two of the antigens were identified as cell wall teichoic acid and a peptidoglycan antigen, respectively. Using this antigen/antibody reference system, cross-reactions between S. epidermidis antigens and antigens from other bacterial species were investigated. Fourteen of the S. epidermidis antigens cross-reacted with antigens from all S. aureus strains investigated. Only few cross-reactions were found between S. epidermidis and bacteria not belonging to the Micrococcaceae. The antigenic relatedness, expressed as a matching coefficient, seems promising for taxonomic work.     

Brucella abortus lipopolysaccharide is mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice.

Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/Hej mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.     

Staphylococcus aureus strains isolated from bovine mastitis: virulence,antibody production and protection from challenge in a mouse model

Septic arthritis in mice was used as a model to evaluate the virulence of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from cases of bovine mastitis. In addition, the model was used to evaluate the cross protection elicited by heterologous antibodies. Mice were intramuscularly inoculated with serial bacterial doses of different strains of S. aureus or CNS, for virulence determination; they were monitored for arthritis, gangrene or death up to 20 days. Antibody response, cross reactivity and resistance to challenge were tested by subcutaneous inoculation with a low dose of one of the S. aureus or CNS strains followed by challenge with two S. aureus strains. S. aureus alpha-hemolysin isolate was the most virulent, followed by alpha+beta-hemolysin and beta-hemolysin isolates. The least virulent isolates were the non-hemolytic S. aureus strains but even they were more virulent than the CNS strains tested. Antibodies against three different S. aureus antigens were detected by the ELISA in all mice that were inoculated with the S. aureus strains but not in any of those with the CNS strains. Immunoblot test against various S. aureus strains as antigens showed high cross-reactivity among the S. aureus strains but only a slight similarity, restricted to the bands above 36 kDa, with the CNS sera. Low-dose inoculation of alpha or alpha+beta strains before challenge with homologous and heterologous strains protected the mice, whereas the two beta strains provided only partial protection. The inoculations of non-hemolytic S. aureus or the CNS strains did not elicit any protection. Our findings demonstrate that pre-exposure of mice to a low dose of certain S. aureus strains could provide protection and that the antibodies produced could have an important protective role.     

Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods

Antimicrobial activity of seven commercial smoke preparations (four liquid and three solid) was studied. The minimum inhibitory concentration (MIC) was determined against a selection of food spoilage and pathogenic micro-organisms. The main smoke components were identified and quantified by gas chromatography/mass spectrometry. The most effective condensate was S2. All strains except Salmonella enteritidis were inhibited by S2 with an MIC <0·5–1·5%. Smoke extract L2 inhibited growth of Vibrio vulnificus, Yersinia enterocolitica, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, L. inocua, Brochothrix thermosphacta and Lactococcus lactis ssp. lactis with an MIC of <0·2–0·8%. The condensate L3 inhibited effectively V. vulnificus, B. subtilis, L. innocua and Staph. aureus. L1, L4, S1 and S3 had no inhibitory effects at levels tested against most micro-organisms. Vibrio vulnificus was the most susceptible micro-organism to test compounds. The antimicrobial activity of smoke preparations was related to the concentration of phenols.     

Staphylococcus aureus isolated from Kawasaki disease patients hyper-releases extracellular protein A.

S. aureus isolates from patients with Kawasaki disease (KD) release high levels of extracellular protein A (SpA), as compared to S. aureus in other diseases. The molecular weight of this released protein A is about 70 kDa. Extracellular KD SpA purified by affinity chromatography possessed the same amino acid sequence at the NH2-terminal IgG binding region and the same antigenic specificity as recombinant and cell-wall-bound SpA preparations. The size of DNA fragments containing the spa gene from S. aureus KD strains was 160-165 kb. All of these DNA fragments contained the igb portion encoding the IgG-binding region of KD SpA. Significantly higher molecular size of the SpA molecules hyper-released in the stationary-phase culture and the lack of production of other exo-proteins allow us to speculate that S. aureus isolated from patients with KD have mutations occurring in the agr locus.     

Cytidine deamination assay to differentiate Staphylococcus aureus from other staphylococci

AIMS: Adoption of the property of cytidine (cytosine-beta-d-riboside) deamination in staphylococci to distinguish Staphylococcus aureus from other staphylococci. METHODS AND RESULTS: A total of 560 staphylococcal strains were examined. The test demonstrated a sensitivity of 97.1% and a specificity of 98.8%. Of the 249 S. aureus strains (115 oxacillin-resistant) 58 strains were coagulase-negative S. aureus and another 16 strains were clumping factor-negative S. aureus. The 74 deficient S. aureus strains were identified by 16S rRNA gene sequencing and further investigated by spa typing and 13 spa types were found. CONCLUSIONS: The cytidine deaminase test (CDT) is useful especially for distinguishing coagulase- and clumping factor-negative S. aureus from other staphylococci and the results correlated well with 16S rRNA sequencing and the polymerase chain reaction (PCR) amplification of the nuc gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Cytidine deamination assay differentiates S. aureus from other staphylococci. This method is fast (6 h) and reliable in distinguishing between non-S. aureus and the defective (coagulase-negative, clumping factor-negative) S. aureus isolates which could have major consequences for therapy.     

Enterotoxins and phage typing of Staphylococcus aureus isolated from clinical material and food in Libya

Enterotoxin was detected in 22 (61.1%) of the 36 S. aureus strains isolated from clinical materials and in 3 (13%) of the 23 S. aureus strains from food samples (P < 0.05). On the basis of individual types of enterotoxin, staphylococcal enterotoxin A (SEA) was produced by 11.1%, SEB by 38.9% and SEC by 22.2% of SS. aureus strains from clinical material. Of the food S. aureus strains, SEC and SED produced by 8.7% and 4.3% respectively. Of the clinical and food S. aureus strains, 52.8% and 39.1%, respectively, were typeable by the 23 phages of International Phage Set. The majority of the typeable S. aureus strains from clinical and food sources belonged to group II being at 22.2% and 17.4% respectively. Furthermore, of the 14 SEB-producing S. aureus, 42.9% were of phage group II. In conclusion, the results obtained indicate that enterotoxin-producing S. aureus strains from clinical materials in Libya are not uncommon; however, certain foods appear not to be the source of such strains. Because of the low susceptibility to bacteriophages shown by S. aureus isolated in Libya, compared to reports from several countries, other methods of typing should be used in conjunction with phage typing in epidemiological investigations concerning this organism.     

Isolation of strains of a new ecological variant of Staphylococcus aureus from asses

47 coagulase-positive staphylococcal strains were isolated from the nasal smears of 175 healthy donkeys. In accordance with the schemes of Akatov--Devriese and Mayer-Witte--Akatov, 10.6% of the cultures were classified with the coagulase-positive species S. hyicus and 89.3%, with the species S. aureus. Out of S. aureus strains, 11.9% were found to have the characteristics of ecovar hominis, while 16.2% of the cultures could not be classified with definite ecovars. Most of the strains (71.4%) were found to differ from the known ecovars of S. aureus in their biological properties. For this reason, the above strains were classified with the new ecovar asinae. The authors propose to make the existing S. aureus identification scheme (the scheme of Mayer-Witte--Akatov) more complete by adding the tests for hyaluronidase and phosphatase.     

Population studies of methicillin-resistant and -sensitive Staphylococcus aureus strains reveal a lack of variability in the agrD gene, encoding a staphylococcal autoinducer peptide

The virulence of Staphylococcus aureus is controlled by the accessory gene regulator (agr) system, including an extracellular inducer encoded by agrD. Variable agr PCR restriction fragment length polymorphism (RFLP) patterns of unique S. aureus strains (n = 192) were determined for a region comprising agrD and parts of the neighboring agrC and agrB genes. Twelve unique RFLP patterns were identified among S. aureus strains in general; these patterns were further specified by sequencing. All sequences could be catalogued in the three current agr groups. A major proportion of the S. aureus strains belong to agr group 1, whereas only 6% of the methicillin-susceptible S. aureus strains and 5% of the methicillin-resistant S. aureus strains belong to agr groups 2 and 3, respectively. The homology between groups varied from 75 to 80%, and within groups it varied from 96 to 100%. Different levels of sequence variability were observed in the different agr genes. agr-related bacterial interference among colonizing S. aureus strains in the noses of persistent and intermittent human carriers was studied. S. aureus strains belonging to different agr groups were encountered in the same individual. This may suggest that the activity of the agrD gene product does not define colonization dynamics, which is further substantiated by the rarity of agr group 2 and 3 strains.     

Classification and biological characteristics of coagulase-positive Staphylococci isolated from animals

A total of 165 coagulase-positive staphylococcal strains of different origin (142 S. aureus strains and 23 S. intermedius strains) were subjected to biological typing in accordance with the schemes of Hajek-Marsalek and Meyer-Witte. The former of these schemes permitted to identify 68% and the latter 18% of S. aureus strains. The cultures isolated from swine and chickens had the most uniform composition: 85-86% of the strains belonged to biotype B. 44% of the strains isolated from cows and sheep belonged to biotypes C (ecovars bovis and ovis) and A (ecovar hominis); the rest of the strains could not be identified. 96% of the strains isolated from minks were made up of S. intermedius, more than a half of them belonging to biotype E (ecovar canis). In 80% of S. aureus strains and 48% S. intermedius cultures protein A was detected. Only 9% of S. aureus strains of animal origin were found capable of producing enterotoxins (A-D). The expediency of working out a unified scheme for the biotyping of coagulase-positive staphylococci is discussed.     

Sensitivity of Staphylococcus aureus strains isolated from the patients of the SP ZOZ Hospital in Nidzica to bacteriocidal agents

The aim of this study was to evaluate a frequency of isolation and antimicrobial susceptibility testing of Staphylococcus aureus strains isolated from clinical specimens obtained from patients hospitalized in different hospital wards (SP ZOZ) in Nidzica from 01. 09. 2000 to 31. 12. 2003. During over three years 716 Staphylococcus aureus strains were cultured out of 15517 clinical specimens supplied to the Bacteriological Laboratory of SP ZOZ in Nidzica. S. aureus strains were isolated from 4.6% of examined samples. Samples were collected from patients hospitalized in all wards (five wards). Analysis of susceptibility to antimicrobial agents of identified S. aureus strains was performed. Seventy strains (9.8%) were metihicillin-resistant (MRSA). One hundred twenty four strains (17.3%) revealed inducible resistance to macrolides, linkosamides and streptogramins B (MLS, mechanism). The greatest activity in vitro against clinical S. aureus strains showed glycopeptide antibiotic--vancomycin (100% of susceptible strains). Clinical S. aureus strains isolated from patients of hospital in Nidzica are in the majority susceptible to antibiotics/chemotherapeutics, except of penicillin. Percentage of methicillin-resistant strains (MRSA) is not high (<10). Nevertheless, constant monitoring of a drug susceptibility of nosocomial S. aureus strains is important, considering the necessity of control of current epidemiological and therapeutic situation.     

Effect of Variations in Conditions of Incubation upon Inhibition of Staphylococcus aureus by Pediococcus cerevisiae and Streptococcus lactis

The effects of pH, temperature, proportion of Staphylococcus aureus in the inoculum, various strains of effector organism, and various strains of S. aureus were examined for their influence on interactions between staphylococci and effector organisms in associative culture. In general, small changes in pH had little effect upon either growth of S. aureus or production of enterotoxin in associative culture. Inhibition of growth of S. aureus caused by effector organisms was much greater at 25 than at 30 C. Proportion of S. aureus in the inoculum greatly affected both growth of the staphylococci and production of enterotoxin. Only slight differences were found between strains of either effector organism or S. aureus which affected the interactions in associative culture.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Antimicrobial resistance pattern of methicillin-resistant Staphylococcus aureus in the food industry

There is increasing concern about the impact on public health of methicillin-resistant Staphylococcus aureus (MRSA) associated with animal food products. MRSA remains a serious problem because of the high incidence and multidrug resistance of the strains, even for strains isolated from foods, food environments and food handlers. The objectives of this study are: (i) to evaluate the susceptibility of S. aureus strains isolated from food, food handlers and food-processing environments to 14 antibiotics currently used in veterinary and human therapy; (ii) to assess the presence of the mecA gene. A total of 1007 samples were collected from food, food handlers, and environments and were analyzed for the presence of S. aureus. S. aureus was present in 165 of the 1007 samples. A total of 157 isolates were methicillin-susceptible S. aureus (MSSA) and 8 isolates were MRSA. In particular, out of 8 MRSA strains detected, 4 strains harboured the mecA gene. All MRSA strains were resistant to at least one of the tested antibiotics and 6 strains demonstrated multi-resistance. Considering the high level of resistances in S. aureus and the isolation of MRSA strains, the surveillance of antimicrobial resistance and the spreading of this pathogen is of crucial importance in the food production chain. These data are useful in improving background data on antimicrobial resistance of S. aureus isolated from food, processing environments and food handlers, supporting the prudent use of antibiotics and the development of international control programs.     

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.