Molecular characterization of methicillin-resistant Staphylococcus aureus isolates from a hospital in Ghana

Background: Methicillin-resistant Staphylococcus aureus (MRSA) are a major cause of hospital- and community-acquired infection. They can colonize humans and cause a wide range of infections including pneumonia, endocarditis and bacteraemia. We investigated the molecular mechanism of resistance and virulence of MRSA isolates from a teaching hospital in Ghana. Methodology: A total of 91 S. aureus isolates constituted the initial bacterial sample. Identification of S. aureus was confirmed by the VITEK 2 system. The cefoxitin screen test was used to detect MRSA and antibiotic susceptibility was determined using the VITEK 2 system. The resistance (mecA, blaZ, aac-aph, ermC, and tetK) and virulence (lukS/F-PV, hla, hld and eta) genes were amplified by polymerase chain reaction (PCR) and positive samples subjected to DNA sequencing. Pulsed field gel electrophoresis (PFGE) was used to ascertain the relatedness of the isolates. Results: Fifty-eight of 91 (63.7%) isolates were putatively methicillin resistant by the phenotypic cefoxitin screen test and oxacillin MICs. However, 43 (47%) of the isolates were genotypically confirmed as MRSA based on PCR detection of the mecA gene. Furthermore, 37.9% of isolates displayed resistance to tetracycline, 19% to trimethoprim-sulphamethoxazole, 15.5% to clindamycin, 12.1% to gentamicin, 13.8% to ciprofloxacin and erythromycin, 6.9% to moxifloxacin and 7.0% to rifampicin. None of the isolates was positive for inducible clindamycin resistance. The prevalence of resistance (mecA, blaZ, aac(6')-aph(2''), tetK, and ermC) and virulence (hla and lukS/F-PV) genes respectively were 74%, 33%, 22%, 19%, 3%, 5% and 3%, with isolates organized in two highly related clades. Conclusion: Results indicate a fairly high occurrence of MRSA, which can complicate the effective therapy of S. aureus infections, necessitating surveillance and stringent infection control programmes to forestall its spread

Bursitis séptica / Septic bursitis

Se reportan seis casos de bursitis séptica vistos en el Hospital Cayetano Heredia entre 1987 y 1991; cinco fueron varones y la edad promedio fue de 51.2 años. Las bursas afectadas fueron: olecraneana (tres casos), prepatelar (dos) e infrapatelar (un caso); el germen más aislado fue S. aureus (cuatro casos), tuvimos además un caso de E. coli y uno en quien se aisló Neumococo. Todos evolucionaron favorablemente con el tratamiento antibiótico

Distribution of capsular materials on the cell wall surface of strain Smith diffuse of Staphylococcus aureus.

The fine structure of the capsule of Staphylococcus aureus Smith diffuse was examined by the technique of freeze-substitution and immunoelectron microscopy. The cell surface was covered with a thick layer consisting of fine fibrous structures which were absent from an unencapsulated strain, Smith compact. Anti-teichoic acid antibody did not react with this surface layer but reacted with the surface of strain Smith compact. Anti-capsular antibody, made from the serum of a rabbit immunized with strain Smith diffuse and specific absorption with unencapsulated strain Wood 46, reacted with the fibrous layer of the Smith diffuse strain. Since the anti-teichoic acid antibody did not react with the encapsulated strain Smith diffuse, the capsular layer acts as a barrier to penetration of the anti-teichoic acid antibody through the capsular layer. A portion of a few cell surfaces of the encapsulated strain remained accessible to the anti-teichoic acid antibody. The capsular layer in this portion of the cell surface was thin, and this surface seemed to be a new cell wall surface created by the cell separation.

Influence of mucin on serum and connective tissue protein binding to Staphylococcus aureus isolated from nasal carriage and clinical sources.

A total of 89 Staphylococcus aureus strains were tested for [125I]-labelled fibronectin (Fn), type I (Cn-I), type II (Cn-II), type IV (Cn-IV) collagens and laminin (Lm) binding, and nasal carriage isolates (54 strains) demonstrated higher degree of interaction than clinical isolates (35 strains). Strains belonging to nasal carriage group, after preincubation with mucin demonstrated a significant decline in binding to Fn (39.4%), Cn-I (44.7%), Cn-IV (42.0%) and Lm (43.5%) compared with inhibition of binding of clinical isolates to Fn (13.3%), Cn-I (8.0%), Cn-IV (9.8%) and Lm (11.2%). S. aureus strain Nig-6 demonstrated a mucin concentration (in the range 0.01 to 100 mg/ml) dependent decrease of [125I]-labelled serum and connective tissue protein binding. Mucin concentrations of 100, 150, 175 and 250 micrograms/ml when incubated with 10(9) cells, caused 50% displacement of [125I]-labelled Lm, Cn-I, Cn-IV and Fn uptake respectively. Mucin interaction with bacterial cells seems probably important in the pathomechanism of staphylococcal adhesion and colonization.

Purification of staphylococcal enterotoxins A and E by immunoaffinity chromatography using a murine monoclonal antibody with dual specificity for both of these toxins.

An immunosorbent column was prepared by coupling a murine monoclonal antibody (MAb) with dual specificity for staphylococcal enterotoxins A (SEA) and E (SEE) to Affi-Gel 10. Purification of both SEA and SEE from culture supernatants was carried out with the immunosorbent column using 0.2 M acetic acid containing 0.15 M NaCl as eluant. The yields obtained were approximately 76% for SEA and 70% for SEE. Purified SEA and SEE were found to be immunologically and electrophoretically homogeneous. Immunoaffinity chromatography using a MAb with dual specificity proved to be valuable in the purification of SEA and SEE, not only from the standpoint of percentage recovery, but also because of the degree of purity and the ease of purification.

Improved purification of leukocidin from Staphylococcus aureus and toxin distribution among hospital strains.

For purification of F and S components of the Panton-Valentine leukocidin, an easy three-step method using fast protein liquid chromatography was developed to replace the time-consuming purification procedures previously published. This technique enabled the recovery of 13 and 17 mg of purified F and S, respectively, per litre of culture supernatant. Affinity-purified neutralizing polyclonal antibodies were obtained against each individual component. One hundred and thirty-nine Staphylococcus aureus strains isolated from various clinical samples of hospitalized patients were screened by immunoprecipitation for Panton-Valentine leukocidin (PVL) production. Only 8 strains produced PVL; all originated from injured superficial soft tissues. Contrary to widespread opinion, the 8 PVL-producing strains were never associated with severe infection.

Caracterización bioquímica de cepas de staphylococcus sp. aisladas de muestras clínicas / Biochemical characterization of strains of Staphylococcus sp isolated from clinical samples

Por el creciente rol en infecciones humanas de cepas de género Staphylococcus, en especial coagulasa negativas y el interés por conocer las especies involucradas, se estudiaron 100 cepas de Staphylococcus sp aisladas de diferentes muestras clínicas de pacientes hospitalizados y llevadas al laboratorio de Microbiología del Hospital Dr. Gustavo Fricke de Viña del Mar, Chile. El estudio abarcó un número considerable de pruebas bioquímicas y morfológicas, según los esquemas propuestos por Schleifer-Kloos y Kloos-Schleifer. Se obtuvieron 5 grupos de cepas bien definidas, un grupo que incluyó a 43 cepas coagulasa positivas se identificó con la cepa tipo de S. aureus, aisladas especialmente de procesos supurativos. Los cuatro grupos restantes fueron todos coagulasa negativos, el primer grupo con 25 cepas fue identificado con S. epidermidis, el segundo grupo con 20 cepas fue identificado con S. hominis, el tercero con 8 cepas se identificó como S. haemolyticus y el cuarto grupo con 4 cepas con S. saprophyticus. Destacándose que la mayoría de las especies coagulasa negativas se aislaron de sangre en especial S. hominis, se hacen consideraciones sobre el presunto rol que estas especies jugarían en los procesos infecciosos y la correcta identificación de las especies del género Staphylococcus

A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods.

A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 10(4) cfu/ml, and at ca. 10(5) cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperometric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.

A new and rapid method for the assay of bacterial fatty acids using high resolution capillary gas chromatography and trimethylsulfonium hydroxide.

Gas-liquid chromatography of cellular fatty acids is a useful tool for the identification of bacteria. Derivatization of bacterial fatty acids to methyl esters by conventional techniques is usually time-consuming and complicated. A new one-step technique using trimethyl-sulfonium hydroxide allows the direct formation of fatty acid methyl esters within 1-2 min. Some random examples of profiles demonstrate that straight, branched, saturated, unsaturated, hydroxy and cyclopropyl fatty acids match conventional preparations well. The method is a very sensitive one, since only a few colonies are sufficient for preparation of fatty acid methyl esters.

Separation and characterization of modified variants of recombinant human insulin-like growth factor I derived from a fusion protein secreted from Escherichia coli.

Human insulin-like growth factor I, IGF-I, was produced in Escherichia coli fused to a synthetic IgG-binding peptide The fusion protein is secreted into the medium during fermentation and was initially purified on an IgG-Sepharose column. After hydroxylamine cleavage, IGF-I was purified to homogeneity. During purification, impurities in the form of modified variants of IGF-I were detected and characterized. The closely related impurities were identified to be a misfolded form of IGF-I, having mismatched disulphide bonds, a form with the single methionine residue in IGF-I oxidized to methionine sulphoxide and a variant in which the methionine residue was substituted by a norleucine residue during protein synthesis. A form proteolytically cleaved between two arginine residue was also detected. These impurities were separated from the major component, native IGF-I, by using reverse-phase h.p.l.c. The modified molecules as well as native IGF-I were characterized both as intact molecules and as fragments, after pepsin digestion, using the techniques of plasma desorption m.s., N-terminal sequencing and amino acid analysis. The oxidized form was 90%, and the norleucine analogue was 70%, as potent as native IGF-I in a biological radioreceptor assay, and the form having mismatched disulphides lacked receptor affinity.

A novel labeling procedure of anteiso-fatty acid-containing lipids in staphylococci for investigating the effect of penicillin on lipid release.

L-[4,5-3H]isoleucine was introduced to label anteiso-fatty acid (AIFA)-containing lipids in Staphylococcus aureus SG 511. After an overnight incubation in peptone broth in the presence of 37 kBq L-[4,5-3H]isoleucine/ml, 8.5-13% of the total radioactivity applied was found to be incorporated into the cells. 22.4-25.6% of the incorporated radioactivity was found in AIFA-containing lipids extracted by chloroform-methanol-water (2:1:0.2, v/v/v) at pH 2. The interphase contained 70-75% of the incorporated radioactivity. Lipoteichoic acid, extracted by phenol-water (80:20, w/v) contained less than 1% of the incorporated radioactivity, as measured after purification by hydrophobic interaction chromatography on octyl sepharose gel. Within 1 h after addition of 10 micrograms/ml penicillin G to exponentially growing cultures of S. aureus, that led to non-lytic death of the cells, 11.9-18.1% of the incorporated L-[4,5-3H]isoleucine label were released. Lipids containing AIFA were excreted to 5.4-8.4% of total incorporated activity; this amount represents more than 1/4 of the labeled cellular lipids.

Empleo del dot-enzimoinmunoensayo en la valoración de la proteína A de Staphylococcus aureus / Dot-immunobinding assay use in valoration of protein A from Staphylococcus aureus

Se describe un método inmunoenzimático, que, por su gran sensibilidad, permite detectar pequeñas cantidades de proteína A presente en la membrana celular de determinadas cepas de Staphylococcus aureus o la liberada en el medio de cultivo. Esta técnica del dot-enzimoinmunoensayo permite poner en evidencia concentraciones de hasta 0,115 ug.ml-1 de proteína. Comparando los resultados obtenidos con la técnica de inmunodifusión radial, se confirma su mayor sensibilidad y la posibilidad de utilizar un método específico y de fácil realización en el dosaje de proteína A. (AU)

Empleo del dot-enzimoinmunoensayo en la valoración de la proteína A de Staphylococcus aureus / Dot-immunobinding assay use in valoration of protein A from Staphylococcus aureus

Se describe un método inmunoenzimático, que, por su gran sensibilidad, permite detectar pequeñas cantidades de proteína A presente en la membrana celular de determinadas cepas de Staphylococcus aureus o la liberada en el medio de cultivo. Esta técnica del dot-enzimoinmunoensayo permite poner en evidencia concentraciones de hasta 0,115 ug.ml-1 de proteína. Comparando los resultados obtenidos con la técnica de inmunodifusión radial, se confirma su mayor sensibilidad y la posibilidad de utilizar un método específico y de fácil realización en el dosaje de proteína A.

Isolations of protein A and protein G from the bacterial surface.

Ten tested cultures each of Staphylococcus aureus (S. aureus) and of Streptococcus belonging to serological group G bound human IgG to a high extent. Protein A could be solubilized from strain Cowan I of S. aureus by lysozyme, mutanolysine, hydroxylammoniumchloride, hot acid extraction or lysostaphin and subsequently purified by affinity chromatography on human IgG-sepharose. The purified protein A preparation had molecular weights between 29,000 and 63,000 D and inhibited binding of 125I-labeled human IgG to S. aureus Cowan I. Protein G could be solubilized from strain 26540 of the G-streptococci with lysozyme or hot acid extraction and purified by affinity chromatography on human IgG-sepharose. The purified protein G revealed a molecular weight of 67,000 D and inhibited binding of human IgG to the G-streptococci.

Direct analysis of bacterial fatty acids by Curie-point pyrolysis tandem mass spectrometry.

Although pyrolysis-mass spectrometry (Py-MS) has been used for bacterial taxonomy, many of the mass spectral peaks used for discrimination of organisms have not been correlated to known biomolecules. This work presents the discrimination of five bacterial species based on Py-MS patterns containing only peaks that can be correlated to fatty acids and fatty acid derivatives. These correlations were confirmed by pyrolysis-tandem mass spectrometry of authentic standards and the organisms. The pattern recognition procedures used gave better results when only the fatty acid peaks were used in the analysis than when full spectra were used.

Peptidoglycan cross-linking in Staphylococcus aureus. An apparent random polymerisation process.

The peptidoglycan of Staphylococcus aureus contains relatively short glycan chains and is highly cross-linked via its peptide chains. The material from wild-type (strain H) and mutants H28, H4B and MR-1 was freed from the teichoic-acid-linked component and then hydrolysed by Chalaropsis muramidase to yield disaccharide-repeating units of the glycan with attached peptides either non-cross-linked (monomer) or joined to similar units by one (dimer), two (trimer) or more (oligomer) peptide cross links. The resulting fragments were separated by high-resolution HPLC so that distinguishable components as large as nonamer could be identified. Extrapolation showed that, in S. aureus H, H28 and MR-1, oligomers at least as large as eicosamer formed part of the smooth distribution of oligomer fragments, whereas in strain H4B (PBP4-) the maximum size was around dodecamer. The oligomer distribution profile was related to the polymerization theories of Flory, which allow a distinction to be made between a monomer addition model, whereby each oligomer can only be synthesized by the addition of a single monomer unit to its next lower homologue, and a random addition model, in which an oligomer can be formed by linkage of any combination of its constituent smaller units. In S. aureus close approximation to the random addition model for oligomer synthesis and hence for peptidoglycan cross-linking was observed, both in PBP4+ and PBP4- mutants. The implications for secondary cross-linking in S. aureus cell wall formation are inescapable, although the possibility of an endopeptidase/transpeptidase providing later modification of the peptidoglycan is not completely ruled out.

Adhesive proteins of haemagglutinating Staphylococcus aureus isolated from bovine mastitis.

Two proteins derived from the cell wall of Staphylococcus aureus, exhibiting apparent molecular masses of 116 kDa and 145 kDa, were found to bind to human buccal and bovine lactiferous sinus epithelial cells. By using antibodies specific for fibronectin-binding protein of S. aureus of human origin, the 116 kDa protein, but not the 145 kDa protein, was identified as a fibronectin-binding protein. The 145 kDa protein bound to bovine fat globule membranes, human buccal epithelial cells, bovine lactiferous sinus epithelial cells and sheep erythrocytes. The properties of the 145 kDa protein suggest that it is an adhesin with a possible role in the early stages of the development of bovine mastitis.

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.

[Adherence properties and production of slime by staphylococci: relation to pathogenicity]. / Propriétés d'adhérence et production de slime des staphylocoques: relation avec la pathogénicité.

Among the S. aureus 25/39 are non producing slime and non adherent isolates. These results did not allow a correlation between these properties and pathogenesis. Several different phenotyping systems (biotyping, phage typing, serotyping, antibiotic susceptibility profiling, and plasmid pattern analysis) have been used in an attempt to identify strains of CNS. There is still a need however, for a simple, rapid, and cost effective method of distinguishing true pathogens from simple contaminants. Our results suggest that testing isolates for slime positivity and for adherence property may fulfill this task.

[Polyamines in representative taxa of coryneform and other bacteria]. / Poliaminy predstavitelei nekotorykh taksonov korinepodobnykh i drugikh bakterii.

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.