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Presence of Staphylococcus aureus with reduced susceptibility to vancomycin in Germany.

A total of 457 Staphylococcus aureus strains from the culture collection of the National Reference Center for Staphylococci in Bonn, Germany, were screened for susceptibility to vancomycin because some Staphylococcus aureus strains are able to form subpopulations that show intermediate resistance to vancomycin. Two methicillin-resistant Staphylococcus aureus strains (isolated in 1993) exhibited intermediate resistance. One of these, Staphylococcus aureus 137-93, which displayed the genomic DNA fragment pattern of the northern German epidemic strain, appeared homogeneously resistant. Neither of these strains had been identified by routine susceptibility testing. The resistance of the German isolates was lower than that of the Japanese isolate Mu50. To determine whether a similar mechanism confers vancomycin resistance in Staphylococcus aureus Mu50 and 137-93, the intracellular cell wall precursor concentration was measured and was not found to be comparably increased in Staphylococcus aureus 137-93. In conclusion, strains showing intermediate resistance have been present in Germany for some time (at least since 1993), but the subpopulations with decreased sensitivity were overlooked during antibiotic susceptibility testing.

Anti-Bacterial Agents↗

Staphylococcus aureus infection on cut wounds in the mouse skin: experimental staphylococcal botryomycosis.

Staphylococcus aureus cells were inoculated on the cut wounds in the skin of cyclophosphamide-treated mice. Biopsy specimens were taken from three mice at 1, 3, 6, 12, 24, 36, 48 and 60 h after the inoculation and were examined by light and electron microscopies. One hour after the inoculation Staphylococcus aureus cells were seen around the cut wound and deeper into the subcutaneous tissue. By 6 h after the inoculation, Staphylococcus aureus cells formed clusters of bacterial colonies. By 36 h after the inoculation inflammatory cells, mainly polymorphonuclear leukocytes and macrophages, were seen around the clusters. Electron microscopic examination revealed fibril-like structures around the Staphylococcus aureus cells at 1 h. The Staphylococcus aureus cells were enclosed in membrane-like structures at 3 h. The membrane-like structures and the fibril-like structures were positive for Ruthenium red. By 12 h after the inoculation, the membrane-like structures increased in thickness and in electron density. Inflammatory cells were seen around but outside of the membrane-like structures at 24, 36 and 48 h. At 60 h the tissues around the membrane-like structures were degenerated and almost necrotic. These results suggest that Staphylococcus aureus cells may form biofilm in dermal or subcutaneous tissues in a neutropenic condition.

Animals↗

[Postoperative mediastinitis due to methicillin resistant Staphylococcus epidermidis with low sensitivity to vancomycin].

BACKGROUND: Vancomycin is the drug of choice for methicillin-resistant Staphylococcus. Antibiotherapy failure is rarely clinically related to Staphylococcus with vancomycin low susceptibility. CASE REPORT: A surgical cure of an aortic stenosis in a neonate was complicated by a Staphylococcus mediastinitis. After initiation of antibiotherapy with vancomycin and rifampin and surgical debridement, there was a rapid improvement. Few days later, failure of therapy was obvious. Despite continuous infusion of vancomycin, with a serum level of 29 mg/L, blood cultures were positive again to Staphylococcus. There was no endocarditis or inadequate surgical drainage. Susceptibility of the Staphylococcus was tested, looking for a tolerant strain. The vancomycin minimum bactericidal concentration was 30 mg/L (above usual value 2 to 8 mg/L), while the minimum inhibitory concentration was 3.75 mg/L. A higher dosage of vancomycin associated with fusidic acid was rapidly efficient, and total recovery was achieved. CONCLUSION: In case of failure of vancomycin therapy, despite correct serum levels, the susceptibility of the Staphylococcus strain has to be determined. A low susceptibility strain prescribes more prolonged combination of two antibiotics.

Anti-Bacterial Agents↗

Antimicrobial susceptibility of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows.

The high prevalence of the great resistance to antimicrobials shown by the aetiological agents of infectious bovine mastitis, often leads to chronic recurrent mastitis, one of the main causes of early culling of dairy cows. The purpose of this study was to determine the in vitro susceptibility pattern of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows, to different anti-microbials. A total of 45 Staphylococcus spp. strains [33 coagulase-negative Staphylococcus (CNS) and 12 Staphylococcus aureus (SA)] were used. These strains were tested with 12 different antimicrobials by the Kirby and Bauer standardized disc diffusion method; 84.44% and 86.66% of the 45 strains were resistant to ampicillin and penicillin, respectively. The highest sensitivity was to cephalothin (84.44%), gentamicin (80%) and to sulphazotrin (77.77%). CNS showed higher resistance (P < 0.05) than SA. The in vitro susceptibility pattern of Staphylococcus spp. strains isolated from mammary parenchymas studied in the present investigation was similar to that observed in recent studies of in vitro and in vivo susceptibility patterns of Staphylococcus spp. isolated from cases of bovine mastitis.

Animals↗

Staphylococcus muscae, a new species isolated from flies.

A new coagulase-negative species of the genus Staphylococcus, Staphylococcus muscae, is described on the basis of the results of a study of four strains that were isolated from flies. 16S rRNA sequences of the type strains of S. muscae, Staphylococcus schleiferi, and Staphylococcus sciuri were determined and used, together with the corresponding sequences of Staphylococcus aureus and Staphylococcus epidermidis, for a comparative analysis. The new species is characterized taxonomically; this species is differentiated from the other novobiocin-susceptible staphylococci by its physiological and biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is strain MB4 (= CCM 4175).

Animals↗

Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus.

The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.

Amino Acid Sequence↗

Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer?

The biofilm-associated protein (Bap) is a surface protein implicated in biofilm formation by Staphylococcus aureus isolated from chronic mastitis infections. The bap gene is carried in a putative composite transposon inserted in SaPIbov2, a mobile staphylococcal pathogenicity island. In this study, bap orthologue genes from several staphylococcal species, including Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus simulans and Staphylococcus hyicus, were identified, cloned and sequenced. Sequence analysis comparison of the bap gene from these species revealed a very high sequence similarity, suggesting the horizontal gene transfer of SaPIbov2 amongst them. However, sequence analyses of the flanking region revealed that the bap gene of these species was not contained in the SaPIbov2 pathogenicity island. Although they did not contain the icaADBC operon, all the coagulase-negative staphylococcal isolates harbouring bap were strong biofilm producers. Disruption of the bap gene in S. epidermidis abolished its capacity to form a biofilm, whereas heterologous complementation of a biofilm-negative strain of S. aureus with the Bap protein from S. epidermidis bestowed the capacity to form a biofilm on a polystyrene surface. Altogether, these results demonstrate that Bap orthologues from coagulase-negative staphylococci induce an alternative mechanism of biofilm formation that is independent of the PIA/PNAG exopolysaccharide.

Adhesins, Bacterial↗

Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food.

AIMS: To develop a multiplex PCR that allows the identification of bacteria belonging to the Staphylococcus genus and in particular to the species Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus isolated from food manufacturing plants. METHODS AND RESULTS: Five primer pairs were used in the multiplex PCR, one specific to the Staphylococcus genus and four specific to S. xylosus, S. saprophyticus, S. epidermidis and S. aureus species. All the 31 Staphylococcus reference strains yielded a specific PCR product with the genus-specific primers. Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus gave a specific PCR fragment with the corresponding species-specific primers. No amplification with the Kocuria, Macrococcus and Micrococcus strains was observed in our conditions. This multiplex PCR was performed on 30 strains of Gram-positive cocci isolated from different workshops and fermented sausages. Among them, 28 belonged to the Staphylococcus genus and 14 were identified to S. saprophyticus, four to S. xylosus, two to S. aureus and one to S. epidermidis. CONCLUSIONS: This multiplex PCR provided reliable and repeatable PCR results. It allowed the identification of a major part of the isolates, highlighting the predominance of the S. saprophyticus species in the workshops studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This tool is a useful way to screen the strains isolated from foodstuff and food environment and to monitor these species during the food processing.

Bacterial Typing Techniques↗

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis.

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.

Animals↗

Preliminary studies on the characterization and distribution of Staphylococcus and Micrococcus species on animal skin.

A total of 221 strains of staphylococci and 98 strains of micrococci isolated from the skins of Eastern gray squirrels, Southern flying squirrels, raccoons, opossums, squirrel monkeys, swine, sheep, horses, cattle, and dogs were characterized in a preliminary attempt to resolve their natural relationships and distribution in nature. Staphylococci demonstrating the widest host range included Staphylococcus xylosus and unnamed Staphylococcus sp. 3. Unnamed Staphylococcus sp. 2 was isolated only from sheep, Staphylococcus sp. 4 only from opossums, Staphylococcus sp. 5 only from squirrel monkeys, and Staphylococcus sp. 6 only from swine. The predominant species isolated from human skin, including S. epidermidis, S. hominis, S. haemolyticus, and S. capitis, were either not isolated or only rarely isolated from animal skin. Micrococcus varians was the predominant Micrococcus species isolated from animal skin. M. luteus was only occasionally isolated. M. lylae, M. sedentarius, M. roseus, M. kristinae, and M. nishinomiyaensis, species occasionally isolated from human skin, were not isolated from animal skin.

Animals↗

Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter.

Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC transporters with proven or putative metal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it may be involved in either siderophore- or transferrin-mediated iron uptake in S. epidermidis.

ATP-Binding Cassette Transporters↗

Recognition of Staphylococcus saprophyticus in urine cultures by screening colonies for production of phosphatase.

Phenolphthalein diphosphate was incorporated into a primary blood agar medium for use in performing quantitative urine cultures. Phosphatase-negative staphylococci, such as Staphylococcus saprophyticus, were differentiated from phosphatase-positive species, such as Staphylococcus epidermidis, by spot testing colonies on filter paper saturated with 1 N NaOH. Phosphatase-positive colonies turned pink within seconds, and phosphatase-negative colonies showed no color. None of 55 S. saprophyticus isolates showed production of phosphatase on this medium. Of 193 consecutive coagulase-negative staphylococci isolated from the urine of 190 adolescent female patients, 84% were phosphatase positive, non-S. saprophyticus species; 16% were phosphatase-negative and indicated S. saprophyticus (22), Staphylococcus haemolyticus (4), Staphylococcus simulans (2), Staphylococcus warneri (1), and Staphylococcus hominis (1). Phosphatase activity was variable in the other flora encountered in the urine cultures. Mixtures of phosphatase-positive and -negative organisms did not cause false-positive reactions.

Adolescent↗

Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection.

Consecutive record review over a 63-month period revealed 229 Staphylococcus lugdunensis isolates, or 10.1% of the staphylococcal species that were not Staphylococcus aureus or Staphylococcus epidermidis. A total of 155 S. lugdunensis specimens were isolated from sites over the entire bodies of the 143 patients studied. The most common clinical diagnoses were skin and skin structure infections (55.4%) and blood and vascular catheter infections (17.4%). For 40% of the reviewed specimens, S. lugdunensis was the sole agent isolated, and for 60% of specimens, S. lugdunensis was isolated as part of mixed flora. In only 15.4% of clinically reviewed specimens was S. lugdunensis clearly a culture contaminant or colonizing organism. The pattern of human infection identified in this study emphasizes the predominance of skin and soft tissue S. lugdunensis infections over deep serious infections such as endocarditis, peritonitis, infected hip prosthesis, and osteomyelitis and vascular-associated infections. S. lugdunensis should be included along with S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus saprophyticus as a coagulase-negative species of Staphylococcus pathogenic for humans.

Adolescent↗

Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp.

The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.

Glyceraldehyde-3-Phosphate Dehydrogenases↗

Reduction in methicillin-resistant Staphylococcus aureus infection rate in a nursing home by aggressive containment strategies.

For the month of October, 1993, the Methicillin-Resistant Staphylococcus aureus nosocomial infection rate in our 42-bed Extended Care Unit/Nursing Home was 33% (8.5% for the 1993 year). Our facility was committed to decrease colonization and infection rates and to prevent the introduction of additional colonized patients into the closed environment. Methicillin-Resistant Staphylococcus aureus containment practices were instituted and consisted of total population and staff surveillance, aggressive containment measures and followed by maintenance containment protocols. The aggressive plan included contact isolation, baths with chlorhexagluconate, treatment of nasal carriers with mupiricin and treatment of both colonized and infected patients. This was followed by maintenance measures of screening new admissions for Methicillin-Resistant Staphylococcus aureus with contact isolation and treatment for positive as described during the aggressive phase. Total population surveillance was repeated after one year. Results showed that no employees were colonized with Methicillin-Resistant Staphylococcus aureus. The initial colonization rate in residents/patients was 52%. After one year the colonization rate dropped to 2% and the infection rate to 1.4%. Molecular epidemiology demonstrated that there was limited acquisition of new strains of Methicillin-Resistant Staphylococcus aureus within the Extended Care Unit. The process was shown to be cost effective. Aggressive containment practices applied to a nursing home with a high Methicillin-Resistant Staphylococcus aureus infection rate not only reduced rates of colonization, but also markedly reduced infections. This reduction was maintained over time.

Aged↗

[Resistant antibiotic analysis of methicillin-resistant staphylococcus].

Methicillin-resistant staphylococcus aureus is one of the important patholonic bacteria which cause nosocomial infection. In order to investigate the resistant antibiotic circumstances of this organism and hence provide foundations of prevention and treatment, we determined the resistant rates of 88 staphylococcus strains for methicillin and for other 14 kinds of commonly used antibiotics, and we determined the engendering beta-lactamase. The results showed that the isolation rate of methicillin-resistant staphylococcus aureus (MRSA) was 56%; the rate of methicillin-resistant staphylococcus coagulase-negative (MRSCoN) was 47.6%; the resistance rate and the multi-resistant rate of methicillin-resistant staphylococcus (MRS) for the 14 kinds of antibiotics were higher as compared with those of methicillin-sensitive (MSSA). Among all the tested strains, MRSA was the highest to engender beta-lactamase (92.9%); Methicillin-sensitive staphylococcus coagulase-negative (MSSCoN) was the lowest (39.4%). MRS was sensitive to vancomycin and furantoin. These suggest that when treating this bacterium, we should select these two antibiotics first.

Anti-Bacterial Agents↗

[Antimicrobial resistance of Staphylococcus aureus isolates from inpatients of departments of internal medicine, surgery, and pediatrics and intensive care unit].

OBJECTIVE: To investigate the antimicrobial resistance of Staphylococcus aureus isolates obtained from inpatients of departments of internal medicine, surgery, and pediatrics and intensive care unit (ICU), and study on the differences of resistant rates among the clinical isolates. METHODS: The strains of Staphylococcus aureus were cultured and their antimicrobial resistance was assayed by disc diffusion test (K-B method) and the data was analyzed by WHO NET 5 software. chi(2) test was made to identify the significance of difference. RESULTS: 2 625 strains of Staphylococcus aureus were obtained from the clinical departments of 60 hospitals all over China, among which 1 669 strains (63.3%) were obtained from the inpatients of departments of internal medicine, surgery, and pediatrics and ICU from 1 January to 31 December 2001. Most of the strains were isolated from sputum (34.3%, 572/1 669), secretion (13.2%, 221/1 669), pus (11.7%, 195/1 669), blood (10.2%, 171/1 669), and wound (5.6%, 94/1 669). The resistant rates of Staphylococcus aureus isolates from the inpatients in department pediatrics of to oxacillin, gentamicin, clindamycin, ciprofloxacin, levofloxacin, and chloramphenicol were 23.3%, 16.1%, 29.3%, 11.2%, 4.0% and 14.4% respectively, all significantly lower than those of the isolates from the inpatients of the departments of internal medicine and surgery, and ICU (all P < 0.001). The resistant rates of Staphylococcus aureus isolates from the inpatients of departments of internal medicine and surgery to oxacillin, gentamicin, clindamycin, ciprofloxacin, and levofloxacin were significantly lower than those of isolates from the patients of ICU (all P < 0.001). CONCLUSION: A high isolation rate of multi-drug resistant Staphylococcus aureus among the inpatients of departments of internal medicine, surgery, and pediatrics and ICU. It is important to select appropriate antimicrobial agents based on the origin of bacterial strains and avoid blindness before the results of drug sensitivity are obtained.

Anti-Bacterial Agents↗

[Drug resistance analysis of staphylococcus infection in our hospital from 2003 to 2005].

OBJECTIVE: To explore the change in mobility of staphylococcus and its drug resistance etiology investigation and clinical treatment. METHODS: The routine biochemical identification was used for staphylococcus differentiation. Minimal inhibitory concentrations was used for drug-resistance determination. Some drug-resistance determination were detected by K-B method. The inducible resistance of erythromycin to clindamycin was checked by D-test. RESULTS: Staphylococcus was in the first place in the hospital infection. The rates of methicillin-resistant staphylococcus were 54.1%. The drug-resistance rates of staphylococcus to penicillin, oxacillin, erythromycin, tetracycline, ciprofloxacin, gentamicin, clindamycin, SMZCO, chloramphenicol, vancomycin, teicoplanin antibacterials were 93.2%, 54.1%, 85.1%, 56.7%, 45.9%, 48.6%, 58.1%. 45.9%, 31.1%, 0%, 0%. D-test positive rate was 37.9%. CONCLUSIONS: The results are helpful in study of pathogenic bacteria and drug resistance characteristics in staphylococcus infection.

Anti-Bacterial Agents↗