Slime production by Staphylococci isolated from prosthesis-associated infections.

Clinical isolates of Staphylococcus epidermidis are frequently referred to produce a biofilm, known as slime, involved in adherence to medical devices and in resistance to host defences. A high frequency of slime producing Staphylococcus aureus strains was never reported, at least in the case of human isolates. In the present study the production of slime by clinical isolates of S. aureus and S. epidermidis from catheter associated infections and from post-surgical infections was studied by a sensitive method based on culturing the isolates on Congo red agar. The study demonstrates that in nosocomial surgical infections, considered separately from catheter-associated infections, S. aureus emerges as a more prevalent etiologic agent than S. epidermidis, with a proportion of slime producing strains markedly high.     

Antimicrobial sensivity and ability to slime production of koagulaze-negative staphylococci

The aim of this study was to assess the ability of slime production ofcoagulase-negative staphylococci (CONS) and evaluate the susceptibility of bacteria to antibiotics. Strains were isolated from clinical specimens obtained from hospitalized patients. The most frequently isolated species were S. epidermidis (51%), S. hominis (18%), S. haemolyticus (13%). The result of this study shows that 61% of S.epidermidis produce slime on CRA (Congo red agar), whereas none of the tested S. haemolyticus strains has this ability. All examined strains were susceptible to vancomycin, linezolid and quinupristin/ dalfopristin. The majority of strains were susceptible to minocycline, fusid acid, nitrofurantoin and rifampicin. Sixty six percent of isolates were determined as methicillin-resistant coagulase-negative staphylococci.     

A novel application of Fourier-transformed infrared spectroscopy: classification of slime from staphylococci

It has been proposed that the virulence of nosocomial Staphylococcus infections associated with indwelling medical devices is related to the ability of the bacterium to colonise these materials by forming a biofilm composed of multilayered cell clusters embedded in a slime matrix. However, the pathogenic role of exopolysaccharide biofilms is not fully understood. A new method was sought for differentiating the structure of slime from two closely related bacterial strains, Staphylococcus aureus and Staphylococcus epidermidis. Using PCR it was confirmed that these strains were positive for the icaA and icaD genes and the complete ica operon (2.7 kb). Monosaccharide analysis by thin-layer chromatography revealed an identical profile for both strains, with xylose and glucose present among the four visible bands. Using Fourier-transformed infrared spectroscopy and hierarchical cluster analysis, three of four S. aureus samples (75%), and four of five S. epidermidis samples were grouped according to species. A novel FTIR approach in classifying slime produced by S. aureus and S. epidermidis is reported.     

A multi-center blinded study on the efficiency of phenotypic screening methods to detect glycopeptide intermediately susceptible Staphylococcus aureus (GISA) and heterogeneous GISA (h-GISA)

Background

Staphylococcus aureus, one of the most frequently isolated pathogens in both hospitals and the community, has been particularly efficient at developing resistance to antimicrobial agents. In developed countries, as methicillin-resistant S. aureus (MRSA) has prevailed and, furthermore, as S. aureus with reduced susceptibility to vancomycin has emerged, the therapeutic options for the treatment of S. aureus infections have become limited. In developing countries and especially African countries very little is known concerning the resistance of S. aureus to antibiotics. In Madagascar no data exist concerning this resistance.

Objective

To update the current status of antibiotic resistance of S. aureus in Antananarivo, Madagascar.

Methods

Clinical S. aureus isolates were collected from patients at the Institut Pasteur of Madagascar from January 2001 to December 2005. Susceptibility tests with 18 antibiotics were performed by the disk diffusion method.

Results

Among a total of 574 isolates, 506 were from community-acquired infections and 68 from nosocomial infections. There was no significant difference in the methicillin resistance rate between community-acquired strains (33 of 506; 6.5%) and nosocomial strains (3 of 68, 4.4%). Many MRSA isolates were resistant to multiple classes of antibiotics. Resistance to tetracyclin, trimethoprim-sulfamethoxazole and erythromycin was more common. Among MRSA isolates resistance rates to rifampicin, fusidic acid, gentamicin and ciprofloxacin were lower than that observed with other drugs easily available in Madagascar. No isolates were resistant to glycopeptides.

Conclusion

The rate of methicillin-resistant S. aureus is not different between community-acquired and nosocomial infections and is still rather low in Madagascar.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from clinical specimens of patients with nosocomial infection: are there unnoticed silent outbreaks?

Bacteriological and epidemiological studies were carried out on 90 isolates of methicillin-resistant Staphylococcus aureus (MRSA) at Turgut Ozal Medical Center of In?nü University, (Malatya/Turkey). MRSA isolates were obtained from patients with nosocomial infections. Staphylococcus aureus clinical isolates were collected between May 2004-May 2005. Isolates were tested for resistance to methicillin. Antimicrobial susceptibility testing and slime production evaluation was performed. Genotype studies were carried out by arbitrarily primed polymerase chain reaction (AP-PCR) and consequent cluster analysis. All of the isolates were mecA-positive in a PCR-based assay; all exhibited resistance to oxacillin, by agar dilution (MICs > or = 4 mg/L) and disc diffusion methods, and multiple antibiotics. Most MRSA isolates were collected in intensive care units. Of 90 samples, 53 were found to be unrelated to the others while the remaining 37 strains were either identical or closely related. Dendrogram analysis identified nine major clusters. These data support the opinion that MRSA are significant nosocomial pathogens in intensive care units and that resistant clones may be transmitted between patients. Molecular epidemiological tools are helpful for understanding transmission patterns and sources of infection, and are useful for measuring outcomes of intervention strategies implemented to reduce nosocomial MRSA.     

Slime-producing Staphylococcus epidermidis and S. aureus in acute bacterial conjunctivitis in soft contact lens wearers

In recent years, an increase in ocular pathologies related to soft contact lens has been observed. The most common infectious agents were Staphylococcus spp. Some strains produce an extracellular polysaccharidic slime that can cause severe infections. Polysaccharide synthesis is under genetic control and involves a specific intercellular adhesion (ica) locus, in particular, icaA and icaD genes. Conjunctival swabs from 97 patients with presumably bacterial bilateral conjunctivitis, wearers of soft contact lenses were examined. We determined the ability of staphylococci to produce slime, relating it to the presence of icaA and icaD genes. We also investigated the antibiotic susceptibility and Pulsed Field Gel Electrophoresis (PFGE) patterns of the clinical isolates. We found that 74.1% of the S. epidermidis strains and 61.1% of the S. aureus strains isolated were slime producers and showed icaA and icaD genes. Both S. epidermidis and S. aureus slime-producing strains exhibited more surface hydrophobicity than non-producing slime strains. The PFGE patterns overlapped in S. epidermidis strains with high hydrophobicity. The similar PFGE patterns were not related to biofilm production. We found scarce matching among the Staphylococcus spp. studied, slime production, surface hydrophobicity and antibiotic susceptibility.     

新生儿耐甲氧西林凝固酶阴性葡萄球菌败血症40例分析

目的：探讨耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）在新生儿败血症中的分布,临床特征及耐药性特点。方法：应用全自动血培养系统（BACTE/9050）,ATB细菌鉴定仪,从80例新生儿血培养阳性瓶中分离出40株MRCNS。应用ATB药敏系统,测定了青霉素等17种抗生素的耐药性。结果：80株细菌中,分离出40株MRCNS（占50%）,其中溶血葡萄球菌17株（42.5%)居首位,表皮葡萄球菌15株（37.5%),其他葡萄球菌8株（20%）。药敏结果显示多重耐药。结论：凝固酶阴性葡萄球菌（CNS）已成为小儿血培养的第一位检出菌,主要为溶血葡萄球菌和表皮葡萄球菌,MRCNS占主导地位（达50%左右）,成为新生儿病房院内感染的主要致病菌。药敏结果显示MRCNS呈多重耐药,万古霉素及其他糖肽类抗生素是治疗MRCNS感染的首选药物,MRCNS在新生儿病房造成院内感染的危害性以及耐药性应引起广泛关注。     

Circulation of methicillin-resistant staphylococci in hospitals with different profiles

Analysis of prevalence of methicillin-resistant staphylococci in hospitals with different specializations was performed. 2584 strains were isolated. Methicillin-resistant (MR) strains were present in different profile hospitals and their total prevalence between isolated strains was 12.3% while significantly varied in different hospitals (from 8% to 37%). Along with MRSA (methicillin-resistant Staphylococcus aureus), MRSE (methicillin-resistant Staphylococcus epidermidis) and MRSS (methicillin-resistant Staphylococcus saprophyticus) were presented in all studied hospitals. The prevalence of MR strains was highest among strains isolated from flame burn wounds (37%), while in samples from newborns in maternity hospital resistant strains represented 12% of isolates, and in general clinical hospital--not more than 9% of isolates. Relationship between rates of isolation of methicillin-resistant staphylococci and specialization of hospital unit was noted. For example, prevalence of MR staphylococci in isolates from newborns in ICU (47.5%) differed from the same one in maternity hospital (11.6%).     

Antibiotic resistance dynamics and isolation rate of staphylococci and enterococci from patients of reconstructive surgery units

The dynamics of isolation of staphylococci and enterococci from clinical material of patients and their antibiotic susceptibility within a 5-year period (2005-2009) was analysed. 5990 isolates were tested: 1250 isolates of Staphylococcus aureus, 3268 isolates of S. epidermidis, 1005 isolates of Enterococcus faecalis and 467 isolates of E. faecium. Grampositive infections were shown to be prevailing within the last 2-3 years, the nosocomial epidermal staphylococci more and more replacing S. aureus (the ratio of S. epidermidis and S. aureus in 2009 was 3.3). The isolation rate of E. faecalis significantly increased (by 3.5 times) and the ratio of E. faecalis and E. faecium in 2009 was 4.3. The microflora composition with respect to the isolation source was analysed and its clinical significance was estimated. The study of the antibiotic susceptibility showed that oxacillin had its own specific niche, while antibiotics active against resistant grampositive cocci, such as rifampicin, fusidic acid, fluoroquinolones (moxifloxacin), cefoxitin, as well as amoxicillin/clavulane in infections due to E. faecalis, might be considered as the drugs of choice. In the treatment of nosocomial infections, when the etiological role of MRSA or VRE is suspected or confirmed, the complex therapy should obligatory include the most active antibiotics (vancomycin or linezolid among them).     

Congo red agar plate method: improved accuracy and new extended application to Staphylococcus aureus.

In the last decade an increasing number of research studies have focused on the role of slime formation in Staphylococcus epidermidis and, more recently, also in S. aureus. In this context, much attention is being paid to evaluating the prevalence of slime production among bacteria strains isolated from clinical infections in an attempt to assess the role and the diagnostic value of this well recognised virulence marker. Such types of investigations require reliable techniques to identify slime producing strains. For years, even though based on a subjective chromatic evaluation, the Congo red agar plate (CRA) represented a reference phenotypic test for S. epidermidis. Only with the new introduction of PCR-based techniques, able to specifically identify the genes necessary for slime production, did the accurate genetic classification of slime producing bacteria become possible. In the present investigation, a comparison with new PCR methods confirmed the validity of the classic CRA test, implemented with minor refinements. Thanks to a few modifications it was also possible to adapt and extend the CRA test, making it also suitable to screen S. aureus strains.     

Phage pattern and antibiotic resistance pattern of coagulase-negative staphylococci obtained from immunocompromised patients.

A total of 152 coagulase-negative staphylococcal strains were isolated from clinical samples of 14 patients hospitalized after bone-marrow transplantation in a specialized hospital ward in Hungary, during an 18-month period between 1987 and 1989. Two species, Staphylococcus epidermidis and Staphylococcus haemolyticus, predominated (each, 45%). Using Pulverer and co-workers' phage set for typing, 68% of the isolates were typable; 16 phage patterns were observed. A characteristic long pattern with phages Ph10/Ph13/Ph15/U4/U15/U16/U20/U33 /U46 appeared only in S. epidermidis, among 5 of 11 colonized patients (8.5% of all strains). Single lysis with phage Ph13 was observed in 7 of the 14 patients (49% of all strains), in species S. capitis, S. epidermidis, S. haemolyticus, S. hominis, and S. warneri. In S. haemolyticus, non-typable strains predominated (66%); this character occurred only in 2% among other species. The strains colonizing the immunocompromised patients differed from each other in phage pattern, antibiotic resistance pattern, and/or slime production. No hospital infection was suggested. On the other hand, high incidence of two well-definable phage patterns raises some relationship between phage receptors or some regulatory systems in phage multiplication and factors responsible for special colonization as common surface properties.     

The effects of magnesium, calcium and EDTA on slime production byStaphylococcus epidermidis strains

Effect of magnesium, calcium and EDTA on slime production by 15 slime-positive and 13 slime-negative Staphylococcus epidermidis strains isolated from various clinical specimens was determined. The slime production on tryptic soy broth was significantly enhanced after addition of 128 mumol/L Mg2+. Similarly, the addition of Ca2+ caused a significant increase in slime production of all tested strains when concentration of Ca2+ exceeded 64 mumol/L. In contrast, in the presence of EDTA the slime production by all strains was significantly reduced. Hence Ca2+ and Mg2+ increase slime production of S. epidermidis. This finding is important in the context of the pathogenesis of biomedical implant infections caused by S. epidermidis.     

Problems of prevalance and resistance of nosocomial Klebsiella pneumonia in hospitals of Russia]

Prevalence of Klebsiella pneumoniae among gramnegative pathogens of nosocomial infections in intensive care units of 33 hospitals of 22 towns in Russia was investigated. Antibiotic susceptibility and extended-spectrum beta-lactamase production were tested in 420 nosocomial K. pneumoniae isolates. Carbapenems (imipenem, meropenem and ertapenem) showed the highest activity. Extended-spectrum beta-lactamase production based on the phenotyping methods was revealed in 342 (81.4%) isolates. The maximum activity against the K. pneumoniae isolates producing extended-spectrum beta-lactamase was observed in imipenem and meropenem (no unsusceptible strains were isolated). 3.2% of the isolates was not susceptible to ertapenem. Differences in the activity of cefoperazone/sulbactam, amikacin, ciprofloxacin and levofloxacin against the extended-spectrum beta-lactamase producing isolates in various hospitals were recorded.     

Species-specific sensitivity of coagulase-negative Staphylococci to single antibiotics and their combinations

The activity of beta-lactam antibiotics (oxacillin, cloxacillin, cephalotin), vancomycin, gentamicin and rifampicin applied in vitro individually and in combination against 37 nosocomial methicillin-resistant strains of coagulase-negative staphylococci (CNS) was assessed to demonstrate the heterogeneity of this group of bacteria and estimate the chance of the efficacy of such therapy. The strains belonged to four species: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus cohnii, Staphylococcus hominis. They originated from a hospital environment and from the skin of medical staff of the intensive care unit of a paediatric ward at a university hospital. All strains were methicillin-resistant, according to CLSI standards, but individual strains differed in MIC(ox) values. Susceptibility to other tested antibiotics was also characteristic for the species. The increased susceptibility to antibiotics in combinations, tested by calculating the fractional inhibitory concentration (FIC) index, concerned 26 out of 37 investigated strains and it was a feature of a particular species. Combinations of vancomycin and cephalotin against S. epidermidis and oxacillin with vancomycin were significant, as well as cephalotin and rifampicin in growth inhibition of multiresistant S. haemolyticus strains.     

Adhesion dependence of coagulase-negative staphylococci to biomaterials and PIA polysaccharide synthesis on icaADBC operon presence

Both Staphylococcus epidermidis and Staphylococcus haemolyticus are important causes of infections associated with catheters and other medical devices. This infections result in significant morbidity, mortality and economic cost. It has recently been shown that not only S. epidermidis but also S. haemolyticus can produce slime and carries the ica operon responsible for and slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. This study is focused on detecting icaA and icaD genes in S. haemolyticus and comparison of these two species. It turned out that strain representatives within the same species behave very differently and a single tested strain from each species is unlikely to be representative of the species as a whole. Contrary to S. epidermidis, S. haemolyticus strain appeared to carry no icaA-like and icaD-like genes, but was able to form biofilm in vitro.     

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.     

Biomaterial-associated staphylococcal peritoneal infections in a neutropaenic mouse model

Abstract Adhesion of staphylococcal cells to polyethylene with end point-attached heparin was quantified by bioluminescence. Staphylococcus epidermidis 3380 and the slime-producing S. epidermidis RP12 adhered to the highest extent, and S. lugdunensis 2342 to the least extent. Preincubation of the polymer with dialysis fluid reduced adhesion of S. epidermidis 3380 and RP12 but enhanced that of S. aureus , and preadsorption of the surface with fibronectin decreased subsequent adhesion of S. epidermidis and S. haemolyticus strains. When staphylococci were grown in the presence of a biomaterial their ability to activate peritoneal cells was decreased. The bactericidal activity was impaired, whereas ingestion of opsonized coagulase-negative staphylococci (CNS) strains was unaffected. With S. epidermidis RP12 the presence of biomaterial did not influence either phagocytosis or bactericidal effect of peritoneal cells. After intra-peritoneal challenge with staphylococcal strains, the organ uptake of S. aureus Cowan 1 was increased in normal mice whereas immunosuppressed mice died. CNS strains increased mainly in the peritoneal cavity of immunosuppressed mice. The uptake of bacteria in liver and kidneys was increased with S. epidermidis 3380, S. lugdunensis 2343 and S. schleiferi 667-88. Generally, CNS strains persisted in the peritoneal cavity of both normal and immunosuppressed mice. These data indicate that host defense mechanisms, mainly polymorphonuclear neutrophils, fail to eliminate CNS infections in the peritoneum, and that initial adhesion to an implanted biomaterial may be of lesser importance in the peritoneal cavity than in e.g. catheter-associated infections. There are strain-specific virulence factors of bacteria, and slime producing strains evade the host defense mechanisms more efficiently than non-slime producing strains.     

Staphylococcal and other Bacterial Species Associated with Intramammary Infections in Danish Dairy Herds

Four thousand six hundred forty– five quarter milk samples from 1179 cows from 20 commercial dairy herds were examined in order to determine the prevalence of bacterial species. A total of 859 isolates from 839 (18.1%) culture positive samples could be assigned to 34 different species and subspecies. Diagnostics of staphylococcal species was based on conventional procedures able to differentiate between all 36 species and subspecies presently acknowledged. Staphylococcus aureus was found in 10.2% of the samples and was the most common species isolated. Streptococcus dysgalactiae (1.6%) and Streptococcus uberis (1.4%) were the second and third most common species isolated. Seventeen different coagulase negative staphylococcal species (CNS) were found in 4.1% of the samples. The most frequently isolated CNS were S. epidermidis (1.3%), S. chromogenes (1.0%) and S. simulans (0.7%). Isolates of S. aureus were phage typed, and isolates of S. epidermidis were investigated by phage typing, antibiogram typing, and biotyping. A total of 378 (79.9%) isolates of S. aureus could be typed by phages, assigning them to 18 different phage types. However, 6 phage types accounted for 92.1% of the typable isolates. One to 2 phage types predominated within each herd. Eleven (18%) isolates of S. epidermidis could be typed by phages, assigning the isolates to 3 different types. Biotyping of S. epidermidis produced a total of 8 different types, the most common accounting for 29.5% of the isolates. A total of 6 different antibiogram types were observed among all isolates of S. epidermidis. Resistance towards penicillin (36.1%), tetracycline (9.8%) and streptomycin (9.8%), were recorded in the isolates of S. epidermidis. However, 35 (57.4%) of the isolates were susceptible to all 12 antibiotics tested.     

Community-acquired methicillin resistantStaphylococcus aureus isolated in dermatology department

Community-acquired methicillin-resistantStaphylococcus aureus (CA-MRSA) strains, known as a nosocomial pathogen, have emerged in the community worldwide. CA-MRSA infects frequently young children and is implicated in skin and soft tissue infections. In the present study, we reported the isolation of CA-MRSA strains from elderly patients admitted to the dermatology department at University Hospital of Monastir. The relatedness of these isolates was investigated by PFGE typing which indicated that all strains were clonally related. MRSA strains were thoroughly characterized by molecular methods which revealed that all isolates possessed the unique sequence type ST80 as well as a single spa type t044. Whole genotypic results suggest that all isolates were PVL producing CA-MRSA and were closely related and belonging to the ST80 clone.