Selected features determine pathogenicity of Staphylococcus haemolyticus

The study have been done on S. haemolyticus strains isolated from patients hospitalized an Surgical Unit. Aim of the study was to determine pathogenic traits of S. haemolyticus: slime producing, adhesion to biomaterials, antibiotics susceptibility and the profiles of surface proteins. Among 44 S. haemolyticus strains, in the test-tube method, there have been 38% labeled as slime producing and 62% as non-producing. In the plate method at 48% slime production was noticed, while 52% strains did not produce slime. It is quite significant that all CNS strains which have an ability to produce mucus, that was proved by means of two methods (test-tube and plate), show a high level of TTC's reduction to formazan. The analysis of resistance to antibiotics in relation to slime production demonstrated more frequent antibiotic resistance of the slime-producing strains.     

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5%) were found to be slime producers by Christensen's test tube method whereas 58 strains (31%) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1%) and erythromycin (47.1%) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2%) and 36 (62.1%) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4%) of 129 S. aureus strains were positive for beta-lactamase enzyme. However, 78 (81.25%) of 96 beta-lactamase positive S. aureus strains were beta-lactamase positive ORSA isolates, but none of them had vancomycin resistance.     

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.     

Detection of Pseudomonas aeruginosa biofilm on medical biomaterials

The adhesion and biofilm formation of Pseudomonas aeruginosa strains on the surface of catheters made of various polymers (PU, SL, PCW) were determined in vitro. It was used the method by Richards et al. with modification of Rózalska et al. (1998), in which soluble colourless TTC is reduced to insoluble red formazan. The results of this study indicate that 80.3% of this strains adhered and 94.6% formed biofilm on the Nelaton catheter, 86% strains adhered and 76.1% formed biofilm on the polyurethane catheter, and 73.2% strains adhered, and 78.9% formed biofilm on the Foley catheter.     

Detection of the intercellular adhesion loci (ica) in clinicalStaphylococcus aureus strains responsible for hospital acquired auricular infection

In clinicalStaphylococcus aureus strains, the presence of theica genes, biofilm formation and susceptibility to antibiotics are considered important factors of virulence. In this study, 35 strains ofS. aureus, isolated from auricular infection, were investigated for slime production using Congo red agar (CRA) method, antibiotic susceptibility, presence ofmecA gene, and presence oficaA andicaD gene. The results show that 60% of strains weremecA positive when tested by PCR although 25.7% of strains were oxacillin resistant when tested with ATB STAPH. Qualitative slime production ofS. aureus using CRA revealed that 74.3% ofS. aureus were slime producers. All the strains carried theica gene.     

Comparison of three methods detection of slime production by Staphylococcus aureus and Staphylococcus epidermidis

In this article, slime production of Staphylococcus aureus and Staphylococcus epidermidis strains from infective skin lesions was evaluated by three different methods: Congo red agar method (CRA), Christensen tube method (CT) and spectrophotometric method (SC). All strains by CT method interpreted as negative (dark-claret or red colonies of the surface). 12 (37.5%) strains of S. aureus, 16 (50.0%) strains of S. epidermidis produced slime as shown by CT method, 6 (18.7%) strains of S. aureus, 8 (25,0%) strains of S. epidermidis by SC method. They also found a correlation of slime production by CT and SC method (p > 0.05).     

Biofilm detection and the clinical significance of<Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> isolates

The ability of Staphylococcus epidermidis to produce biofilm was compared in 147 clinically significant strains repeatedly isolated from blood cultures of patients with bloodstream infection and in 147 strains isolated from skin. The strains were examined for the presence of ica operone, for the ability to form biofilm by Christensen's test-tube method and for the production of slime by Congo Red agar method. The ica operone was found in 92 (62.6 %) blood isolates and in 44 (29.9) isolates from skin. Christensen's test-tube method was positive in 79 (53.7) and 33 (22.4), Congo Red agar method in 64 (43.5) and 31 (21.1) of blood and skin isolates, respectively. All three methods were more frequently positive in clinically significant isolates from blood than in strains isolated from skin. The detection of ica operone and the Christensen's test-tube method showed better correlation with the clinical significance than the Congo Red agar method.     

Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci

Biofilm is one of the known virulence factors of staphylococci, a human and animal pathogen and commensal. Some of the strains become invasive under favorable conditions while others do not cause disease. Early detection and management of potentially pathogenic staphylococci is the essential step to prevent device-associated infections. There is also a need to evaluate one simple method for the detection of potential pathogens. Hence this study was planned to study the difference in potential of commensal, colonizing and invasive strains of staphylococci to produce biofilm. We used one qualitative (Congo red agar) and one quantitative (microtiter plate) method for detection of biofilm production and evaluated the sensitivity and specificity of Congo red agar method by using microtiter plate method as a gold standard. We consecutively enrolled staphylococcal strains isolated from peripheral intravenous device (IVD), venous blood, site of IVD insertion and nasal mucosa of patients admitted to pediatric ward with peripheral intravenous devices in place for more than 48 h. Total 100 invasive, 50 colonizing and 50 commensal isolates were studied. Of 100 invasive isolates 74% (74/100) were biofilm positive while only 68% (34/50) colonizing and 32% (16/50) commensal isolates were biofilm positive. The difference in biofilm production by commensal, colonizing and invasive strains was statistically significant (p<0.0001). Sensitivity and specificity of Congo red agar test for detection of biofilm producers were 90.63% and 90.79% for Staphylococcus aureus and 75.86% and 96.88% respectively for coagulase negative staphylococci. CRA is a method that could be used to determine whether an isolate has the potential for biofilm production or not.     

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.     

Influence of slime production and adhesion of Candida sp. on biofilm formation

The increase of fungal infections in recent years is connected with the progress in medicine. The vast usage of biomaterials is an inseparable element of contemporary medicine but it also leads to development of infections. Yeast-like fungi Candida albicans are still the main pathogen of candidiasis. The ability to slime production and adhesion to polystyrene of Candida sp. on different surfaces can cause to form biofilm on surfaces of biomaterials used in production of catheters, drains and prosthesis. The aim of the study was to evaluate the influence of slime production and adhesion to polystyrene, of Candida sp. on biofilm formation on different biomaterials. 50 strains of Candida sp. were examined. They isolated from ill to Clinics of Anesthesiology and Intensive Therapy University Hospital No 1 of dr. A. Jurasza in Bydgoszcz. The ability to slime production was evaluated by Christensen method in modification Davenport and Branchini methods. The adhesion to polystyrene was evaluated by Richards et el method. The ability to produce biofilm biomaterials by the studied fungi was measured after 72 hours of incubation at 37 degrees C on different biomaterials. Yeast-like fungi Candida sp. fabricating slime and adhesion forming frequently biofilm on surface researched of biomaterials. Influence of chosen biological specificity ascertain on the ability to produce biofilm on surfaces of siliconized latex and polyvinylchloride.     

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.     

In vitro effect of pH and ethanol on biofilm formation by clinicalica-positiveStaphylococcus epidermidis strains

Biofilm production is an important step in the pathogenesis ofStaphylococcus epidermidis associated biomaterial infections.Staphylococcus epidermidis strains isolated from dialysis fluid (n=9) and needle cultures (n=14) were phenotyped and genotyped for extracellular polysaccharide production and were examined for their ability to produce slime in a medium at various pH levels (3, 5, 7, 9 and 12) and with ethanol supplementation (0, 2, 5, 10 and 15%) using a semi-quantitative adherence assay. A total of 23 clinicalicaADBC positiveS. epidermidis, one reference strain (S. epidermidis CIP 106510) used as positive control, and oneicaADBC negative strain (E21) were investigated. Qualitative biofilm production analysis revealed that 15 of the 23icaADBC positive strains (65.21%) produced slime on Congo Red agar plates. Quantitative biofilm was determined by measuring the optical density at 570 nm (OD₅₇₀). The results show that the slime production depended on the pH value of the medium and the ethanol concentration. At highly acidic (pH 3) and alkaline (pH 12) levels, the OD₅₇₀ was lower, while at pH 7 the adhesion was moderate. In addition the cells adhered strongly with 2% ethanol than with the other concentrations. Our results suggest that pH and ethanol were stress factors that led toS. epidermidis biofilm formation and also play a possible role in the pathogenesis of biomaterial-related infections.     

Capillary isoelectric focusing--useful tool for detection of the biofilm formation in Staphylococcus epidermidis

The biofilm formation is an important factor of S. epidermidis virulence. Biofilm-positive strains might be clinically more important than biofilm-negative ones. Unlike biofilm-negative staphylococci, biofilm-positive staphylococci are surrounded with an extracellular polysaccharide substance. The presence of this substance on the surface can affect physico-chemical properties of the bacterial cell, including surface charge. 73 S. epidermidis strains were examined for the presence of ica operon, for the ability to form biofilm by Christensen test tube method and for the production of slime by Congo red agar method. Isoelectric points (pI) of these strains were determined by means of Capillary Isoelectric Focusing. The biofilm negative strains focused near pI value 2.3, while the pI values of the biofilm positive strains were near 2.6. Isoelectric point is a useful criterion for the differentiation between biofilm-positive and biofilm-negative S. epidermidis strains.     

Evaluation of biofilm production and prevalence of the icaD gene in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains isolated from patients with nosocomial infections and carriers

Staphylococcus aureus is one of the most important etiological agents responsible for healthcare-associated infections and is capable of producing many virulence factors including biofilm. The aim of the present study was to analyze the correlation between the presence of the icaD and icaA genes and the ability to produce biofilm in vitro in 302 methicillin-resistant (MRSA) and 268 methicillin-sensitive S. aureus (MSSA) strains isolated in the Provincial Hospital in Gdansk. Presence of the icaD and icaA genes was detected by PCR and the ability to produce biofilm in vitro was measured both spectrophotometrically and via Congo Red Agar plate culture methods. We found that 91% of MRSA strains harbored the icaD gene. Moreover, all icaD-negative strains were icaA-positive. Of MRSA and MSSA strains, 47% and 69%, respectively, produced biofilm in vitro. The level of consistency between the two applied phenotypic methods was 96%. Additionally, we found that strains with the same biofilm status may be present in asymptomatic carriers and cause infections.     

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.     

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.     

Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinase activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).     

静脉插管相关感染的病原学分析

目的调查静脉插管相关感染的病原微生物分布及其体外药敏试验的情况,为临床诊疗提供依据。方法回顾性分析我院2006年1月至2007年12月期间实施静脉插管的患者发生感染的情况,同时对分离的病原微生物及其耐药性进行分析。结果106例阳性标本中革兰阳性球菌53株（50％）,革兰阴性杆菌33株（31．1％）,真菌16株（15．1％）;革兰阳性球菌中以凝固酶阴性葡萄球菌（31．1％）、金黄色葡萄球菌（11．3％）为主,在葡萄球菌感染中耐苯唑西林金黄色葡萄球菌（MRSA）占75％,耐苯唑西林凝固酶阴性葡萄球菌（MRSCON）占87．9％;革兰阴性杆菌中以鲍曼不动杆菌（9．4％）、铜绿假单胞菌（6．6％）为主;真菌以白色念珠菌（4．7％）及热带念珠菌（3．8％）为主。革兰阳性球菌对万古霉素、利奈唑胺及替考拉宁敏感性高;革兰阴性杆菌较敏感的药物为头孢哌酮／舒巴坦、美罗培南、亚胺培南及阿米卡星,耐药率分别为5％、9．1％、14．8％及15．4％。结论静脉插管相关感染病原微生物以凝固酶阴性葡萄球菌多见,细菌耐药比较严重,应重视抗生素的合理使用。     

The evaluation of amylolytic activity of strains of coagulase negative staphylococci

Amylase activity of 30 strains of Staphylococcus spp. was determined by Tryptic Soy Agar on supplemented with 1.0% starch as the substrate. After incubation (time incubation 24 h or 168 h), the plates were flooded with Lugol solution. A clear zone around the colonies indicated amylase activity. The 23 (76.7%) strains CNS demonstrated the amylase activity. It was observed that 17 (80.9%) strains of S. epidermidis, and 6 (66.7%) strains non-S. epidermidis, starch hydrolyzed. Amylase production depends of time incubation (frequently 168 h) and growth atmosphere (frequently oxygen atmosphere)