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.     

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).     

Microbiological External Quality Assurance Program (MEQAO)--evaluation of results obtained in 2001 of sanitary-epidemiological station laboratories

The aim of this study was to evaluate reliability of identification and determination of sensitivity to antibiotics and chemotherapeutics of some Enterobacteriaceae strains in 49 sanitary-epidemiological stations. All laboratories engaged in this study received 4 strains each (S. marcescens phenotype ESBL+, K. pneumoniae phenotype ESBL+, S. Typhimurium and S. Newport) previously identified in the Department of Bacteriology of National Institute of Hygiene. The laboratories included in the survey were asked to determine the genus (serovar) of each control strain using the lowest number of biochemical test recommended by Institute and to determine of the sensitivity of the identified strains to antibiotics in accordance with the guidelines of the NCCLS and domestic recommendations. Only in 2 participating laboratories the identification of one of studied strains (K. pneumoniae) was completely bad and in 9 laboratories the serological identification of S. Newport was incomplete or serological type was not correctly recognised. In the determination of the sensitivity of the control strains to chemotherapeutic agents abnormalities were found in the technique of antibiogram and test for ESBL performing and incorrect selection of disc for antibiograms as well as erroneous interpretation of the results.     

Antibiotic sensitivity of staphylococci isolated from the surface of the skin of the breasts in pregnant women

Nine hundred and fourty coagulase-positive and coagulase-negative strains of staphylococci isolated from the skin surface of the mammary glands of 94 pregnant women were tested by the disc agar diffusion method for their sensitivity to five antibiotics. The highest number of the isolates were sensitive to erythromycin and lincomycin (87.7 and 89.7 per cent, respectively). The highest number of moderately resistant strains were detected with respect to methicillin. 19 out of 42 cultures of Staph. aureus were resistant to benzylpenicillin and 24 cultures were resistant to tetracycline. Among staphylococci 130 strains or 13.8 per cent were polyresistant.     

Antimicrobial resistance of Enterococcus sp. isolated from the intestinal tract of patients from a university hospital in Brazil

This study reports the results about antimicrobial resistance of Enterococcus spp. isolated from intestinal tract of patients from a university hospital in Brazil. The identification of strains at species level was performed by conventional biochemical tests, API 20 Strep (bioMérieux), and polymerase chain reaction assay. The species distribution was E. faecium (34%), followed by E. faecalis (33%), E. gallinarum (23.7%), E. casseliflavus (5.2%), E. avium (1%), and E. hirae (1%). Intrinsic resistance to vancomycin characterized by presence of vanC genes was found in E. gallinarum and E. casseliflavus. The high prevalence of VanC phenotype enterococci is very important because these species have been reported as causing a wide variety of infections. Vancomycin-resistant E. faecium or E. faecalis were not found and no one isolate of these species was a beta-lactamase producer. Thirteen clinical isolates of enterococci (13.4%) showed multiresistance patterns, which were defined by resistance to three classes of antibiotics plus resistance to at least one aminoglycoside (gentamicin and/or streptomycin). The resistance to several antimicrobials shown by enterococcal strains obtained in this study is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.     

Dynamics of antibiotic resistance of purulent septic pathogens at an emergency facility]

The predominant pathogens at the emergency medicine hospital were analysed and its resistance to antibiotics was investigated in dynamics. The susceptibility of the pathogens was analysed by the method of dynamic equalibration. Statistically significant reduction of resistant bacteria isolation was demonstrated for 2 species of 7--that is S. aureus and E. faecalis regarding 4 antibiotics. The ratio of staphylococci resistant to cefazolin, ceftazidime, doxycyclin reduced, the ratio of enterococci resistant to chloramphenicol and doxycyclin also diminished. No correlation was demonstrated between the pathogen isolation frequency and ratio of resistant strains.     

Virulence factors and<Emphasis Type="Italic">in Vitro</Emphasis> adherence of<Emphasis Type="Italic">Enterococcus</Emphasis> strains to urinary catheters

The ability to adhere in vitro to urinary catheters and the presence of enterococcal virulence factors was determined in 30 Enterococcus urinary isolates (12 E. faecalis, 12 E. faecium, 3 E. casseliflavus, 3 E. gallinarum). Silicone, siliconized latex and polyvinyl chloride (PVC) were examined by sonication quantitative culture technique and scanning electron microscope. As compared to E. faecalis and E. faecium, E. casseliflavus and E. gallinarum displayed lower adhesion to all synthetic materials. All the tests performed showed higher adherence of all tested strains to siliconized latex and silicone than to PVC. Biofilmforming ability was observed in 5 E. faecalis but in none of the remaining strains. The gene coding enterococcal surface protein (Esp) was detected in 7 E. faecalis and 6 E. faecium strains. Gelatinase was found in 1 E. faecalis, 2 E. faecium and hemolysins were found in 6 E. faecalis and 1 E. faecium strains. All E. casseliflavus and E. gallinarum strainswere negative for these traits. Hydrophobic type of cell surface (measured by its affinity for n-hexadecane) was shown in a few isolates. Bacterial adherence was not significantly associated with the above pathogenic factors.     

Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Detection of methicillin and mupirocin resistance in staphylococcal hospital isolates with a touchdown multiplex polymerase chain reaction

Staphylococcal hospital isolates (n = 166) were tested in a touchdown multiplex-polymerase chain reaction assay for the identification of methicillin and mupirocin resistance and discrimination of S. aureus (femA gene) from coagulase negative staphylococci and other bacteria. All isolates harbored the 16SrDNA (Staphylococcus genus specific internal control) gene, and 130 (78 %) the mecA (methicillin resistance) gene. Fifty-seven (44 %) of these were determined as methicillin-resistant S. aureus, while the remaining 73 (56 %) were methicillin-resistant coagulase-negative staphylococci. Seventy-five (45 %) isolates harbored the ileS-2 (high-level mupirocin resistance) gene and were determined as mupirocin-resistant. This assay represents a simple, rapid, reliable approach for the detection and discrimination of methicillin-and mupirocin-resistant staphylococci.     

Production of staphylococcal enterotoxin in mixed cultures.

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Evaluation of susceptibility to antibiotics of Staphylococcus aureus strains resistant to methicillin]

Methicillin-resistant strains of Staphylococcus aureus (MRSA) constitute a serious diagnostic and therapeutic problem. Over 500 strains of Staphylococcus aureus were tested for susceptibility to methicillin. By application of a screening method, 13.7% of these strains were classified as methicillin-resistant. Over 95% of these strains were isolated from hospital infections. Applying criteria of belonging of these strains to methicillin-resistance classes it was found that 49.3% belonged to class II, 31.2% to class III and 19.5% to class IV. Analysis of susceptibility to antibiotics of MRSA strains demonstrated significant differences between class II and between class III and IV in resistance to imipenem, gentamycin, erythromycin and tetracycline. All tested strains were susceptible to ciprofloxacin, ofloxacin, vancomycin and teicoplanin. The screening method (25 mg methicillin/l of TSA medium) results in obtaining of univocal results of determination of methicillin-resistance in S. aureus.     

Staphylococci Isolated from Carriage Sites and Infected Sites of Dogs as a Reservoir of Multidrug Resistance and Methicillin Resistance

The aim of this study was to compare the spread of multidrug-resistant (MDR) and methicillin-resistant (MR) staphylococci in healthy dogs and in dogs with evident symptoms of infection. The samples from 172 healthy and 197 infected dogs were examined. The staphylococci were identified with conventional methods and by means of the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method (MboI). Susceptibility to 15 antibiotics from 10 different antimicrobial classes was tested. Resistance to methicillin was confirmed by the presence of Staphylococcus aureus mecA and S. sciuri mecA genes. Multidrug resistance was defined as resistance to three or more antimicrobial classes. The oral mucosa to be the most frequent site of staphylococcal colonization (55.8 %), followed by nasal cavity (44.2 %), and anus (32.6 %). The prevalence of MDR staphylococci in infected dogs was significantly higher than in the healthy animals (74/137 vs. 34/95, P = 0.006). The MR strains of S. pseudintermedius (2.9 %) originated solely from infected dogs. In contrast, the MR coagulase-negative strains (7.4 %) were isolated solely from healthy dogs. S. aureus strains originated from nasal swabs, MRSA strains were not isolated. MDR staphylococci and MR S. pseudintermedius are more common among infected dogs, but coagulase-negative staphylococci (mostly S. sciuri) seem to be a reservoir of methicillin resistance in healthy dogs.     

Therapeutic effect of some antibiotics on experimental staphylococcal infection and its correlation with in vitro activity of antibiotics in sub-inhibitory concentration against Staphylococcus aureus strains

The aim of the study was to demonstrate of whether the therapeutic effects of antibiotics depend on their in vitro activity in sub-inhibitory concentrations against staphylococci. Cloxacillin, gentamicin and lincomycin were used in the study. Groups of S. aureus strains, containing 6 strains with similar MIC values each but different sensitivity to sub-inhibitory antibiotic concentrations (sub-MIC) were selected (a total of 36 trains): i. strains increasing their sensitivity to phagocytosis and bactericidal activity of rabbit leukocytes after incubation with an antibiotic in 0.1 MIC concentration, ii. strains with sensitivity to the above factors unaffected by incubation with an antibiotic in 0.5 MIC concentration. The doses of staphylococci causing death of 90-100% of Swiss albino mice 10 days after i.p. infection were determined. The injected doses (LD 90-100) and various doses of antibiotics were used to determine ED50 values as well as the survival rate of the mice with experimental staphylococcal infections after treatment with these antibiotics. It was demonstrated that effective doses (ED 50) of the antiboitics were significantly lower when the antibiotics were administered once to mice infected with strains S. aureus sensitive to sub-MIC concentrations of the investigated antibiotics than for mice infected with strains resistant to their sub-MIC concentrations. Similar correlations were observed in mice which were given the antibiotics several times (for 7 days): the percentage of the surviving mice was higher in the group infected with sub-MIC sensitive strains. The therapeutic effect of cloxacillin, gentamicin and lincomycin demonstrated a significant correlation with the S. aureus strains used to induce the infections and their sensitivity, or lack of sensitivity in vitro, to phagocytosis and bactericdal activity of leukocytes in the presence of antibiotics in sub-MIC concentrations.     

伤口分泌物的病原菌分布及耐药性分析

目的调查福建省龙岩市第二医院伤口分泌物病原菌的分布及耐药情况,为合理应用抗生素提供依据。方法收集2012年1月至2013年5月患者伤口分泌物标本,采用常规方法进行分离培养,用VITEK-2 Compact全自动微生物分析仪系统进行鉴定及药敏分析。结果送检503份标本,培养阳性272份,阳性率为54. 1% ;病原菌检出343株,其中革兰阴性菌189株占55. 1%,革兰阳性菌151株占44.0%,真菌3株占0.9% ;前5位的病原菌分别为金黄色葡萄球菌、凝固酶阴性葡萄球菌、铜绿假单胞菌、大肠埃希菌、鲍曼不动杆菌。耐甲氧西林金黄色葡萄球菌（MRSA）和耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的检出率分别是30.4%、82. 1%。粪肠球菌中耐高浓度氨基糖苷类肠球菌（HLAR）的检出率为55%。革兰阳性菌对万古霉素、替加环素、利奈唑胺未出现耐药菌株。铜绿假单胞菌、肠杆菌科细菌对亚胺培南的耐药率分别为5. 6%、0。大肠埃希菌中超广谱P-内酰胺酶（ESBL）的检出率是42.9%。鲍曼不动杆菌对头孢哌酮/舒巴坦的耐药率最低为25.0%,对其他抗菌药物耐药严重,多数抗菌药物的耐药率均〉45%。结论伤口感染的主要病原菌是革兰阴性菌,多重耐药菌株比例较高,临床应根据药敏结果合理选用抗生素,减少新的耐药菌株出现。     

Drug sensitivity of Enterococcus sp. strains isolated from clinical material or from healthy persons

The aim of this study was to evaluate the drug susceptibility of Enterococcus sp. strains isolated in 2000-2001, from patients of five Warsaw's hospitals (154 strains) and from fecal samples of healthy persons (33 strains). On biochemical reaction profiles species of clinical enterococci were identified as: E. faecalis--66.2%, E. faecium--29.2%, E. hirae--1.9%, E. gallinarum--1.3%, E. casseliflavus--0.6% and E. avium--0.6%. The species of enterococci from stool's samples were identified as: E. faecalis--28 strains, E. durans--2 strains and single strains: E. faecium, E. gallinarum and E. casseliflavus. Susceptibility to 20 antibiotics was tested by disc diffusion method. None of these 187 enterococcal strains was vancomycin resistant; 3 strains of E. gallinarum and 1--E. casseliflavus demonstrated intermediately susceptibility to vancomycin, but they were susceptible to teicoplanin--phenotype Van C. Among clinical strains were resistant to penicillin--33.3% of E. faecalis and 100% of E. faecium, to ampicillin--over 80% of E. faecium and 1 strain of E. faecalis. None of these strains produced beta-lactamase. High-level resistance to aminoglicoside was expressed by 48 strains (47.1%) E. faecalis and 36 (80%) E. faecium isolated from clinical specimens. Both--HLR to streptomycin and gentamycin were found in 28.3% of E. faecalis and 68.9% of E. faecium. Among 33 strains isolated from fecal samples of healthy persons--3 of E. faecalis were resistant to streptomycin and one was resistant to both gentamicin and streptomycin. In general, enterococcal strains isolated from samples of healthy persons were susceptible to the most of used antibiotics. But to rifampicin none of these strains were susceptible. There were about 40% of E. faecalis strains isolated from healthy persons, resistant to tetracyline.     

Susceptibility to selected chemotherapeutics of Staphylococcus aureus strains resistant to methycyline isolated from clinical materials in the years 1991-1992 and 1997

The susceptibility to selected chemotherapeutic agents was determined in 100 strains of Staphylococcus aureus methicillin-resistant (MRSA) isolated from clinical materials in 1991-1992 (50 strains) and in 1997 (50 strains). Two methods were used for the determination: disc method and antibiotic dilution in agar. The minimal inhibitory concentration (MIC) was determined for vancomycin, teicoplanin, furazolidone, nitrofurantoin, ofloxacin, gentamicin, netilmicin and trimethoprim. The concentrations of the chemotherapeutics in the substrate ranged from 0.125 to 512 mg/l. The obtained results served for drawing of the following conclusions: all studied MRSA strains isolated in 1991-1992 and in 1997 were sensitive to glycopeptide antibiotics: vancomycin and teicoplanin, to nitrofurans: nitrofurantoin and furazolidone, and to fusidic acid. MRSA strains isolated in 1991-1992 were sensitive to ofloxacin, but in 1997 about 80% of the strains were resistant to that antibiotic, and this resistance was noted in S. aureus strains with homogeneous resistance to methicillin. Increasing frequency of resistance to mupirocin was found, in 1991-1992 4% of the strains were resistant, and in 1997 the resistance of MRSA to that antibiotic was found in 12%. No changes occurred in the sensitivity of staphylococci to trimethoprim/sulfamethoxazole (cotrimoxazole). About 94% of strains in 1991-1992 and 1997 were sensitive to that drug. The sensitivity to cotrimoxazole is connected with one of its components (trimethoprim), with 94% of MRSA strains sensitive to it.     

Characterization of Staphylococcus aureus strains isolated from cystic fibrosis patients by conventional and molecular typing

The phenotypic and genotypic characteristics of Staphylococcus aureus strains isolated from respiratory tract of cystic fibrosis (CF) patients were investigated. Slime production, cell-surface hydrophobicity, type of capsular polysaccharide, profile of heteroresistance to methicillin and Sma I restriction profiles were evaluated. S. aureus CF strains have been shown to be heterogeneous in respect to several important features. All of them were slime producing with variation in colony morphology. High or moderate cell-surface hydrophobicity (CSH) was found for, respectively, 16.2% and 83.8% strains. Thirty strains were resistant to methicillin, 60% of them showed heteroresitance and 40% were homoresistant. It was found that 59.6% of strains produced capsular polysaccharides (CP) of 5 or 8 type. Among CP5/CP8 strains, CP8 was the predominant type (81.1%). Typing of 62 CF strains by macrorestriction analysis of chromosomal DNA revealed several major types, differing in their SmaI profiles with a similarity coefficient lower than 0.4. Some of the strains isolated from the same patient at different times of hospitalization, as well as strains isolated at the same time from the relatives, were identical in their PFGE pattern.     

Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture

The notification of "Gram-positive cocci, possibly staphylococcus" in a blood culture drawn from a seriously ill patient is responsible for a large amount of vancomycin prescribing in institutions where methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of bacteraemia. A duplex real-time TaqMan polymerase chain reaction targeting the species-specific nuc gene, and the mecA gene encoding methicillin-resistance, was developed as a tool for rapid identification and detection of S. aureus and methicillin-resistance, and optimised for immediate as-needs testing. Three different DNA extraction methods achieved varying DNA quality, with PCR inhibition the main problem. Serial blood cultures (n=120) identified as possible staphylococci on Gram stain from our clinical laboratory were examined. There was one false negative result for a methicillin-resistant Staphylococcus epidermidis, which was positive on repeat testing, and one false negative result due to DNA extraction failure for MRSA from peritoneal dialysate inoculated into blood culture medium. Sensitivity and specificity of 97% and 100%, respectively, were obtained for mecA; and sensitivity and specificity of 98% and 100%, respectively, for nuc. Detection of slow-growing coagulase-negative staphylococci as co-infecting strains may be reduced. The assay quickly and reliably identified S. aureus in mixed infection, and identified methicillin resistance in both S. epidermidis and S. aureus strains.     

Influence of testing conditions on the susceptibility results of Staphylococcus cohnii to beta-lactams

The high occurence of coagulase-negative staphylococci among bacteria responsible for hospital infections is unquestioned. Studies on the poorly-known novobiocin-resistant, coagulase-negative Staphylococcus cohnii were undertaken. The possibilities of optimizing conditions for determination of susceptibility to beta-lactam antibiotics of this species were researched. In the case of S. cohnii the new cefoxitin test for detection of methicillin resistant strains, introduced by the National Reference Centre for Antibiotics in Poland was found as a good and of credible quality. It was also shown, that application in in vitro examination conditions stimulating the mechanisms of resistance to beta-lactam antibiotics, supplies credible results relating to their true susceptibility. The necessity of establishing individual conditions for susceptibility determination in different species of coagulase-negative staphylococci was suggested.     

Synthesis and potent antimicrobial activity of some novel methyl or ethyl 1H-benzimidazole-5-carboxylates derivatives carrying amide or amidine groups

A series of benzimidazole-5-carboxylic acid alkyl ester derivatives carrying amide or amidine substituted methyl or phenyl groups at the position C-2 were synthesised and evaluated for antibacterial and antifungal activities against S. aureus, methicillin resistant S. aureus (MRSA), S. faecalis, methicillin resistant S. epidermidis (MRSE), E. coli and C. albicans. The results showed that while all simple acetamides are essentially inactive, aromatic amides and amidines have potent antibacterial activities. Aromatic amidine derivatives 13 f-h exhibited the best inhibitory activity with 1.56-0.39 microg/mL MIC values against MRSA and MRSE.