Application of normative documents for determination of biocidal activity of disinfectants and antiseptics dedicated to the medical area: a narrative review.

Disinfectants and antiseptics are important weapons to reduce the number of micro-organisms and thus limit the number of infections. Different methods of antimicrobial activity testing, often not standardized, without appropriate controls and not validated, are applied. To address these issues, several European Standards (EN) have been developed, describing the test methods to determine whether chemical disinfectants or antiseptic products have appropriate bactericidal, sporicidal, mycobactericidal or tuberculocidal activity; fungicidal or yeasticidal activity; or virucidal activity. In this narrative review, the 17 ENs concerning evaluation of the above-mentioned antimicrobial activity of preparations dedicated to the medical area are briefly reviewed, together with recent publications on this topic. Suspension and carrier tests have been performed in clean and dirty conditions simulating the medical area. In addition, a wide range of applications of these standards has been presented in the research of biocides for hand antisepsis, surfaces disinfection, including airborne disinfection as well as medical device and medical textile disinfection. The role of normative documents in the investigation of antimicrobial activity of disinfectants and antiseptics to limit infections has been underestimated. This narrative review aims to persuade researchers to conduct antimicrobial activity testing in line with validated ENs and highlights an existing gap in ongoing research. It also aims to raise awareness of the wide range of biocidal activity tests with standardized methods in the medical area. We also pay attention to the recently developed European Pharmacopoeia monography concerning the testing of bactericidal and fungicidal activity of antiseptics classified as medicinal products.

Significant increase in the isolation of glycopeptide-resistant enterococci from patients hospitalized in the transplant surgery ward in 2004-2005.

Enterococci despite their low pathogenicity are the third cause of hospital infections. Enterococci resistant to glycopeptides present special risks. The aim of this work was to determine the frequency of isolates of all enterococci versus enterococci resistant to glycopeptides from patients in the Transplant Surgery Ward. Moreover, vancomycin-resistant enterococci (VRE) were characterized with respect to the type of van and ddl genes as well as vancomycin and teicoplanin MIC values. Among 160 enterococcal strains isolated in 2004, only 2 were resistant to glycopeptides (1.3%). In 2005, among 244 enterococci, 44 strains were resistant (18%). All resistant strains were Enterococcus faecium, as confirmed by detection of the ddl gene specific for E. faecium. Moreover, among all enterococci isolated from these patients, E. faecium dominated (over 50% in 5 subsequent years). All examined VRE possessed VanA type of resistance with high vancomycin and teicoplanin MIC values. All of them possessed the vanA ligase gene. The investigated VRE were characterized by high resistance to most antibiotics: penicillin and amoxicillin, rifampicin, ciprofloxacin, and high concentrations of streptomycin, but susceptible to linezolid and quinupristine/dalfopristine. Strains differed in their susceptibility to tetracycline, nitrofurantoin, and high concentrations of gentamicin.

Occurrence of glycopeptide-resistant enterococci in transplant medicine internal wards in 2001-2005.

The appearance of vancomycin-resistant enterococci (VRE) has caused serious therapeutic problems. In Poland, the frequency of VRE isolation is lower than in the United States or some other European countries. The aim of our work was to analyze the occurrence and characterization of VRE isolated from patients of 2 transplant medicine wards. These wards contained liver or kidney transplant patients. This study examined 5 years, including 235 to 313 enterococcal isolates per year. In 2001-2002, none of the isolated enterococci was confirmed as VRE, which appeared in 2003 (11 strains) and continued on a similar level (from 4% to 6%) in the next 2 years. Among all isolated enterococci, Enterococcus faecalis predominated. In 2003 and 2004, the numbers of E. faecium and E. faecalis among isolated VRE strains were similar, but in 2005, we observed significant predominance of E. faecium. Among VRE strains examined by polymerase chain reaction for the presence of vanA, vanB, vanD, vanE, and vanG ligases, only vanA was found in all cases. The examined strains represented several patterns of resistance to other antibiotics.

Purification and some properties of the staphylococcal extracellular lipase.

Staphylococcal lipase has been purified by application of a multistep procedure involving ammonium sulfate precipitation, and hydrophobic interaction chromatography followed by gel filtration through Sepharose CL-4B. A purified enzyme was obtained which appeared to be homogeneous by molecular sieving, polyacrylamide gel electrophoresis and sucrose gradient centrifugation. The enzyme was then subjected to physicochemical analysis. It has been found that staphylococcal lipase appears in two molecular forms: light (45 kDa) and heavy (300 kDa). Amino acid analysis indicates that lipase contains 17 amino acids with a prevalence of hydrophobic amino acids. No sulfur-containing amino acid was found in the enzyme molecule. The lipase contains about 2% sugars and some amount of lipids. The lipase preparation is stable within pH 5.0 to 9.0 and exhibits maximal activity at pH 8.0. The optimal temperature for the enzymatic reaction was established at 55 degrees C.

Radioimmunoassay of delta-toxin from B. thuringiensis: correlation with bioassay.

A radioimmunoassay procedure, developed for delta-toxin of B. thuringiensis, is applicable to the assay of the toxin's bioactivity. When delta-toxin is inactivated to different degrees by heat treatment, the amount of toxin as determined by radioimmunoassay is proportionally decreased. It thus appears that the delta-toxin antibody may recognize and bind to the conformation in the molecule associated with its insecticidal activity.

Adaptation of capillary electrophoresis to the determination of selected cephalosporins for injection.

The migration behaviour of cephazolin, cefuroxime sodium, ceftriaxone sodium, cefoperazone sodium and ceftazidime in a mixture was studied. Phosphate-borate buffer pH 5-8 alone and with addition of sodium dodecylsulfate (SDS) was used. In capillary zone electrophoresis of all research compounds separation was not achieved. It was observed that supplementation buffer pH 6.5 with SDS (10 g/l) improved resolution of cephalosporins, but addition of pentanesulfonic acid (17.4 g/l) to the running buffer at pH 6.5 results in separation of each cephalosporin. In this condition good repeatability of migration times as well as repeatability of peak area were confirmed.

Analysis of bacterial meningitis during 1992-1996 in Poland.

Retrospective study of bacterial meningitis cases was performed by analysis of filled questionnaires received from 38 different hospitals located in 27 out of 49 provinces of Poland. Obtained data allowed to indicate that S. epidermidis, N. meningitidis, S. pneumoniae and H. influenzae were the most common bacterial strains isolated from cerebrospinal fluid of meningitis patients during the last five years. Besides data analysis performed, some strains of N. meningitidis (n = 97), H. influenzae (n = 28) and S. pneumoniae (n = 39) isolated from cerebrospinal fluid of meningitis patients hospitalized in different places in Poland sent by cooperating hospital laboratories (1995-1996), were phenotypically characterized. Neisseria meningitidis B:22:P1.14 was the most common isolate phenotype during the investigated time period. Streptococcus pneumoniae of twenty different serological types were isolated. Type 1 was the dominant--18% of strains. All but one Haemophilus influenzae strains isolated from cerebrospinal fluid belonged to serological type b. Biotyping showed presence of only two types: I and II, 39.3% and 60.7% of isolates respectively. The sensitivity of collected strains to selected antimicrobial agents (penicillin, cefotaxime, ceftriaxone, sulphametoxazol, chloramphenicol, tetracycline, ciprofloxacin, rifampin and erythromycin) used in treatment and prophylaxis of bacterial meningitis was investigated. Minimum inhibitory concentrations were determined by agar dilution method or with the use of E-tests. All investigated strains but one N. meningitidis, were not resistant to penicillin (MIC < or = 1 microgram/ml).

Active surveillance of meningococcal meningitis in Poland.

Starting from 1970, the notification of N. meningitidis cases in Poland was compulsory and separated from other cases of meningitis purulenta. Based on the experience of European Monitoring Group on Meningococci, the active surveillance of meningococcal meningitis in Poland was initiated in April 1995. It was the first time that such study was conducted to recognise the actual situation of meningococcal meningitis infections in our country. Ninety seven N. meningitidis strains were isolated (31 in 1995 and 66 in 1996) from cerebrospinal fluid (CSF) of meningitis patients hospitalized in 54 hospitals located in 33 out of 49 provinces of Poland. Most patients were below 2 years of age and 43% belonged to infant group. Meningococcal strains were phenotypically characterized as follow: identification of N. meningitidis was performed by Gram staining, oxidase and catalase tests as well as latex or diagnostic sera agglutination assays. Meningococcal serotypes and subtypes were determined by whole-cell ELISA with monoclonal antibodies. The predominant meningococcal serogroup during 1995 and 1996 was B (80% of all isolates tested), the serogroup C (12.6%) and W-135 (3.5%). Only two non-groupable and two serogroup A strains were isolated in Poland. Active surveillance allowed to determine B:22:P1.14 to be the most prevalent N. meningitidis phenotype in Poland. Two isolates of N. meningitidis phenotype C:2a:P1.2,5, which caused emergency situation in Czech Republic since 1993, were isolated from CSF of patients in October 1996 in southern Poland. All strains were susceptible to cefotaxime, chloramphenicol, ciprofloxacin, rifampin and tetracycline; some strains were resistant to sulphonamides (60.6% - MIC = 32 mg/l and 14.8% - MIC = 128 mg/l). Only one of the tested strains in two years surveillance study in Poland was resistant to penicillin (MIC = 2 mg/l).

Antimicrobial activity of selected non-antibiotics--activity of methotrexate against Staphylococcus aureus strains.

Some non-antibiotic drugs, e.g. cytostatics, anaesthetics or vasodilators may also show the antimicrobial activity. The aim of this study was to detect and characterise the antimicrobial activity expressed by selected non-antibiotic drugs analysed during state control performed in the Drug Institute. Over 60 drugs were randomly chosen from different groups. The surveillance study was performed on standard microbial strains: S. aureus ATCC 6538P, E. coli ATCC 8739, P. aeruginosa ATCC 15442 and C. albicans ATCC 10231. It was found that the drugs listed below inhibited growth at least one of the examined strains: Acesan 0.075 tabl., Benuron 500 mg tabl., Chlorchex 0.5 aerosol, Methotrexat-Ebewe 500 mg amp., Naproxen 500 mg tabl., Nospa Forte 60 mg tabl., Platamine 50 mg amp., Platidiam 50 mg amp., Sensit 50 mg drag., Septofervex 2 mg tabl., Seractil 400 mg tabl., Sermion 4 mg amp., Sinemet 125 mg tabl., Tarproxen 500 mg tabl. and Zyban 150 mg tabl. It was interesting, that strong antimicrobial activity of methotrexate was limited to Staphylococcus aureus strain. Minimal inhibitory concentration of methotrexate was determined by agar dilution method with the use of 54 clinical S. aureus strains (MRSA-32 and MSSA-22). For MSSA strains, MIC50 and MIC90 were 10 micrograms/ml and 20 micrograms/ml, respectively (range: 10-20 micrograms/ml). For MRSA strains, MIC50 and MIC90 were 20 micrograms/ml and 100-> 100 micrograms/ml, respectively (range: 10-< 100 micrograms/ml).

[Characterization of Neisseria meningitidis strains isolated from carriers]. / Charakterystyka szczepów Neisseria meningitidis izolowanych od nosicieli.

Neisseria meningitidis carriers strains were isolated from 17-19 teenagers (n = 14) and recruits (n = 267). The longitudinal study comprises three meningococcal carriage trials performed on healthy young men during two--six months of their service in Polish military units. Altogether 54, 124 and 89 meningococcal strains were obtained during spring 1998 and autumn 1998, 1999 trials. Sixty two percent of meningococcal carrier strains were non-groupable, however among the remaining strains, serogroup B was predominant (29.5%). During spring 1998 and autumn 1999 trials the predominant phenotypes were N. meningitidis NG:21:P1.7, but during the autumn 1998 NG:21:P1.7 or NG:NT:P1.5. Ribotyping of type 21 and/or subtype P1.7 strains (n = 27) showed presence of 2 main ribotypes. Pulsed Field Gel Electrophoresis of consecutive isolates recovered from the same carrier showed great similarity of the patterns.

[Characteristics of Neisseria meningitidis strains isolated from patients with symptoms of meningococcal meningitis in Poland in 1995-2000]. / Charakterystyka szczepów Neisseria meningitidis izolowanych od osób z objawowym zakazeniem meningokokowym w latach 1995-2000 w Polsce.

Phenotype and genotype identification of 179 Neisseria meningitidis strains isolated from cerebrospinal fluid or blood of patients with meningococcal infection, hospitalized in Poland, was performed. This is the first analysis of that type conducted in Poland. We analyzed strains collected in 1995-2000 from laboratories located all over the country. Phenotype Neisseria meningitidis B:22:P1.14 was the predominant among analyzed invasive strains in Poland. Type 22 is characteristic to most of the strains isolated in our country. No strain from analyzed group belonged to known epidemic clusters. One penicillin resistant strain (MIC = 2 mg/l) and about 27% strains with decreased susceptibility to penicillin (0.1 = < MIC < 1.0 mg/l) were present among 166 N. meningitidis tested. All strains were susceptible to ciprofloxacin and rifampicin.

[Tests for Neisseria meningitidis in adolescents and adults (recruits)]. / Badania nosicielstwa Neisseria meningitidis u mlodziezy i osób doroslych (poborowych).

No N. meningitidis strain was isolated from 168 children 14 years old. However, among 17-19 years old young people 14% meningococcal carriage rate was demonstrated. In three surveillance trials performed in military units in spring 1998 and autumn 1998 and 1999; 81, 225 and 153 isolates of N. meningitidis, respectively were obtained. A change in N. meningitidis carrier status was observed. At the autumn study, two times more N. meningitidis intermittent carriers were found than in the spring investigations. Among these population we found short-term and long-term carriers--last day of the longest trial--162 day. Meningococcal carriage rate increases among recruits in overcrowded living conditions.

[Susceptibility to macrolide antibiotics of Bordetella pertussis and Bordetella parapertussis strains isolated from whooping cough patients in 1968 and in 1997-99]. / Wrazliwosc na antybiotyki z grupy makrolidów paleczek Bordetella pertussis i Bordetella parapertussis wyizolowanych od chorych na krztusiec w 1968 roku i w latach 1997-99.

The activity of erythromycin, azithromycin, clarithromycin, dirithromycin, oleandomycin, roxithromycin, spiramycin and josamycin against 21 and 34 B. pertussis strains and against 6 and 8 B. parapertussis strains isolated respectively in the years 1968 and 1997-99 was examined. The antibiotic agar dilution method was used. The minimum concentration of macrolides which inhibited growth of B. pertussis and B. parapertussis was calculated for 50% (MIC50) and 90% (MIC90) of isolates. The susceptibility to macrolides of B. pertussis and B. parapertussis strains isolated in the years 1968 and 1997-99 did not differ significantly. The MIC90 values of erythromycin were the same for B. pertussis (MIC90 = 0.125 mg/l) and B. parapertussis strains (MIC90 = 0.25 mg/l) recovered in 1968 as for those recovered in the years 1997-99. The most active antibiotic against all strains was azithromycin (MIC90 = 0.06 mg/l). The least active antibiotics were oleandomycin (MIC90 = 2-4 mg/l) and spiramycin (MIC90 = 8 mg/l). The study showed that erythromycin remains the antibiotic of choice for treatment of whooping cough and in case of emergence of B. pertussis and/or B. parapertussis strains erythromycin resistant, can be replaced by azithromycin.

[Use of western-blotting technique and computer analysis for comparing cellular antigens of coagulase-negative staphylococci]. / Zastosowanie techniki western-blotting i analizy komputerowej do porównania antygenów komórkowych szczepów gronkowców koagulazo-ujemnych.

Western-Blotting technique and computer programme have been used to analyse cellular antigenic patterns of coagulase-negative staphylococci mainly of S. epidermidis and S. saprophyticus species. It was shown that antigenic pattern among strains of one species was similar. Intra strains differences are mainly quantitative. The qualitative differences concern not quantitatively dominant antigens. The main antigens of S. epidermidis and S. saprophyticus strains were of 28 and 44 kD molecular weight respectively. The antigens of molecular weight around 44, 48, 89 and 105 kD were detected in antigenic extract of strains from different species. Only one of the quantitatively dominant antigen of m. w. 48 kD, was localised in all analysed materials. The method presented here was used for the first time to analyse staphylococcal antigens. Such analysis could be performed not only for bacterial antigens.

[Preparation of Staphylococcus aureus antigens for evaluation of their immunological reactivity with the human sera by the western blot method]. / Przygotowanie antygenów Staphylococcus aureus do oceny odpowiedzi immunologicznej w surowicy metoda western-blott.

Extracellular antigens as well as cell wall extracts of 4 S. aureus strains isolated from different kinds of infection were analysed by Western-Blott technique. Materials obtained in two systems of bacteria cultivation (with and without aeration) were compared. Four systems of PAGE (native conditions, with 8.0 M urea, with SDS and SDS after previous reduction of the material with 2-mercaptoethanol) were compared in order to get the best differentiation of proteins and antigens. Immunological reactivity of the antigens mixture with two human sera: highly positive (with three S. aureus antigens in ELISA) from patient with staphylococcal sepsis and negative (from blood donor) were analysed. The best results were obtained after reduction of the cell wall extracted material in SDS-PAGE. The different protein patterns depending on the strain and the method of bacteria cultivation were observed. The standardisation of Western-Blott technique was performed, including titration of the sera to get the best differentiation of the antigens. The difference in immunological reactivity of the positive and negative sera with staphylococcal antigens mixture showed rather quantitative than qualitative character.

[Humoral response to Staphylococcus aureus antigens evaluated by the western blotting method]. / Humoralna odpowiedz na antygeny Staphylococcus aureus oceniana metoda western-blott.

Cellular antigens extracted from the cells of four Staphylococcus aureus strains from different kinds of infections (sepsis, osteomyelitis, furunculosis) were analysed by the western blotting technique. Antibiotic sensitivity pattern of the strains was compared. One isolate was found to be MRSA strain. Sera samples from patients of whom strains were isolated and four sera from blood donors (as a control) were used in the investigation. IgG levels for purified staphylococcal antigens (lipase, alpha-toxin and teichoic acid) were estimated. Interaction between extracted bacterial antigens and serum antibodies of IgG class were analysed in homologous and heterologous systems. The most strong immunological reaction of the investigated sera with staphylococcal antigens was observed in the case of homologous system. Serum from sepsis patient was found to be the most reactive serum with all staphylococcal antigens mixtures.

[Evaluation of different methods for detecting resistance of staphylococcus aureus strains to methicillin]. / Ocena wiarygodnosci metod oznaczania opornosci szczepów Staphylococcus aureus na metycyline.

Resistance to methicillin of 70 Staphylococcus aureus isolates from infection and 6 standard strains was evaluated by screening and disc diffusion techniques. Amongst wild S. aureus isolates 28 were identified as methicillin-susceptible (MSSA), 18 as heterogeneously resistant and 24 as homogeneously resistant to methicillin (MRSA). The best detection of methicillin-resistance was obtained by two screening techniques 1) on Mueller-Hinton agar with 6 mg oxacillin per litre and incubation for 40 hours at 37 degrees C. The evaluation of commercial test Crystal MRSA ID System proved its high usefulness in proper detection of MRSA and allowed to get results in 4 hours. Disk diffusion with 1 microgram oxacillin disk and incubation for 20 hours at 30 degrees C prove to be the most reliable out of all disc diffusion techniques studied although inferior to screening techniques.

[Evaluation of susceptibility to antibiotics of Staphylococcus aureus strains resistant to methicillin]. / Ocena wrazliwosci na antybiotyki szczepów Staphylococcus aureus opornych na metycyline.

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.

[Meningococcal infections in Warsaw's district]. / Zakazenia meningokokowe w województwie stolecznym warszawskim.

The epidemiological situation of meningococcal meningitis in Warsaw's district in comparison to the situation in Poland in the years 1980-1997 in discussed. In September 1997, the local population of Zielonka--small city in Warsaw's district, was alarmed by two meningococcal septicaemia cases in girls attending to the same kindergarten. Anti-epidemic measures undertaken were described.

[The study of carrier of Neisseria meningitidis performed in Zielonec and characteristics of meningogocci isolated from patients at the Wolomin hospital, Warsaw district]. / Badania nosicielstwa N. meningitidis przeprowadzone w Zielonce oraz charakterystyka meningokoków izolowanych od chorych z terenu TSSE-Wolomin, woj. stol. warszawskie.

Meningococcal carriage study, performed after N. meningitidis outbreak in Zielonka included 130 persons (111 children). No N. meningitidis strain was isolated. Phenotype and genotype analysis of 6 meningococcal isolates obtained from blood and CSF, showed their heterogeneity with exception of 2 isolates from Zielonka's cases of meningococcal sepsis which were identical.