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.

Molecular and phenotypic characteristics of methicillin-resistant Staphylococcus aureus strains isolated from hospitalized patients in transplantation wards.

OBJECTIVES: Hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) frequently causes therapeutic problems and provides information about the epidemiological condition of the ward. MATERIALS AND METHODS: HA-MRSA isolated from patients on transplantation wards in 1991, 1994, 1996, and from 2005 to 2007 were compared using molecular methods such as restriction fragment length polymorphism-pulse field gel electrophoresis, multilocus sequence typing (MLST), multiplex polymerase chain reaction (PCR) for detection type of staphylococcal chromosomal cassette mec, and PCR for detection. RESULTS: The analysis covered HA-MRSA strains, each from a different patient. All organisms were typed using molecular methods. MLST results were compared with an international base. The examined strains belonged to five different worldwide known clonal complexes: CC8 (78%), CC5 (12%), CC1 (4%), CC30 (2%), and CC51 (4%). All could be recognized as representatives of a clonal complex CC8 clones: ST239-III (sequence type 239 and SCCmec type III named EMRSA-1, -4, -11, Brasilian, Hungarian) occurred with a frequency of 35.9%, ST254-IV (EMRSA-10, Hannover) occurred in 33.3%, ST247-I (EMRSA-5,-7, Iberian) occurred in 20.5%, ST241-III (Finland-UK) occurred in 5.15%, and ST8-IV (EMRSA-2,-6) occurred in 5.15%. CONCLUSION: The predomination of different clones of HA-MRSA in the particular years was observed. In 1991, the EMRSA-10 (Hannover) clone predominated (53.3%). The Brasilian-Hungarian (EMRSA-1, -4, -11) clone predominated in 1994 (50%) as well as from 2005 to 2007 (41.3%), whereas in 1996 the Iberian clone was most frequent (53.9%).

The dominant sequence types of vancomycin-resistant Enterococcus faecium among transplantation ward patients.

OBJECTIVES: Vancomycin-resistant enterococci (VRE) frequently cause therapeutic problems and provide information about the epidemiological condition of the ward. MATERIALS AND METHODS: VRE isolated from patients on transplantation wards in 2007-2008 were compared using 2 molecular methods: RFLP-PFGE (restriction fragment length polymorphism-pulse field gel electrophoresis) and MLST (multilocus sequence typing). RESULTS: The analysis covered 29 Enterococcus faecium strains resistant to glycopeptides, each from a different patient. All organisms were typed using 2 molecular methods. MLST results were compared with an international base. The 30 examined strains belonged to 8 different worldwide known sequence types. All could be recognized as representatives of a single clonal complex CC17. CONCLUSION: Both methods of typing appeared to be useful to asses the epidemiological condition of the investigated wards.

Extremely long time stability study of selected antibiotic standards.

A study was conducted to investigate the quality of reference substances which were produced long before the introduction of advanced analysis and purification techniques. The quality and antimicrobial activity of WHO and EP oxytetracycline, doxycycline, colistin and spiramycin reference standards were assessed. The oldest substance was stored for 54 years in a freezer. Assay and purity tests were conducted according to Ph. Eur. 6.0. Additionally, antibacterial activity was tested with the microbiological method according to Ph. Eur. 4.0 (agar diffusion method). The results of the study show that several of the tested substances remained stable for over 40 years and one for over 50 years of storage. In most cases, the determined potency is close to the declared one, regardless of the method used (HPLC or microbiological). Composition analysis of multi-substance antibiotics (colistin and spiramycin) showed important differences compared with new reference substances. Results also indicate that no excessive degradation occurred during the entire storage period and impurity levels have not changed significantly.

Molecular epidemiology of vancomycin-resistant Enterococcus faecalis among patients of transplantology wards.

OBJECTIVE: Vancomycin-resistant enterococci (VRE) require epidemiological monitoring especially in transplantation wards. The aim of our work was to perform a molecular analysis of vancomycin-resistant Enterococcus faecalis (VREfl) strains among solid organ recipients during various years. MATERIALS AND METHODS: Strains were examined for the presence of different genes determining vancomycin resistance: vanA, vanB, vanD, or vanG by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP)-pulsed field gel electrophoresis (PFGE) was performed on bacterial DNA digested with SmaI enzyme. RESULTS: From 2003 to 2006, we isolated 12 strains of VREfl from 8 patients (2 liver and 6 kidney transplantations). All strains harbored the vanA gene. Among the strains, 5 displayed patterns similar to each other, despite being isolated from different patients, and were susceptible to ampicillin with high resistance to aminoglycosides. CONCLUSIONS: These results suggested that a single strain of VREfl was present for 3 years in closely related hospital wards, but it disappeared in the following years.

Molecular epidemiology of vancomycin-resistant Enterococcus faecium infecting recipients of solid organs in the transplant surgery ward in 2005 and 2006.

BACKGROUND: The aim of the investigations was to compare the vancomycin-resistant Enterococcus faecium (VREfm) strains obtained from our patients. MATERIALS AND METHODS: Strains were compared using restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE) of bacterial DNA. RESULTS: VREfm infected 26 liver recipients, 22 kidney recipients, and 9 other surgery or nephrology patients. Only five strains possessed the vanB determinant. The PFGE analysis revealed two large and several small groups of related strains. CONCLUSIONS: The PFGE analysis enabled the investigation of VRE epidemiology among patients after transplantation. Strains with similar patterns most probably originated from one source and clearly suggested an outbreak.

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.

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.

Phenotypical and genotypical characterization of Neisseria meningitidis carrier strains isolated from Polish recruits in 1998.

The aim of this study was to investigate the Neisseria meningitidis carriage rate among two cohorts of Polish recruits upon entry to the military and during the first 2 months of their service, i.e. in the spring and autumn of 1998, and to characterize the meningococcal strains isolated. Pharyngeal swabs were taken four and five times from 151 and 168 men, respectively. Altogether, 81 and 180 meningococcal isolates representing 54 and 102 different strains were recovered. The overall rates of carriage in the spring and in the autumn were 36% and 61%, and, among recruits who submitted to sampling on at least three occasions, 39% and 55%. Eighty-three of 156 (53%) meningococcal carrier strains were nongroupable; among the remaining strains, serogroup B was predominant (32% of all carrier strains). In both surveys the predominant phenotype was Neisseria meningitidis NG:21:P1.7.

Cluster analysis of Neisseria meningitidis type 22 strains isolated in Poland.

The aim of this study was to investigate the genetic relationships among strains belonging to Neisseria meningitidis serotype 22 recovered in Poland from 1995 to 1998. Phenotypical characterisation of meningococcal strains isolated from patients with meningococcal disease has been performed since 1995. Strains belonging to Neisseria meningitidis type 22 were most frequently recovered from severe cases of meningitis and septicaemia. The type 22 strains were separated into clusters by serosubtyping and DNA analyses by pulsed-field gel electrophoresis and ribotyping. However, the grouping according to serosubtypes was quite different from that obtained by ribotyping and pulsed-field gel electrophoresis. Furthermore, the two genotyping methods seldom identified the same clusters, nor were clusters identified among 26 Neisseria meningitidis type 22 strains recovered from patients hospitalised in five cities. Determination of the susceptibility to penicillin showed that a single strain was resistant (MIC, 2 mg/l) and that one-third (24/71) of the strains tested had reduced susceptibility (MIC range, 0.1-1 mg/l).

[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.

[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.

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.

[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.

[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.

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).

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).

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).

[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.