Application of EN 16615 (4-Field Test) for the Evaluation of the Antimicrobial Activity of the Selected Commercial and Self-Made Disinfectant Wipes.

The purpose of disinfectants is to reduce microorganisms on a contaminated surface and to prevent the spread of microorganisms. The relatively new EN 16615 simulates disinfection by wiping and allows for assessing the recovery of microorganisms from the surface and, importantly, the degree of spread of microorganisms when the surface is disinfected by wiping. For the first time, using this standard, the tested products in the form of commercial disinfectant wipes were compared with self-made wipes soaked in respective disinfectant liquids. The disinfected surfaces were simulated by homogeneous polyvinyl chloride plates. The studies were carried out not only with the standard, but also with clinical multidrug-resistant microbial strains. Based on the research, it can be concluded that the most effective products in the disinfection process (log10 reduction of ≥5) with the shortest contact time (1 min) were products containing ethanol, propanol, and quaternary ammonium compounds (self-made wipes) and propanol (commercial wipes). The least effective products (log10 reduction of <5) in terms of the contact time declared by the manufacturer were products containing ethanol and sodium hypochlorite (commercial wipes). Much better antimicrobial activity of self-made wipes was observed in comparison to the activity of the commercial wipes.

Occurrence of antimicrobial agents, drug-resistant bacteria, and genes in the sewage-impacted Vistula River (Poland).

Antimicrobial agents (antimicrobials) are a group of therapeutic and hygienic agents that either kill microorganisms or inhibit their growth. Their occurrence in surface water may reveal harmful effects on aquatic biota and challenge microbial populations. Recently, there is a growing concern over the contamination of surface water with both antimicrobial agents and multidrug-resistant bacteria. The aim of the study was the determination of the presence of selected antimicrobials at specific locations of the Vistula River (Poland), as well as in tap water samples originating from the Warsaw region. Analysis was performed using the liquid chromatography-electrospray ionization-tandem mass spectrometry method. In addition, the occurrence of drug-resistant bacteria and resistance genes was determined using standard procedures. This 2-year study is the first investigation of the simultaneous presence of antimicrobial agents, drug-resistant bacteria, and genes in Polish surface water. In Poland, relatively high concentrations of macrolides are observed in both surface and tap water. Simultaneous to the high macrolide levels in the environment, the presence of the erm B gene, coding the resistance to macrolides, lincosamides, and streptogramin, was detected in almost all sampling sites. Another ubiquitous gene was int1, an element of the 5'-conserved segment of class 1 integrons that encode site-specific integrase. Also, resistant isolates of Enterococcus faecium and Enterococcus faecalis and Gram-negative bacteria were recovered. Multidrug-resistant bacteria isolates of Gram-negative and Enterococcus were also detected. The results show that wastewater treatment plants (WWTP) are the main source of most antimicrobials, resistant bacteria, and genes in the aquatic environment, probably due to partial purification during wastewater treatment processes.

Evaluation of mycobactericidal activity of selected chemical disinfectants and antiseptics according to European standards.

BACKGROUND: The history of the investigation of standardized mycobactericidal activity of disinfectants and antiseptics is not very long. There is growing interest among the manufacturers of disinfectants in carrying out research on the antimicrobial activities in accordance with European standards (EN). This research could facilitate the introduction of high-quality disinfectants to the market. The aim of this study was to evaluate the mycobactericidal activity of selected chemical disinfectants and antiseptics used in the medical and veterinary fields. MATERIAL AND METHODS: This study included 19 products submitted to the National Medicines Institute in Poland for evaluation of mycobactericidal activity. These products contain in their composition active substances belonging to different chemical groups, including aldehydes, alcohols, amines, quaternary ammonium compounds, phenols, guanidine, and oxidizing compounds. This study, conducted according to the manufacturers' description of the preparations, was carried out in accordance with European standards, which also met the Polish standards: PN-EN 14204: 2013, PN-EN 14348: 2006, and PN-EN 14563: 2012. RESULTS: Tested products for disinfection and antiseptics containing active substances from different chemical groups showed high mycobactericidal activity and met the requirements of the appropriate European standards in most cases. In the case of products containing guanidine and amine compounds, the concentration of active ingredients used in the test and the test conditions specified by the manufacturer did not provide the mycobactericidal activity required by the standards. CONCLUSIONS: Prior to the launch of a new product on the market, it is important to establish the appropriate usage and testing conditions of the preparation, such as its practical concentration, contact time, and environment condition (clean or dirty).

Reduction of the neutralisation time during antimicrobial activity testing of disinfectants according to European Standards.

BACKGROUND: Evaluation of the biocidal activity of chemical disinfectants and antiseptics according to European Standards (EN) is based on determination of the reduction of the number of viable test microorganisms under defined conditions. OBJECTIVE: The objective of this study was to investigate whether reducing the neutralization time required following declared product contact times for the tested microorganisms yields method validations. MATERIAL AND METHODS: This study was conducted on 14 products containing active substances from different chemical groups: alcohols, aldehydes, biguanides, quaternary ammonium compounds, phenols, amines derivatives, oxidizing agents. These products were tested according to phase 1 tests: EN 1040:2005 and EN 1275:2005 and then according to phase 2, step 1 tests: Draft EN 13727:2005 and EN 13624:2003. Biocidal activity was evaluated using the following test organisms: S. aureus ATCC 6538, P. aeruginosa ATCC 15442, E. coli NCTC 10538, E. coli ATCC 10536, E. hirae ATCC 10541, C. albicans ATCC 10231 and A. brasiliensis ATCC 16404. RESULTS: Validation C results for all products and tested microorganism strains were at least half of the density of the suspension for validation (Nvo) after only 10 s of neutralization. Furthermore, results from test procedures performed in parallel were also positive except 5 products toward A. brasiliensis. CONCLUSIONS: The results of our study confirm that the contact time described in the European Standards phase 1: EN 1040 and EN 1275, as well as phase 2, step 1: Draft EN 13727 and EN 13624 can be precisely determined in spite of reducing the neutralization time from 5 minutes to even 10 seconds.

Antibacterial activity of selected commercial products for mouth washing and disinfection, assessed in accordance with PN-EN 1040.

BACKGROUND: Currently, there is a wide range of products for mouth washing on the Polish market. They have different qualitative and quantitative compositions, and they differ particularly in the concentration of active substances. In antisepsis and disinfection, the significant reduction in number of cells of microorganisms in a particular environment is very crucial. The chemical agents should provide a significant decrease in number of microorganisms in a relatively short time. The purpose of this study was to examine the bactericidal activity of selected herbal products used for treatment of inflammation, and disinfection and washing of the mouth, having antibacterial activity as declared by the manufacturers. MATERIAL AND METHODS: The study included 28 products for mouth washing and disinfection available in Poland. Bactericidal activity was studied using a quantitative suspension test according to the standard PN-EN 1040. RESULTS: Only 1 of 4 tested herbal products, registered as medicinal products, showed satisfactory antibacterial activity when they were used according to the manufacturer's recommendations. A total of 13 preparations (48%) complied with the standard requirements against all tested strains. Up to 19% of products showed no bactericidal activity against bacterial strains, and up to 33% were only effective against certain microorganisms. CONCLUSIONS: The informational literature accompanying most antiseptics should be corrected by the manufacturers, providing information about antimicrobial activity consistent with the requirements of applicable standards. The information on the packaging or in the leaflets for antiseptic products should be corrected by the manufacturers to include accurate information on antimicrobial activity.

[Microbiological characteristics of selected liquid soaps for hands washing]. / Mikrobiologiczna charakterystyka wybranych plynnych produktów stosowanych do mycia rak.

INTRODUCTION: According to common belief, supported by the authority of the World Health Organization - WHO, the common (social) hand washing is the simplest, cheapest and the most effective way of reduction the hospital-acquired infections. For this purpose products of"liquid soaps", present in a large number on the market, are most often applied. Microbiological status (microbiological purity and antimicrobial activity) of"liquid soaps" available on the Polish market is not known, because relevant routinely studies have not been performed. Only the antibacterial and / or antifungal activity of certain formulations is sometimes assessed, especially when the manufacturer suggests the standardized application of the products for surgical or hygienic procedures. The aim of this study was to determine the microbiological quality, especially microbiological purity and antimicrobial activity of the selected hands washing products, presents on the Polish market. METHODS: The 12 selected commercial products, available on the market in Poland, dedicated for hands washing were included into study. Microbiological purity test was carried out in accordance with the Polish Pharmacopoeia (FP) monograph (FP monograph numbers correspond to numbers of the European Pharmacopoeia monograph- Ph. Eur.) No 2.6.12 "Microbiological examination of non-sterile products: microbial enumaration tests", and the monograph of FP No. 2.6.13 "Microbiological examination of non-sterile products: test for specified microorganisms". The following physico-chemical properties of soaps were examined: the pH of the formulations was measured according to the monograph FP No. 2.2.3. "Potentiometric determination of pH", the density of products was assayed according to the monograph FPNo. 2.2.5. "Relative density" and determination the water activity was performed by monograph FP No 2.9.39 "Water-solid interactions: determination of sorption-desorption isotherms and of water activity". Next, antibacterial and antifungal protection was determined in accordance with the monograph FP No 5.1.3. "Efficacy of antimicrobial preservation". The study of antimicrobial activity was carried out in accordance with PN-EN 1040 "Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics - Test method and requirements (phase 1)". Finally, using the "time-kill" method the survival of microorganisms after different contact times of the products with bacteria and fungi were determined. RESULTS: All the examined products showed a very high microbiological purity. None of the formulations was characterized by a high acidity or alkalinity. All the analyzed products were slightly thicker than water, but such density of the preparation does not seem to be important parameter in the growth of microorganisms. The results of water activity estimation - the parameter indicating the presence of free, not chemically bound water stimulating microbes growth - do not show that low water content in the preparation may inhibit bacteria and fungi growth. Taking into consideration the antimicrobial protection of the products demonstrated in the tests carried out in accordance within FP monograph No 5.1.3. and PN-EN 1040, and analysing curves indicating killing rate of bacteria and fungi obtained by "time-kill" method, the microorganisms contaminating the products generally should not multiply in their environment, and gradually they die - what can take many hours or even days. CONCLUSIONS: The cases of bacterial infections connected with the usage of non-medical liguid soaps, applied in the health care units and described in the literature, should be considered as related rather to contamination of plastic packaging and dosage system, then to contamination of preparation itself inside the package. It was proved, that in all tested products amount of contaminating microbes diminishes in time. The dynamics of this process depends on the microorganisms character - bacteria dies quicker then fungi. The special attention should be given to washing, cleaning and disinfection of preparation dispensing systems, to avoid microbial contamination of product doses applied directly on the hands. It should be emphasized that only formulations containing antimicrobial agents in an appropriate amount, eliminate microorganisms from the skin surface fast and effectively. In case of hygienic and surgical procedures following the standardized manner in order to obtain required reduction rate of microorganisms in a short time - only products complying with appropriate EN standards are suitable. For these puroposes, the popular "liquid soaps" should not be used.

[Genetic similarity of vancomycin resistant strains of Enterococcus faecium isolated from clinical specimens]. / Genetyczne podobienistwo opornych na wankomycyne szczepów Enterococcus faecium izolowanych z materialu klinicznego.

Twenty vancomycin resistant E. faecium strains (VRE) isolated from patients of three different hospital wards in 2005-2008 were examined. The strains originated from patients of intensive therapy, urological and internistic wards. The chosen wards differ significantly in their specificity. In all cases the presence of o vanA and lack of vanB, vanD, vanE and vanG genes and were found. Strains were compared by using RFLP-PFGE, the reference method for molecular typing of VRE. One group including fourteen strains showing similarity higher than 79.5% was distinguished. This group was divided into subgroups. The greatest similarity was found among strains from patients of intensive therapy ward. Two subgroups of strains showing similarity more than 93.3%, of four strains each were identified. The similarity between these two subgroups was 79.5%. Most strains from other two wards showed less than 79.5% similarity and they could be recognised as not related. Only one strain from internal ward and two strains from urologic ward were similar in 82.1 - 86.4% to one of subgroups of strains originated from intensive therapy.

Antimicrobial activity of glucoprotamin-containing disinfectants.

Introduction of a new antimicrobial agent as a drug--for treatment of infections or as a disinfectant and antiseptic, may result in the occurrence of resistance mechanisms against this agent among microorganisms. Two disinfectants of different composition--Incidin Plus for surface disinfection and Sekusept Plus for medical devices disinfection, both containing glucoprotamin as the active substance, were investigated in this study in order to analyze their antimicrobial activity. Standard bacterial and fungal strains recommended by European Standards, established by European Standardization Committee for testing bactericidal and fungicidal activity of chemical disinfectants were used in the study. Furthermore, 60 clinical bacterial strains with different susceptibility to antibiotics and chemotherapeutics, mostly multiresistant, isolated from different specimens from hospitalized patients were analyzed. In addition, 184 fungal clinical strains isolated from hospitalized patients and outpatients were also included in this study. Antimicrobial activity was evaluated according to EN 1040:2005 --using bacterial strains and according to EN 1275:2005--using fungal strains. Glucoprotamin proved to be a very effective and rapidly acting bactericidal and fungicidal agent. Low concentration of glucoprotamin--0.5% showed to be very effective (1 min) against clinical bacterial isolates. Incidin Plus was also very effective (5 min) against clinical fungal isolates.

[Estimation of activity of pharmakopeal disinfectants and antiseptics against Gram-negative and Gram-positive bacteria isolated from clinical specimens, drugs and environment]. / Wrazliwosc wybranych szczepów bakterii Gram-ujemnych i Gram-dodatnich izolowanych z materialu klinicznego, leków i srodowiska, na farmakopealne zwiazki dezynfekcyjne i antyseptyczne.

The MIC of nine different disinfectants and antiseptics were determined for the Gram-negative and Gram-positive bacteria. Strains originated from clinical specimens, drugs and environment. A sensitivity was determined against chlorhexidinum digluconate (Gram-negative: 0,625-80 mg/L, Gram-positive: 0,3-10 mg/L), benzalconium chloride (Gram-negative: 2,5-1280 mg/L, Gram-positive: 1,25-20 mg/L), salicilic acid (Gram-negative and Gram-positive: 400-1600 mg/L), benzoic acid (Gram-negative: 800-1600 mg/L, Gram-positive: 400-1 600 mg/L), boric acid (Gram-negative: 800-12 800 mg/L, Gram-positive: 1 600-6400 mg/L), chloramine B (Gram-negative: 1600-6400 mg/L, Gram-positive:800- 6400 mg/L), jodine (Gram-negative: 200-1600 mg/L, Gram-positive: 200-1600 mg/L), etacridine lactate (Gram-negative: 40 do > 20480 mg/L, Gram-positive: 40-1280 mg/L) and resorcine (Gram-negative: 1600-6400 mg/L, Gram-positive: 800-6400 mg/L). Diversified values of MIC for different strains were obtained, especially in the case of benzalconium chloride, etacridine lactate, chlorhexidinum digluconate, boric acid and iodine. Strains isolated from environment were usually more susceptible to examined compounds than clinical strains. The biggest diversification of sensitivity was observed among strains originated from drugs where besides sensitive appeared strains characterizing by very high MIC values of some substances, eg. boric acid.

[The resistance to glycopeptide antibiotics among strains of Enterococcus sp. isolated in 1998-2005 from patients of one of the university hospitals in Warsaw]. / Opornosc na glikopeptydy szczepów Enterococcus sp. izolowanychw latach 1998-2005 od chorych hospitalizowanych.

Infections due to vancomycin resistant enterococci (VRE) constitute a serious therapeutic problem in some cases. Up to date there is only poor knowledge about the frequency of isolation of VRE inthe Polish hospitals. The aim of our investigations was to compare the number and the assortment of VRE isolated during eight years (1998-2005) in the one big clinical hospital in Warsaw. All resistant strains were checked for possessing the vanA or van B ligase genes in the PCR reaction. The identification of the strains was proven by PCR with using of the ddl primers. The significant increase in the number of VRE was observed in the 2005 compared with recent years. All strains isolated in recent years possessed vanA ligase genes. Although among all enterococci E. faecalis made majority (over 70%), among VRE predominated E. faecium.

[Comparison of the checkerboard and E-test methods used for the analysis of two antibiotics combination]. / Porównanie metod szachownicy i E-testu stosowanych do badania przeciwbakteryjnej aktywnosci zestawien dwóch antybiotyków.

The aim of this study was to compare checkerboard method with E-test assay for interaction analysis of aminoglycosides in combination with other antibiotics on selected clinical bacterial strains. In the first step, MIC values of analysed antibiotics, against particular bacterial strain were established. In the next step, antibiotics interaction was analysed by checkerboard technique and E-test. We found some difficulties while comparing these two methods. The checkerboard and E-test results corresponded in about 55%. Twenty-one percentage of results obtained by both methods showed some discrepancies. In 15% of cases, because of high MICs values, comparison of the results was impossible. Some investigators declare FIC indexes, from over 0.5 up to 4, for neutral effects. Sharing this point of view, above 21% of discrepancies results agreed. In such situation, definite disagreement was observed only in 8% of obtained results. In this investigation, additive and neutral effects were dominant. The E-test technique is less-laborous than standard agar method. In this study the E-test assay indicated synergy in only one case. Because of the manner in which the E-test strips were placed on the agar (scales intersecting at the MICs) only dramatic cases of antagonism were detected. Mild cases were undetected because the inhibition zone run under the crossed strips and was therefore unreadable and interpreted as indifference. On the basis of these results, examination of interactions between antibiotics by E-test, appears to be possible alternative to checkerboard method with mentioned limitations.

[Interaction of neomycin with other antibiotics on selected bacterial strains]. / Ocena oddzialywania neomycyny z antybiotykiem innej grupy na wybrane szczepy bakteryjne.

Antimicrobial combinations are used most frequently to provide broad-spectrum empirical coverage in the treatment of bacterial infections. However, combination of two antibiotics may not influence their activity, may lead to synergy or antagonism in the activity. Neomycin may be combined with one of the following antibiotics: ampicillin, procaine penicillin, gramicidin, bacitracin, polymyxin B, lincomycin, oxytetracycline, and erythromycin in some human and veterinary multiantibiotic drugs distributed in Poland. The checkerboard method has been one of the traditional assays for the measurement of antibiotic interactions. The aim of this study was to analyse the activity interaction of neomycin with second antibiotic in multiantibiotic drugs distributed in Poland on standards and clinical bacterial strains. Checkerboard results for all strains demonstrated synergism for 2.5% of combinations, only for standards strains. In one case Salmonella Enteritidis, in combination of neomycin with bacitracin, inhibition effect was observed. Additive effects were predominant--49%. In 18% neutral effects were shown, but in 26% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. In combination of aminoglycoside (neomycin) with beta-lactams antibiotics (ampicillin, procaine penicillin) in vitro, no synergy was observed for all examined strains. The best results were achieved for combinations of neomycin with peptide antibiotics (polymyxin, gramicidin and bacitracin)--5 for all 6 synergy effect observed.

[Comparison of checkerboard and time-kill methods for the analysis of two antibiotics combined]. / Porównanie metod szachownicy i "time-kill" stosowanych do badania zestawienia dwóch antybiotyków.

Activity interaction analysis of two antibiotics by two methods: checkerboard and "time-kill" was compared during this study. Combinations of procaine penicillin, polymyxin B and bacitracin with neomycin and procaine penicillin with dihydrostreptomycin were examined. Checkerboard method is the most widely used technique for antimicrobials interactions analyses. The "time-kill" method, performed by the broth macrodilution technique, provides a dynamic picture of antimicrobial action and interaction over time (based on serial colony counts). Differences of "time-kill" method and the checkerboard technique, allow single visual examination (after 16 to 24 hours of incubation). Additive and inhibition effects were observed in combinations of neomycin with beta-lactam antibiotic (procaine penicillin) and peptide antibiotics (bacitracin and polymyxin B) on clinical strain S. Enteritidis IL 35 "Time-kill" method also confirmed observations mentioned above. In combinations of procaine penicillin with dihydrostreptomycin on strains E. coli IL 531 and E. coli IL 256 synergy effects on checkerboard technique were noticed. Such observation was not confirmed by the "time-kill" method. The methodologies and definitions of synergism are variable and not standardized. This situation should be improved, because comparison of the results obtained by different methods becomes a very difficult task.

[Interaction of aminoglycosides and the other antibiotic on selected bacterial strains]. / Ocena oddzialywania aminoglikozydu z antybiotykiem innej grupy na wybrane szczepy bakteryjne.

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.