Assessment of the Microbiological Status of Probiotic Products.

The aim of this study was to perform the microbiological analysis of quality of 25 probiotic products, available on the Polish market. Analysis of bacterial viability in probiotic products showed that not all of these preparations possess a suitable number of bacteria. Moreover, some of the tested probiotic products contained bacterial strains other than those declared by the manufacturer. All tested strains recovered from probiotic products were found to be resistant to metronidazole and susceptible to nitrofurantoin. The susceptibility to other antibiotics was strain specific. Probiotic products should be subject to regular and thorough inspection by appropriate institutions.

[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.

[The evaluation of bacteria penetration by medical textiles for multiple use and disposable multilayer surgical drapes, according to the PN-EN ISO 22610 standard]. / Badanie przenikania bakterii przez tkaniny medyczne przeznaczone do wielokrotnego uzycia oraz wielowarstwowe oblozenia chirurgiczne jednorazowego uzytku, wedlug normy PN-EN ISO 22610.

INTRODUCTION: Cotton as well as synthetic textile medical products are widely used as barrier materials and individual protection against displacement of biological infectious factors. The required level of protection of these products for multiple use and disposable multilayer laminates against the penetration of microbes depends on the risk connected with type of surgical procedure defined in normative documents. METHODS. Cotton and syntetic medical textiles for multiple use, 30-times subjected to processes simulating conditions of the use as well as disposable multilayer surgical drapes were tested. Resistance to microbial wet penetration was conducted according to the PN-EN ISO 22610: 2007 standard. RESULTS: The barrier of cotton fabrics was reduced after first washing and then systematically grew after each often cycles to the value close to the value at the beginning. From the twentieth cycle of simulated conditions of the use, barrier index was reduced. The barrier of the synthetic textile stayed on the average level, while multilayer disposable products ensured the full impermeability for the bacteria. CONCLUSIONS: Natural cotton textiles for multiple use could be apply on operative blocks in limited range because of the changes of the cotton structure caused by repeated laundering process and sterilization. Synthetic materials also have limited application, although are more resistant to cleaning and sterilization processes. Disposable synthetic laminates with many layers use guarantee impermeability for bacteria and may be applied in operative blocks without restrictions.

[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.

[The estimation of penetrating of microbes by the protective clothing and textile medical devices, according to obligatory norms]. / Ocena, wedlug obowiazuj4cych norm, przenikania drobnoustrojów przez odziez ochronna i tkaniny medyczne.

Textile medical products can be widely used as barrier materials and individual protection against biological threats. Rules of introducing such products to market are regulated by the Directive 93/42/ EEC. Detailed requirements and testing methods of textile medical products are presented in obligatory norms. The required level of protection of these products against the penetration of microbes depends on the risk connected with planned type surgical procedure, the duration of the surgical intervention, risks of bleeding or presences of other body liquids of the patient and susceptibilities of the patient to infection. The aim of the study was to establish resistance of medical textiles to wet bacterial penetration. Materials were examined by the apparatus dedicated to this type of testing and obtained results were rated with reference to obligatory contracted requirements. assured Textiles laminated with foils possessed best protective proprieties, whereas medical products made from the cotton do not provide the sufficient level of the protection against microbes.

Molecular survey of beta-lactamases conferring resistance to newer beta-lactams in Enterobacteriaceae isolates from Polish hospitals.

The first national survey of resistance to newer beta-lactams in nosocomial populations of Enterobacteriaceae in Poland was performed. The study covered all nonrepetitive enterobacterial isolates cultured from specimens from inpatients in 13 regional secondary-care hospitals from November 2003 to January 2004. Among 2,388 isolates, the predominant species was Escherichia coli (59.6%), followed by Proteus mirabilis (14.5%) and Klebsiella spp. (8.5%). The frequency of extended-spectrum beta-lactamases (ESBLs) was very high, with ESBLs present in 11.1% of all isolates and 40.4% of Klebsiella pneumoniae isolates, the latter value greatly exceeding that for E. coli (2.5%). The contribution of outbreak isolates was significant, resulting, for example, in a particularly high rate of ESBL producers among Serratia marcescens isolates (70.8%). The pool of ESBL types was overwhelmingly dominated (81.7%) by CTX-M-like beta-lactamases CTX-M-3 (80.6%) and CTX-M-15, with SHV types (17.5%; SHV-2, SHV-5, and SHV-12) and sporadic TEM-like enzymes (0.7%; TEM-19 and TEM-48) being the next most frequent. Acquired AmpC-type cephalosporinases were observed exclusively in P. mirabilis, in 20.5% of the isolates of this species (compared with the frequency of ESBL producers of 11.5% of P. mirabilis isolates). All these cephalosporinases (CMY-12, CMY-15, and a novel variant, CMY-38) originated from Citrobacter freundii. Four isolates of E. coli (two isolates), K. pneumoniae (one isolate), and P. mirabilis (one isolate) produced class A inhibitor-resistant beta-lactamases (TEM-30, TEM-32, TEM-37, and SHV-49), being the first of such producers identified in Poland. The survey documented both specific and more global characteristics of the epidemiology of the beta-lactamase-mediated resistance in enterobacteria from Polish hospitals and demonstrated that the ESBL frequency has reached an alarming level.

The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods.

BACKGROUND: Most patients with bacterial infections suffer from fever and various pains that require complex treatments with antibiotics, antipyretics, and analgaesics. The most common drugs used to relieve these symptoms are non-steroidal anti-inflammatory drugs (NSAIDs), which are not typically considered antibiotics. Here, we investigate the effects of NSAIDs on bacterial susceptibility to antibiotics and the modulation of bacterial efflux pumps. METHODOLOGY: The activity of 12 NSAID active substances, paracetamol (acetaminophen), and eight relevant medicinal products was analyzed with or without pump inhibitors against 89 strains of Gram-negative rods by determining the MICs. Furthermore, the effects of NSAIDs on the susceptibility of clinical strains to antimicrobial agents with or without PAßN (Phe-Arg-ß-naphtylamide) were measured. RESULTS: The MICs of diclofenac, mefenamic acid, ibuprofen, and naproxen, in the presence of PAßN, were significantly (≥4-fold) reduced, decreasing to 25-1600 mg/L, against the majority of the studied strains. In the case of acetylsalicylic acid only for 5 and 7 out of 12 strains of P. mirabilis and E. coli, respectively, a 4-fold increase in susceptibility in the presence of PAßN was observed. The presence of Aspirin resulted in a 4-fold increase in the MIC of ofloxacin against only two strains of E. coli among 48 tested clinical strains, which included species such as E. coli, K. pneumoniae, P. aeruginosa, and S. maltophilia. Besides, the medicinal products containing the following NSAIDs, diclofenac, mefenamic acid, ibuprofen, and naproxen, did not cause the decrease of clinical strains' susceptibility to antibiotics. CONCLUSIONS: The effects of PAßN on the susceptibility of bacteria to NSAIDs indicate that some NSAIDs are substrates for efflux pumps in Gram-negative rods. Morever, Aspirin probably induced efflux-mediated resistance to fluoroquinolones in a few E. coli strains.

Susceptibility testing and resistance phenotypes detection in bacterial pathogens using the VITEK 2 System.

A set of well characterized strains, collected in Polish hospitals, including Gram-negative (n = 93) and Gram-positive (n = 90) isolates was used in the study. The VITEK 2 AST-cards were used in the analysis according to the manufacturer's recommendations. Comparison of the susceptibility data obtained by the standard method and by VITEK 2 cards proved concordant in 99% of cases. Clinically important mechanisms were revealed by the VITEK 2 AES with >95% agreement with reference data including methicillin resistance in staphylococci (98%), high-level aminoglycoside resistance in enterococci (100%), VanA and VanB phenotypes in enterococci (100%), and ESBLs in Enterobacteriaceae (93.8%). The VITEK 2 AES System appears a reliable tool for the detection and interpretive reading of clinically important mechanisms of resistance and can be recommended for routine work.

Outbreak of Pseudomonas aeruginosa infections with PER-1 extended-spectrum beta-lactamase in Warsaw, Poland: further evidence for an international clonal complex.

Forty-one Pseudomonas aeruginosa isolates with extended-spectrum beta-lactamases (ESBLs) from a hospital in Warsaw, Poland, were analyzed. Thirty-seven isolates from several wards were collected over 9 months in 2003 and 2004. The isolates were recovered from patients with multiple types of infections, mostly respiratory tract and postoperative wound infections. All 41 isolates produced the PER-1 ESBL, originally observed in Turkey but recently also identified in several countries in Europe and the Far East. The bla(PER-1) gene resided within the Tn1213 composite transposon, which was chromosomally located. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) revealed the presence of three separate clones among the isolates. Two of these, corresponding to sequence types (STs) ST244 and ST235, were responsible for parallel outbreaks. Apart from PER-1, all the isolates produced OXA-2 oxacillinase. ST235 isolates additionally expressed a novel enzyme, OXA-74, differing by one amino acid from the OXA-17 ESBL identified originally in PER-1- and OXA-2-positive P. aeruginosa isolates from Ankara, Turkey, in 1992. These earlier Ankara isolates with PER-1, OXA-2, and OXA-17 were also classified into ST235, which is a single-locus variant of two other STs, ST227 and ST230. ST227, ST230, and ST235 all correspond to the recently described clonal complex BG11, which seems to be internationally distributed, having spread in Turkey, Greece, Italy, Hungary, Poland, Sweden, and much of Russia. It is associated with various beta-lactamases, including PER-1 and VIM metalloenzymes. This work further demonstrates the value of MLST of P. aeruginosa.

Molecular epidemiology of acquired-metallo-beta-lactamase-producing bacteria in Poland.

We have analyzed 40 metallo-beta-lactamase (MBL)-producing isolates of Pseudomonas aeruginosa (n = 38), Pseudomonas putida (n = 1), and Acinetobacter genospecies 3 (n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced beta-lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The bla(VIM) genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained bla(VIM-2) and two of which contained bla(VIM-4). The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.

Evolution of TEM-type extended-spectrum beta-lactamases in clinical Enterobacteriaceae strains in Poland.

Seventeen extended-spectrum beta-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae recovered from 1998 to 2000 in hospitals of five different cities in Poland were analyzed. They expressed several TEM-type ESBLs, TEM-4, TEM-29, TEM-85, TEM-86, TEM-93, and TEM-94. TEM-85 (L21F, R164S, E240K, T265M), TEM-86 (L21F, R164S, A237T, E240K, T265M), TEM-93 (M182T, G238S, E240K), and TEM-94 (L21F, E104K, M182T, G238S, T265M) were identified for the first time. Including the enzymes described earlier, TEM-47, TEM-48, TEM-49, and TEM-68, the group of known ESBLs of the TEM family produced by enterobacteria in Polish hospitals has increased to 10 variants. Comparative sequence analysis of the genes coding for all these beta-lactamases revealed a view of their possible evolution, which, apart from the gradual acquisition of various mutations, could also have involved recombination events. Two different bla(TEM-1) gene alleles were precursors of the ESBL genes: bla(TEM-1A), which was the ancestor of bla(TEM-93), and bla(TEM-1F), from which all the remaining genes originated. The evolution of the bla(TEM-1F)-related genes most probably consisted of three major separate lineages, one of which, including bla(TEM-4), bla(TEM-47), bla(TEM-48), bla(TEM-49), bla(TEM-68), and bla(TEM-94), was highly structured itself and could have been initiated by the bla(TEM-25) gene, identified exclusively in France so far. Plasmid fingerprinting analysis revealed a high degree of diversity of plasmids carrying related bla(TEM) genes, which suggested either the intense diversification or transposition of bla(TEM) genes between different plasmids or some contribution of convergent evolution. The results of this study clearly demonstrate that the environment of Polish hospitals has been highly favorable for the rapid evolution of ESBLs.