Tomasz J. Wąsik

Susceptibility of Staphylococcus aureus Clinical Isolates to Propolis Extract Alone or in Combination with Antimicrobial Drugs

The objective of this study was to assess in vitro the antimicrobial activity of ethanolic extract of Polish propolis (EEPP) against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. The combined effect of EEPP and 10 selected antistaphylococcal drugs on S. aureus clinical cultures was also investigated. EEPP composition was analyzed by a High Performance Liquid Chromatography (HPLC) method. The flavonoid compounds identified in Polish Propolis included flavones, flavonones, flavonolols, flavonols and phenolic acids. EEPP displayed varying effectiveness against twelve S. aureus strains, with minimal inhibitory concentration (MIC) within the range from 0.39 to 0.78 mg/mL, determined by broth microdilution method. The average MIC was 0.54 ± 0.22 mg/mL, while calculated MIC50 and MIC90 were 0.39 mg/mL and 0.78 mg/mL, respectively. The minimum bactericidal concentration (MBC) of the EEPP ranged from 0.78 to 3.13 mg/mL. The in vitro combined effect of EEPP and 10 antibacterial drugs was investigated using disk diffusion method-based assay. Addition of EEPP to cefoxitin (FOX), clindamycin (DA), tetracycline (TE), tobramycin (TOB), linezolid (LIN), trimethoprim+sulfamethoxazole (SXT), penicillin (P), erythromycin (E) regimen, yielded stronger, cumulative antimicrobial effect, against all tested S. aureus strains than EEPP and chemotherapeutics alone. In the case of ciprofloxacin (CIP) and chloramphenicol (C) no synergism with EEPP was observed.

Biofilm Formation and Antimicrobial Susceptibility of Staphylococcus epidermidis Strains from a Hospital Environment

The hospital environment microflora comprise a wide variety of microorganisms which are more or less pathogenic and where staphylococci are one of the most common types. The aim of the presented study was to evaluate the prevalence of the biofilm forming coagulase-negative staphylococci (CoNS) in a hospital environment as a risk factor for nosocomial infections. Among 122 isolated and tested strains of CoNS the most frequent were: S. epidermidis—32 strains, S. haemolyticus—31 strains, S. capitis subsp. capitis—21 strains, S. hominis—11 strains, S. cohnii subsp. cohnii—nine strains. In case of CoNS, the main molecule responsible for intercellular adhesion is a polysaccharide intercellular adhesin (PIA), encoded on the ica gene operon. The analysis revealed the presence of the icaADBC operon genes in 46.88% of S. epidermidis isolates. IcaA and icaD were present in 34.38% and 28.13% of strains respectively while IcaC gene was present in 37.50% of strains. IcaB gene was found in 21.88% of S. epidermidis strains. In 15 (63%) strains all icaADBC operon genes were observed. The assessment of antibacterial drugs susceptibility demonstrated that analyzed CoNS strains were highly resistant to macrolides and lincosamides and more sensitive to rifampicin and linezolid. Our data indicates that the hospital environment can be colonized by biofilm forming coagulase-negative staphylococci and transmission of these strains can cause an increased risk of serious nosocomial infections.

In Vitro Antimicrobial Activity of Ethanolic Extract of Polish Propolis against Biofilm Forming Staphylococcus epidermidis Strains

The aim of the presented study was to examine the antimicrobial activity of ethanol extract of Polish propolis (EEPP) against biofilm-forming CoNS strains in vitro. Our results revealed that EEPP displayed varying degrees of activity against CoNS with MIC values ranging from 1.56 to 0.78 mg/mL. The average MIC was 1.13 ± 0.39 mg/mL while calculated MIC50 and MIC90 values were 0.78 mg/mL and 1.56 mg/mL, respectively. The biofilm formation ability by all tested S. epidermidis strains was inhibited at EEPP concentrations ranging from 0.39 to 1.56 mg/mL. The degree of reduction of AlamarBlue was directly associated with the proliferation of S. epidermidis strains. The increased proliferation of S. epidermidis strains was observed after 12 and 24 hours of incubation in the presence of EEPP concentrations ranging from 0.025 to 0.39 mg/mL. These results suggest that antimicrobial activities of EEPP against S. epidermidis expressed as the reduction of bacterial growth, reduction of biofilm formation ability, and the intensity of proliferation were significantly affected by incubation time and EEPP concentration used as well as the interactions between these factors.

Catechin Hydrate Augments the Antibacterial Action of Selected Antibiotics against Staphylococcus aureus Clinical Strains

Synergistic effects between commonly used antibiotics and natural substances may be an alternative to conventional antibacterial therapies. The objective of the presented study was to assess the in vitro antibacterial activity of catechin hydrate (CH) and evaluate the interactions of CH with selected antibiotics using Staphylococcus aureus clinical and reference strains. CH displayed diverse activity towards examined S. aureus strains, with minimal inhibitory concentrations (MICs) ranging from 256 to 2048 µg/mL. The interaction between CH and antibiotics was assessed by an E-test. The most significant synergistic effects were noticed for CH in combination with clindamycin and erythromycin. For cefoxitin and vancomycin a decrease of MIC values in the presence of CH was also observed, but it did not reach statistical significance. The obtained results demonstrate that CH shows antimicrobial activity against Staphylococcus aureus clinical strains. What is more, we proved a synergistic effect of CH with erythromycin and clindamycin.

Molecular epidemiology of recent HIV‐1 infections in southern Poland

The genetic diversity of human immunodeficiency virus type 1 (HIV‐1) offers an opportunity to track the development of the epidemic across different populations. Viral pol gene fragments from 55 individuals of Polish origin with recent HIV‐1 infection identified in 2008–2010 in four Polish cities were analyzed. Viral sequences were compared with sequences from 100 individuals (reference group) infected before 2004. Viral spread among groups with different HIV transmission categories was compared using a phylogenetic approach. The majority of sequences from individuals with recent infection were subtype B (93%) within which four transmission clusters (18% of samples) were detected. Samples from men infected through sex between men and from persons infected through injecting drugs were broadly separated (P < 0.0001), while samples from individuals infected by heterosexual contacts were dispersed uniformly within phylogenetic tree (P = 0.244) inferred from viral sequences derived from individuals infected recently and the reference group. The percentage of samples from persons infected by heterosexual contacts which clustered with samples from men infected through sex between men was not significantly higher for those with recent infection (47%), compared to the reference group (36%). In conclusion, men infected by sex between men and individuals infected through injecting drugs appear to form separate HIV transmission networks in Poland. The recent spread of HIV‐1 among persons infected with subtype B by heterosexual contacts appears to be linked to both these groups. J. Med. Virol. 84:1857–1868, 2012.

An Analysis of the Effects of Vancomycin and/or Vancomycin-Resistant Citrobacter freundii Exposure on the Microbial Community Structure in Soil

The occurrence of antibiotics and antibiotic resistance genes in the environment has become a subject of growing concern. The extensive use of vancomycin and other pharmaceuticals may alter the biodiversity of soil microbial communities and select antibiotic-resistant bacteria. Therefore, the purpose of the study was to evaluate the impact of vancomycin and/or vancomycin-resistant Citrobacter freundii on soil microbial communities using the denaturing gradient gel electrophoresis (DGGE) and the phospholipid fatty acid (PLFA) approaches. The experiment had a completely randomized block design with the following treatments: control soil (C), soil with vancomycin (1 mg/kg soil—VA1), soil with vancomycin (10 mg/kg soil—VA10), soil with C. freundii (Cit), soil with vancomycin (1 mg/kg soil) and C. freundii (VA1+Cit), and soil with vancomycin (10 mg/kg soil) and C. freundii (VA10+Cit). A bacterial strain resistant to vancomycin was isolated from raw sewage collected from the municipal sewage treatment plant. The obtained results indicated that the antibiotic and/or the bacterial strain exerted a selective pressure that resulted in qualitative and quantitative changes in the population of soil microorganisms. However, a multivariate analysis showed that the genetic and structural diversity of the soil microbial community was primarily affected by the incubation time and to a lesser extent by the antibiotic and introduced bacteria. DGGE analysis clearly showed that certain species within the bacterial community were sensitive to vancomycin as was evidenced by a decrease in the values of S (richness) and H (Shannon-Wiener) indices. Moreover, a PLFA method-based analysis revealed alterations in the structure of the soil microbial community as indicated by changes in the biomass of the PLFA biomarkers specific for Gram-positive and Gram-negative bacteria as well as fungi. The changes observed in the community of soil microorganisms may decrease the rate of microbial-mediated processes, which can lead to a disturbance in the ecological balance of the soil ecosystem.

Prevalence of tick-borne encephalitis virus in a highly urbanized and low risk area in Southern Poland

The knowledge of the exact distribution of tick-borne encephalitis virus (TBEV) endemic foci is crucial to plan and implement the optimal prevention of tick-borne encephalitis (TBE), including a vaccination program. In Poland, however, there is still no data on the local distribution of TBEV in many areas of the country. Silesian agglomeration area (Southern Poland) is a highly urbanized and industrialized region of the country, where TBE cases are only sporadically recorded. In this study, a total of 4350 adult Ixodes ricinus were collected between September 2010 and June 2012 at twelve locations. The screening using real-time PCR was carried out on 854 tick pools of five specimens, and the positive pools were verified by pyrosequencing. TBEV was identified in 13 pools (1.52%) at 4 sites, of which 9 pools were verified by pyrosequencing. An overall pool prevalence was estimated at 0.31% ranging from 0.19% to 1.11% for positive locations [95% CI 0.16-0.52], which is comparable with regions with high number of TBE cases reported annually.

Association between the PRNP 1368 polymorphism and the occurrence of sporadic Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare transmissible neurodegenerative disorder. The etiology of sporadic form of CJD remains unsolved. In addition to the codon 129 polymorphism, polymorphisms in the non-coding region of PRNP are considered as important factors in sCJD development. To assess a possible association between PRNP 1368 SNP and sCJD, we compared the genotype, allele and haplotype frequencies of the 1368 SNP among 46 sCJD patients of Dutch origin with the respective frequencies in healthy controls. We detected a significant association between sCJD and 1368T/T genotype. A significant difference was also observed in 1368 alleles’ distribution. In the haplotype analysis, haplotype 1368C-129G was associated with decreased risk of sCJD in Dutch population. Our findings support the hypothesis that genetic variations in the regulatory region of the PRNP gene may influence the pathogenesis of sCJD.

Antibacterial Activity of Protocatechuic Acid Ethyl Ester on Staphylococcus aureus Clinical Strains Alone and in Combination with Antistaphylococcal Drugs

The aim of the presented study was to examine in vitro the antibacterial activity of protocatechuic acid ethyl ester (ethyl 3,4-dihydroxybenzoate, EDHB) against Staphylococcus aureus clinical isolates alone and in the combination with four selected antibiotics. The EDHB antimicrobial activity was tested against twenty S. aureus strains isolated from the clinical samples, and three reference strains. The phenotypes and genotypes of resistance to methicillin for the tested strains were defined as well as the phenotypic resistance to macrolides, lincosamides and streptogramin B (MLSB). EDHB displayed diverse activity against examined S. aureus strains with the minimal inhibitory concentration (MIC) within the range from 64 to 1024 µg/mL. Addition of ¼ MIC of EDHB into the Mueller-Hinton Agar (MHA) resulted in augmented antibacterial effect in the presence of clindamycin. In the case of cefoxitin no synergistic effect with EDHB was noted. For erythromycin and vancomycin the decrease of mean MICs in the presence of EDHB was observed but did not reach statistical significance. The results of the present study showed that in vitro EDHB possesses antibacterial activity against S. aureus clinical strains and triggers a synergistic antimicrobial effect with clindamycin and to the lesser extent with erythromycin and vancomycin.

Current and future assays for identifying recent HIV infections at the population level.

Summary The precise diagnosis of recent human immunodeficiency virus (HIV) infection is crucial for estimating HIV incidence, defined as the number of new infections in a population, per person at risk, during a specified time period. Incidence assessment is considered to be a tool for surveillance, public health and research. Differentiating recent from long-term HIV infections is possible thanks to the evaluation of HIV-specific immune response development or viral markers measurement. Several methods that enable the recognition of recent HIV-1 infection with the use of a single blood specimen have been developed, and their value for use in population level studies has been demonstrated. However, they are still inadequate due to a variable window period and false recent rates among HIV clades and across populations. Application of these assays at an individual level is far more questionable because of person-to-person variability in the antibody response and the course of HIV infection, and because of the prospective regulatory approval requirements. In this article we review the principles and the limitations of the currently available major laboratory techniques that allow detection of recent HIV infection. The assays based on the alteration of serological parameters, as well as the newest method based on an increase of HIV genetic diversity with the progress of infection, are described.