The analysis of Lactobacillus spp. distribution in the vaginal microbiota of Polish women with abnormal Pap smear result.

Introduction: A healthy vaginal microbiota is represented mainly by Lactobacillus spp. and plays a vital role in maintaining the functional balance in the vaginal environment. Scientists have drawn attention to possible correlations between the vaginal microbiome and gynecological neoplasms. Several recent studies have shown a potential link between the vaginal microbiome and the risk of developing cervical cancer from human papillomavirus (HPV) infection. This study aimed to compare the prevalence and abundance of various lactic acid bacteria species (LABs) in vaginal swabs from healthy controls and patients with abnormal Pap smear results. Methods: The study included 100 women (79 patients with abnormal cervical Pap smear results and 21 controls) from whom vaginal swabs were collected. Real-time quantitative PCR was used to determine seven lactic acid bacteria (LAB) species and their quantities. Results: Most patients were colonized by two Lactobacillus species, primarily Lactobacillus gasseri (93%) and L. crispatus (83%). Patient age and place of residence were associated with the diversity of LAB in the vaginal microbiota. The abundance of L. delbrueckii in the vaginal microbiota increased, whereas the abundance of L. gasseri abundance decreased, with patient age. Lactobacillus acidophilus and Limosilactobacillus fermentum were significantly more often detected in patients living in rural versus urban areas. Statistical analysis did not show any significant differences in LAB between groups of patients with various changes on smear tests. Discussion: The degree of dysplastic changes in the endothelium or the presence of a group of atypical cervical stratified epithelial cells was not associated with significant changes in the studied vaginal bacteria.

Antibiotic Resistance and Genotypes of Helicobacter pylori Strains in Patients with Gastroduodenal Disease in Southeast Poland.

The aim of this study was to investigate genetic diversity of Helicobacter pylori virulence markers to predict clinical outcome as well as to determine an antibiotic susceptibility of H. pylori strains in Poland. Gastric biopsies from 132 patients with gastrointestinal disorders were tested for presence of H. pylori with the use of rapid urease test, microbial culture, and polymerase chain reaction (PCR) detection. The genetic diversity of 62 H. pylori positive samples was evaluated by detection of cagA and PCR-typing of vacA and iceA virulence-associated genes. Most common H. pylori genotypes were cagA(+)vacAs1m2 (27.4%) and cagA(-)vacAs2m2 (24.2%). In logistic regression analysis, we recognized the subsequent significant associations: gastritis with ureC, i.e., H. pylori infection (p = 0.006), BMI index (p = 0.032); and negatively with iceA1 (p = 0.049) and peptic ulcer with cagA (p = 0.018). Thirty-five H. pylori strains were cultured and tested by E-test method showing that 49% of strains were resistant to at least one of the tested antibiotics. This is the first study that reports the high incidence and diversity of allelic combination of virulence genes in gastroduodenitis patients in Poland. Genotyping of H. pylori strains confirmed the involvement of cagA gene and vacAs1m1 genotype in development and severity of gastric disorder.

Resistant Streptococcus pneumoniae strains in children with acute otitis media- high risk of persistent colonization after treatment.

BACKGROUND: Despite advances in the development of pneumococcal conjugate vaccines, acute otitis media (AOM) is a common childhood infection, caused mainly by Streptococcus pneumoniae. It has been suggested that persistence of pneumococcal nasopharyngeal carriage is a risk factor for subsequent recurrent infections. METHODS: In this study we evaluate the relationship between 55 pneumococcal strains obtained from nasopharynx/oropharynx (NP/OP) and middle ear fluid (MEF) of 62 children, aged between 1 and 16 years, during AOM (including recurrent/treatment failure AOM, and post-treatment visits), based on their phenotypic and genotypic characteristics performed by analyses of serotype, antibiotic susceptibility patterns and multilocus sequence typing. RESULTS: S.pneumoniae was isolated from 27.4% of MEF samples; it constituted 43.6% of all positive bacterial samples from MEF samples. There was statistically significant concordance between isolation from the MEF sample and NP/OP colonization by S. pneumoniae (p < 0.0001). During post-treatment visits S.pneumoniae was isolated from 20.8% of children; 91% of them were positive in pneumococcal NP/OP culture during AOM. The serotypes belonging to 10- and 13-valent pneumococcal conjugated vaccines constituted 84% and 92% of the strains, respectively. Multidrug resistance was found in 84% of the strains. According to multivariate analysis, pneumococcal colonization after antibiotic therapy was significantly associated with shorter length of therapy in children with bilateral AOM. CONCLUSIONS: High persistent prevalence of antibiotic-resistant S.pneumoniae strains in children with AOM after unsuccessful bacterial eradication may presumably be regarded as a predisposing factor of infection recurrence.

Molecular diagnostics of periodontitis.

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host's health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

Molecular Epidemiology of Streptococcus pneumoniae Isolates from Children with Recurrent Upper Respiratory Tract Infections.

A total of 125 isolates were recovered from adenoids and/or nasopharynx of 170 children aged 2 to 5 from south-east Poland; they had undergone adenoidectomy for recurrent and/or persistent symptoms of upper respiratory tract infections. Pneumococcal isolates were analyzed by phenotyping (serotyping and antimicrobial resistance tests) and genotyping together with the clonality of the pneumococcal isolates based on resistance determinants, transposon distribution and multilocus sequence typing (MLST). Serotypes 19F, 6B and 23F constituted 44.8% of the isolates. Among all of the strains, 44.8% showed decreased susceptibility to penicillin and resistance to co-trimoxazole (52.8%), tetracycline (38.4%), erythromycin (53.6%), clindamycin (52.8%) and chloramphenicol (27.2%) was observed. Tn6002 was found in 34.8% of erythromycin-resistant isolates while composite Tn2010-in 16.7% of erm(B)-carrying isolates that harboured also mef(E) gene. Tn3872-related elements were detected in 27.3% of erythromycin-resistant strains. In the majority of chloramphenicol-resistant catpC194-carrying isolates (79.4%), ICESp23FST81-family elements were detected. The genotyping showed that pneumococcal population was very heterogeneous; 82 sequence types (STs) were identified, and the most frequent contributed to not more than 8% of the isolates. Nearly 44% STs were novel, each of them was recovered only from one child. Four STs belonged to one of the 43 worldwide spread resistant pneumococcal clones currently accepted by Pneumococcal Molecular Epidemiology Network (PMEN), i.e. Spain 9V-3, Spain 23F-1, Norway NT-42 and Poland 6B-20, accounting for 12 (16.7%) of the 75 nonususceptible isolates, and five STs were single-locus variants of PMEN resistant clones (England 14-9, Spain 9V-3, Spain 23F-1, Greece 21-30, Denmark 14-32), accounting 9 (12%) of nonsusceptible isolates. A few MDR clones belonging to 6B and 19F serotypes found among preschool children emphasizes rather the role of clonal dissemination of local strains in the community than international clones spreading in the increase of resistance among pneumococcal strains.

Fluoroquinolone-resistance mechanisms and phylogenetic background of clinical Escherichia coli strains isolated in south-east Poland.

Fluorochinolones are a class of broad-spectrum antimicrobials in the treatment of several infections, including those caused by Escherichia coli. Due to the increasing resistance of bacteria to antimicrobials, an understanding of fluoroquinolone resistance is important for infection control. The aim of this study was to determine susceptibility of clinical E. coli strains to fluoroquinolones and characterize their mechanisms of quinolone resistance. Totally, 79 non-duplicate clinical E. coli isolates included in this study were mainly from skin lesion -36 (45.6%) isolates; 54 (68.4%) isolates were assigned to phylogenetic B2 group. Resistance to ciprofloxacin was found in 20 isolates. In the quinolone resistance-determining region (QRDR) region of gyrA and parC, 4 types of point mutations were detected. Mutations in parC gene were found in all strains with gyrA mutations. Predominance of double mutation in codon 83 and 87 of gyrA (90%) and in codon 80 of parC (90%) was found. Moreover, plasmid-mediated quinolone resistance (PMRQ) determinants (qnrA or qnrB and/or aac(6')-Ib-cr) were present in 5 (25%) out of 20 fluoroquinolone-resistant isolates. Resistance to fluoroquinolones in all of the tested clinical E. coli isolates correlated with point mutations in both gyrA and parC. The majority of fluoroquinolone-resistant strains belonged to D and B2 phylogenetic groups.

Resistance determinants and their association with different transposons in the antibiotic-resistant Streptococcus pneumoniae.

Multiple resistance of Streptococcus pneumoniae is generally associated with their unique recombination-mediated genetic plasticity and possessing the mobile genetic elements. The aim of our study was to detect antibiotic resistance determinants and conjugative transposons in 138 antibiotic-resistant pneumococcal strains isolated from nasopharynx of healthy young children from Lublin, Poland. These strains resistant to tetracycline and/or to chloramphenicol/erythromycin/clindamycin were tested by PCR using the specific genes as markers. The presence of Tn916 family transposons, carrying tet(M) and int/xisTn916, was observed in all of the tested strains. Tn916 was detected in 16 strains resistant only to tetracycline. Tn6002 and Tn3872-related element were found among 99 erm(B)-carrying strains (83.8% and 3.0%, resp.). Eight strains harbouring mef(E) and erm(B) genes were detected, suggesting the presence of Tn2010 and Tn2017 transposons. Among 101 chloramphenicol-resistant strains, two variants of Tn5252-related transposon were distinguished depending on the presence of int/xis5252 genes specific for cat gene-containing Tn5252 (75.2% of strains) or int Sp23FST81 gene, specific for cat-containing ICESp23FST81 element (24.8% of strains). In 6 strains Tn916-like and Tn5252-like elements formed a Tn5253-like structure. Besides clonal dissemination of resistant strains of pneumococci in the population, horizontal transfer of conjugative transposons is an important factor of the high prevalence of antibiotic resistance.