Agnieszka Chmielarczyk

Studies on the effects of probiotic Lactobacillus mixture given orally on vaginal and rectal colonization and on parameters of vaginal health in women with intermediate vaginal flora

OBJECTIVES The vaginal microflora is composed of many bacterial species and plays a major role in maintaining the balance of this complex environment. This study was conducted in order to assess the degree and persistence of the colonization of vaginal epithelium by strains from an orally administered mixture of lactobacilli, containing Lactobacillus fermentum 57A, Lactobacillus plantarum 57B and Lactobacillus gasseri 57C. We also monitored its effects on parameters of vaginal health, especially total lactobacilli counts, vaginal pH and Nugent score. STUDY DESIGN The patient group in this open study consisted of clinically healthy women with intermediate vaginal flora. Altogether 37 women were included in the study; 25 finished the full cycle consisting of 8 visits during 70 days. Lactobacillus mixture was administered as 1×10(8) c.f.u. once a day for 60 days. Lactobacillus isolates collected from vaginal and rectal samples from studied women during all visits were typed using molecular methods (PFGE for L. fermentum and L. gasseri and MLST for L. plantarum). Total lactobacilli counts, vaginal pH and Nugent score were also determined during the visits. RESULTS We confirmed that the ingested strains were able to reach and colonize both sites within the third and eighth visits, i.e. between the 20th and 70th days of the study. Maximal colonization was recorded between the fifth and seventh visits (31st-60th days). Moreover, ingestion of the Lactobacillus mixture was related to normalization of vaginal parameters (within 28-60 days after the initiation of the treatment). This was demonstrated by a decrease of vaginal pH and Nugent score together with an increase of total numbers of lactobacilli in the vagina and rectum. No adverse events were noted during the course of the study. CONCLUSIONS Oral application of the combination of the three probiotic strains derived from vaginal microbiota of healthy woman with high adherence abilities to both vaginal and colonic epithelium in vitro shows that both individual strains and their mixture can colonize vagina for some weeks, the effect of which is correlated with significant improvement of such parameters like pH and Nugent score values and total numbers of vaginal lactobacilli. This indicates that the mixture may be a good candidate for the planned double-blind, placebo-controlled randomized studies involving larger numbers of women.

Conversion of OXA-66 into OXA-82 in clinical Acinetobacter baumannii isolates and association with altered carbapenem susceptibility

OBJECTIVES Three clinical Acinetobacter baumannii isolates (A-C) were isolated from three separate patients during an outbreak in a hospital in Krakow, Poland. Isolate A was recovered first and was susceptible to carbapenems, whereas isolates B and C were resistant. The aim of this study was to investigate the differences in carbapenem susceptibility in these outbreak-related isolates. METHODS Clonal relatedness was determined using rep-PCR-based DiversiLab. The bla(OXA-51-like) genes and their upstream regions were sequenced. Expression of the genes encoding OXA-51-like and the three major porins CarO, OprD-like and 33-36 kDa Omp were investigated by semi-quantitative RT-PCR. Comparison of outer membrane protein (OMP) profiles was performed using SDS-PAGE. ISAba1-bla(OXA-82) was cloned into the shuttle vector pWH1266 and transferred into A. baumannii ATCC 17978. RESULTS The isolates were identical by rep-PCR and clustered with international clonal lineage 2. Sequencing of bla(OXA-51-like) revealed a conversion of OXA-66 (isolate A) into OXA-82 (isolates B and C). bla(OXA-82) was also associated with ISAba1. Expression analysis revealed overexpression of bla(OXA-82). There was no difference in OMP expression between the isolates. ISAba1-bla(OXA-82) conferred carbapenem resistance in ATCC 17978. CONCLUSIONS Carbapenem resistance in outbreak-related isolates was mediated by conversion of OXA-66 into OXA-82 and its subsequent overexpression. This further highlights the genome plasticity of A. baumannii, leading to carbapenem resistance.

Possible role of Escherichia coli in propagation and perpetuation of chronic inflammation in ulcerative colitis

BackgroundThis study investigated a possible role of Escherichia coli in propagation and perpetuation of the chronic inflammation in ulcerative colitis (UC). The lesions of UC are located superficially on the rectal and/or colonic mucosa. It is suggested that the commensal bacteria of the digestive tract may play a role in the pathogenesis of UC. Several studies have demonstrated proliferation of E. coli in the gut of UC patients. An increase in the number of E. coli in the inflamed tissue is most probably related to the abundance of iron ions produced by the bacteria.MethodsColon mucosal biopsies were collected from 30 patients with acute-phase UC, both from tissues with inflammatory changes (n = 30) and unchanged tissue with no inflammatory changes (n = 30) from the same patient. Biopsies were also taken from 16 patients with irritable bowel syndrome diarrhea who comprised the control group. Quantitative and qualitative analysis of the biopsy specimens was performed using culture methods and real-time polymerase chain reaction (PCR). Genotyping of the E. coli isolates was done using pulsed-field gel electrophoresis. Multiplex PCR was used to compare the E. coli strains for the presence of genes responsible for synthesis of iron acquisition proteins: iroN, iutA, iha, ireA, chuA, and hlyA.ResultsWe demonstrated that there was a significant increase in the number of E. coli at the sites of inflammation in patients with UC compared to the control group (P = 0.031). Comparative analysis of the restriction patterns of E. coli isolated from inflammatory and unchanged tissues showed that the local inflammatory changes did not promote specific E. coli strains. There was a significant difference in the frequency of the iroN gene in E. coli isolated from patients with UC as compared to the control group.ConclusionsThe increase in the numbers of E. coli in the inflammatory tissues is related to the presence of chuA and iutA genes, which facilitate iron acquisition during chronic intestinal inflammatory processes.

Molecular characterization and drug resistance of Escherichia coli strains isolated from urine from long-term care facility residents in Cracow, Poland.

Background The aim of this study was to assess the prevalence of multidrug-resistant Escherichia coli and extended-spectrum β-lactamases (ESBL) pathogens isolated from asymptomatic bacteriuria and urinary tract infections (UTIs), and the relationship between the phylogeny, antimicrobial resistance, and virulence among isolates in residents of 3 long-term care facilities (LTCF) in Krakow, Poland. Material/Methods This was point prevalence study and prospective infection control in a group of 217 people. Urine samples were examined with standard microbiological methods and screened for the presence of blaCTX-M, blaSHV, and blaTEM. E. coli isolates were screened for 6 common virulence factors (VFs) and classified according to the rapid phylogenetic grouping technique. Results Among all the strains tested, 14 isolates (13.9%) expressed ESBL activity. A significant proportion of isolates were resistant to ciprofloxacin (32.7%, n=33). Resistance to trimethoprim/sulfamethoxazole was identified among 45 isolates (44.5%). Independent risk factors for the presence of an ESBL-producing strain were: UTI, urinary and/or fecal incontinence, bedridden, and low values of the Barthel and Katz Indexes. Gene sequencing identified 8 blaCTX-M-15, 1 blaCTX-M-3, 9 blaTEM-1, and 1 blaSHV-12. Among E. coli, no relationship between number of VF genes and phylogeny was found. The most prevalent virulence factor was fimH (82.1%). Conclusions The findings of this study emphasize the need for further research on the epidemiology of multi-drug resistant organisms (MDRO) and ESBL in LTCF, including transmission patterns, rates of infection, and factors associated with infections. It may be necessary to extend the requirements and precautions to MDRO and ESBL-producers.

The application of genetics methods to differentiation of three Lactobacillus species of human origin

In recent decades, the interest in probiotics as diet supplements or drugs has increased. In order to determine a specific bacterial isolate to be probiotic, it is necessary to describe precisely its probiotic characteristics and taxonomic properties, including the strain level. Most of the well-known genotyping methods were designed for the commonly-found pathogenic bacteria. The objective of this study is to undertake an attempt at standardization of FISH, RAPD and PFGE methods to genotype and identify the bacteria belonging to Lactobacillus fermentum, L. gasseri and L. plantarum species. The FISH probes have been designed and tested for Lactobacillus fermentum, L. gasseri and L. plantarum species and an endeavor has been made at standardization of RAPD and PFGE methods for these bacterial species. Moreover, the MLST method was applied to differentiate Lactobacillus plantarum strains. L. plantarum isolated from humans could not be genetically diversified with the use of RAPD, PFGE or MLST methods; only the strains originating from plants have displayed diversification among themselves and have been different from the strains of human origin.

Colonisation of the gastrointestinal tract by probiotic L. rhamnosus strains in acute diarrhoea in children

Lactobacillus rhamnosus (573L/1-3) strains are considered effective in the treatment of rotaviral diarrhoea in children. The colonisation of the gastrointestinal (G.I.) tract by the Lactobacillus rhamnosus strains and the determining factors are discussed reporting data of a prospective, double-blinded, placebo-controlled randomized study in children between the 2nd month and 6th year of life with acute diarrhoea lasting not longer than 5 days. The examined strains were detected in 37/46 (80.43%) patients after 5 days and in 19/46 (41.3%) patients after 14 days since the start of the treatment. L. rhamnosus 573L/1 strain colonised the G.I. tract more persistently. L. rhamnosus strains are effective in colonising the G.I. tract during acute diarrhoea. Persistence of colonisation is dependent on the properties of administered probiotic strains.

Molecular epidemiology, plasmid analysis, virulence, and resistance of Escherichia coli isolated from neonatal intensive care units in Poland

We investigated the set of Escherichia coli isolates originating from newborns in relation to resistance, virulence factors (VFs), phylogenetic groups, plasmid replicon typing, and genotypes. The most isolates were clustered in ECOR group B2. Extended-spectrum beta-lactamase phenotype was found in 27.7% of isolates. The ST131 clone was detected among 33 strains, 12 of which carried the CTX-M-15 gene. Most VFs were detected among ST131 isolates and in the B2 group. IbeA gene was found more frequently in the blood isolates, while the iha gene, in the urine isolates. The 3 most prevalent replicon types were IncFIB, IncF, and IncFIA.

Molecular Epidemiology and Drug Resistance of Acinetobacter baumannii Isolated from Hospitals in Southern Poland: ICU as a Risk Factor for XDR Strains

The objectives of the present study were to investigate the carbapenemase and metallo-beta-lactamase genes of Acinetobacter baumannii clinical isolates by polymerase chain reaction (PCR) and real time PCR and to determine the molecular epidemiology of the strains using the DiversiLab tool. From these data, correlations between drug resistance, resistance genes, and epidemiological clones may be revealed. The study was conducted on 125 A. baumannii collected over the 2013 year. The majority of the isolates from both intensive care unit (ICU) and non-ICU cases originated from pneumonia infections (79.2%), isolates from blood infections accounted for 17.6% and 3.2% were from meningitis infections. In the ICU cases compared with the non-ICU cases, bloodstream infections were more frequently diagnosed (19.2% vs. 11.5%). Sixty percent of A. baumannii strains were resistant to all the antimicrobials tested with the exception of colistin. All strains were susceptible to colistin and polymyxin B. Extensively drug-resistant (XDR) strains accounted for 80.8% of the isolates tested and these XDR strains were more frequently isolated from ICU cases than from non-ICU cases (93.9% vs. 30.8%). Among the 101 isolates of A. baumannii exhibiting the XDR pattern of resistance, 80 possessed the blaOXA-24 gene and 29 had the blaOXA-23 gene. Only two isolates possessed the blaVIM gene. The presence of the ISAba1element was confirmed among 10 strains from patients hospitalized in the ICU. Using repetitive extragenic palindromic sequence PCR (DiversiLab typing), six clones and 12 unique strains were identified, of which two clones dominated. Most isolates belonging to clone 1 (66.7%) and clone 2 (85.5%) were susceptible only to colistin. In summary, it is clear from our findings and those of other studies that carbapenem resistance among A. baumannii strains presents a serious clinical problem worldwide. Furthermore, the presence of XDR international clone II in ICUs poses a potential risk for future outbreaks of A. baumannii infection and controlling A. baumannii infections in hospitals presents a serious challenge.

Enterobacteriaceae infections of very low birth weight infants in Polish neonatal intensive care units: resistance and cross-transmission.

Background: The aims of our study were analysis of the occurrence of infections by members of the Enterobacteriaceae family in 6 Polish neonatal intensive care units in 2009, their drug resistance, the epidemiology of extended-spectrum &bgr;-lactamase (ESBL)-producing strains and the possibility of using modern tools of microbiology diagnosis in infection control, especially for the reduction of antimicrobial resistance. Methods: A prospective surveillance covered 910 newborns. Case patients were defined as neonates with very low birth weight who had clinical signs of septicemia, pneumonia or necrotizing enterocolitis. Early-onset infection was defined as infection diagnosed within 3 days after delivery. Results: The incidence of Enterobacteriaceae infections was 2.6/1000 patient-days. The risk of Enterobacteriaceae pneumonia increased with the length of hospitalization (P = 0.0356). The most common pathogen was Escherichia coli (12.4% of all strains, in early-onset infection 18.5%) and Klebsiella spp. (9.1% of all). The ESBL phenotype was found in 37% of isolates, of which 89.3% were producing CTX-M-type, 70.2% TEM-type and 8.5% SHV-type. Epidemic clones were detected in the 2 studied neonatal intensive care units: 6 of the 9 ESBL-positive Enterobacter cloacae and 16 of the 18 ESBL-positive Klebsiella pneumoniae strains were classified into 1 epidemic clone, which showed resistance to penicillin without inhibitors, amoxycillin/clavulanic acid, cephalosporins, aztreoname, aminoglycosides and trimethoprim/sulfamethoxazole. Conclusions: Enterobacteriaceae bacilli are a significant problem in neonatal intensive care units, especially in early-onset infection and for long hospitalized very low birth weight infants. The observed high drug resistance was in large part related to the dominance of epidemic strains as a result of horizontal transmission. The best way to reduce drug resistance would be adequate procedures of isolation and hand hygiene.

Epidemiology, antibiotic consumption and molecular characterisation of Staphylococcus aureus infections – data from the Polish Neonatology Surveillance Network, 2009–2012

BackgroundOur aim was to determine and characterize S. aureus (SA) isolated from infections in newborns for antibiotic resistance, virulence factors, genotypes, epidemiology and antibiotic consumption.MethodsProspective surveillance of infections was conducted. Data about antibiotic treatment were analyzed. Antimicrobial susceptibility was assessed. PCR amplification was used to detect resistance and virulence genes. Typing methods such as PFGE, spa-typing and SCCmec were used.ResultsSA was found to be associated with 6.5% of infections. Methicillin-Resistant Staphylococcus aureus accounted for 32.8% of SA-infections. An incidence of MRSA-infections was 1.1/1000 newborns. MRSA-infections were diagnosed significantly earlier than MSSA-infections in these newborns (14th day vs. 23rd day (p = 0.0194)). MRSA-infections increased the risk of newborn’s death. Antibiotic consumption in both group was similar, but a high level of glycopeptides-usage for MSSA infections was observed.In the MRSA group, more strains were resistant to erythromycin, clindamycin, gentamicin and amikacin than in the MSSA group. Hla gene was present in 93.9% of strains, and seg and sei in 65.3% of strains, respectively. One dominant clone was found among the 14 MRSA isolates. Fifteen strains belonging to SCCmec type IV were spa-t015 and one strain belonging to SCCmec type V was spa-t011.ConclusionsResults obtained in the study point at specific epidemiological situation in Polish NICU (more detailed studies are recommended).High usage of glycopeptides in the MSSA infections treatment indicates the necessity of antimicrobial stewardship improvement and introducing molecular screening for early identification of infections.