Małgorzata Bulanda

Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease

AIM To determine the features of Enterococcus that contribute to the development and maintenance of the inflammatory process in patients with inflammatory bowel disease (IBD). METHODS Multiplex polymerase chain reaction (PCR) was applied to assess the presence of genes that encode virulence factors [surface aggregating protein (asa1), gelatinase (gelE), cytolysin (cylA), extracellular surface protein (esp) and hyaluronidase (hyl)] in the genomic DNA of 28 strains of Enterococcus isolated from the intestinal tissues of children with IBD (n = 16) and of children without IBD (controls; n = 12). Additionally, strains with confirmed presence of the gelE gene were tested by PCR for the presence of quorum sensing genes (fsrA, fsrB, fsrC) that control the gelatinase production. Gelatinase activity was tested on agar plates containing 1.6% gelatin. We also analysed the ability of Enterococcus strains to release and decompose hydrogen peroxide (using Analytical Merckoquant peroxide test strips) and tested their ability to adhere to Caco-2 human gut epithelium cells and form biofilms in vitro. RESULTS A comparison of the genomes of Enterococcus strains isolated from the inflamed mucosa of patients with IBD with those of the control group showed statistically significant differences in the frequency of the asa1 gene and the gelE gene. Furthermore, the cumulative occurrence of different virulence genes in the genome of a single strain of Enterococcus isolated from the IBD patient group is greater than in a strain from the control group, although no significant difference was found. Statistically significant differences in the decomposition of hydrogen peroxide and adherence to the Caco-2 epithelial cell line between the strains from the patient group and control group were demonstrated. The results also showed that profuse biofilm production was more frequent among Enterococcus strains isolated from children with IBD than in control strains. CONCLUSION Enterococcus strains that adhere strongly to the intestinal epithelium, form biofilms and possess antioxidant defence mechanisms seem to have the greatest influence on the inflammatory process.

Genetic characterization and diversity of Streptococcus agalactiae isolates with macrolide resistance.

Macrolide resistance in 169 Streptococcus agalactiae [group B streptococcus (GBS)] isolates originating from pregnant carriers was investigated. Using multiplex PCR the presence of genes encoding erythromycin resistance and capsular polysaccharides, as well as surface proteins, was determined. Random amplification of polymorphic DNA (RAPD) and PFGE were used to characterize specific clones among the isolates. In the examined population of women, erythromycin-resistant strains were found in 4.5 % of patients, whereas clindamycin-resistant strains were found in 3 % of patients, which was 16 % of strains resistant to erythromycin and 10 % of strains resistant to clindamycin among GBS isolates, respectively. Among the isolates, the largest percentage was represented by the constitutive macrolide-lincosamide-streptogramin B (cMLS(B)) phenotype (63 %), then the inductive macrolide-lincosamide-streptogramin B (iMLS(B)) phenotype (26 %) and the macrolide resistance (M) phenotype (11 %). The ermB gene was indicated in all isolates with the cMLS(B) phenotype and V serotype, whereas mefA/mefE genes were found in isolates with the M phenotype and Ia serotype. Among resistance isolates, serotype V was predominant (67 %), followed by serotypes II (15 %), Ia (11 %) and III (7 %). The most common surface protein encoding genes were alp3 (70 %), then rib (11 %), epsilon (7.5 %), bca (7.5 %) and alp2 (4 %). A statistically significant relationship between macrolide resistance, serotype V and the alp3 gene was demonstrated. PFGE, in comparison to the RAPD method, gave better genetic discrimination of GBS isolates. A relatively high genetic diversity among investigated strains was shown. In addition, the largest genetic homogeneity was found in serotype V.

Comparison of SSI Rates in Endoarthroplasty of Hip and Knee in a Cracow Patient Population and the Importance of Postdischarge Surveillance

Background:The aim of the study was to analyse the epidemiological and microbiological analysis of surgical site infections in patients that underwent knee or hip endoarthroplasty procedures.Materials and Methods:The epidemiological and microbiological surveillance was carried out by the local infection control team in cooperation with the Department of Bacteriology, at the Chair of Microbiology, Jagiellonian University Medical College in Cracow.Results:A total of 651 patients operated in the Department of Orthopedics, Trauma Surgery and Rehabilitation of Cracow Rehabilitation Center in Poland were analyzed. Twenty-three cases of SSI were detected. The cumulative incidence after hip prosthesis (HPRO) procedures was 2.3%, while for knee prosthesis (KPRO) it was 7.0. Standardized risk index, comparing the incidence in our study to German hospitals, shows a statistically significant, higher incidence in patients with knee replacement procedures in our study (p = 0.004). Among etiological agents of SSIs, we demonstrated the dominating role of Gram-positive cocci to be 75% (30% methicillin resistant). This resistance was confirmed only in case of coagulasenegative staphylococci (no MRSA were cultured). Gramnegative rods were isolated with a frequency of 25%: 41.6% in SSI after hip endoarthroplasty and 15% after knee endoarthroplasty. Postdischarge surveillance encompassed 59% of operated patients.Conclusion:The incidence of SSIs of hip prosthesis in our study was comparable to the incidence in the German KISS program, where surveillance is integrating a highly sensitive postdischarge detection. On the other hand, we observed a higher, statistically significant cumulative incidence in case of knee endoarthroplasty. Our microbiological data show effective control of methicillin-resistant Staphylococcus aureus and are also in agreement with the data found in literature referring to coagulasenegative multi-resistant staphylococci as an important problem in the orthopaedic surgery of the knee joint.

Quantitative evaluation of fungi of the genus Candida in the feces of adult patients with type 1 and 2 diabetes - a pilot study

BackgroundGastrointestinal tract microbiota, particularly bacterial microflora, seem to have a different qualitative and quantitative composition in both type 1 (T1DM) and type 2 diabetes (T2DM) mellitus cases as compared to non-diabetic individuals. So far, there are no data from diabetes research concerning the prevalence of fungi, particularly the most common genus, i.e. Candida, which are important components of human colon microflora.We aimed to examine whether there are quantitative changes of Candida fungi in the feces of patients with T1DM and T2DM as compared to healthy controls.FindingsOverall, we included 44 diabetic patients (27 patients with T1DM and 17 with T2DM) as well as 17 healthy, non-diabetic controls. Feces and blood samples were collected from all study individuals. DNA was isolated from fecal samples and quantitative real time PCR (qPCR) was applied in order to determine the number of fungal cells. Statistical association with selected clinical and biochemical features was examined.There was a difference in the amount of Candida in the feces among the three examined groups (p = 0.007). Candida spp. populations in T1DM and T2DM subjects were larger as compared to controls (p = 0.017 and p = 0.037, respectively). However, no difference was found between T1DM and T2DM. No association was identified between the quantity of fungi and examined patients’ characteristics, except for negative correlation with blood lipid parameters in T2DM group.ConclusionsCandida fungi appear to be more prevalent in the feces of patients with T1DM and T2DM. Their amount seems to be associated with serum lipids in T2DM patients. This initial finding requires further confirmation.

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.

Comparison of Methods for Isolation of Bacterial and Fungal DNA from Human Blood

AbstractThe study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus. Additionally, the five commercial kits for microbial DNA isolation from the blood were tested. The developed procedure of DNA isolation consisted of three consecutive steps, i.e., mechanical disruption, chemical lysis, and thermal lysis. Afterward, DNA was isolated from the previously prepared samples (erythrocyte lysis) with the use of five commercial kits for DNA isolation. They were compared paying heed to detection limit, concentration, DNA purity, and heme concentration in samples. The isolation of DNA without preliminary erythrocyte lysis resulted in far higher heme concentration than when lysis was applied. In the variant with erythrocyte lysis, two of the commercial kits were most effective in purifying the DNA extract from heme. Designed procedure allowed obtaining microbial DNA from all four groups of pathogens under study in the amount sufficient to conduct the rtPCR reaction, which aimed at detecting them in the blood.

Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis

BackgroundMicrobiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT® 3D blood culture system (bioMérieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit.ResultsUsing qPCR, FISH, SF, and culture, microbial presence was found in 71.8%, 29.6%, 25.3%, and 36.6% of samples, respectively. It was demonstrated that qPCR was significantly more likely to detect microorganisms than the remaining methods; qPCR confirmed the results obtained with the SF kit in all cases wherein bacteria were detected with simultaneous confirmation of Gram-typing. All data collected through the FISH method were corroborated by qPCR.ConclusionsThe qPCR and FISH methods described in this study may constitute alternatives to blood culture and to the few existing commercial molecular assays since they enable the detection of the majority of microbial species, and the qPCR method allows their identification in a higher number of samples than the SF test. FISH made it possible to show the presence of microbes in a blood sample even before its culture.

Multilocus sequence types of invasive and colonizing neonatal group B streptococci in Poland.

Objectives: The present study aimed to investigate the molecular characterization of Streptococcus agalactiae (group B streptococcus; GBS) strains isolated from newborns with invasive neonatal infections and healthy newborns in Poland. Materials and Methods: Forty-two GBS isolates were characterized by combining different typing methods, i.e. multilocus sequence typing (MLST), molecular serotyping and protein gene profiling. Results: Using MLST, a total of 16 sequence types (STs) were identified, and among these, 11 were clustered into the following 5 clonal complexes (CCs): CC23 (20; 49%), CC19 (7; 17%), CC17 (4; 10%), CC10 (4; 10%) and CC1 (1; 2%). A statistically significant relationship between ST-17 and invasive isolates (p = 0.0398) and ST-23 and colonizing strains (p = 0.0034) was detected. Moreover, 2 novel STs were detected (ST-637 and ST-638). Molecular serotyping showed that in the invasive isolates serotype III was predominant (11; 50%), followed by serotypes II (6; 27%), V (3; 14%) and Ia (2; 9%). In healthy newborns, serotype III was also dominant (12; 60%), followed by serotypes Ia (4; 20%), II (2; 10%), V (1; 5%) and Ib (1; 5%). Protein gene profiling indicated that the rib gene was predominant in the invasive strains (11; 59%), followed by bca (5; 22%), alp2 (2; 9%), alp3 (1; 5%) and epsilon (1; 5%), while in colonizing strains the alp2 gene was most common (10; 50%), followed by epsilon (5; 25%), rib (2; 10%), bca (2; 10%) and alp3 (1; 5%). A statistically significant relationship was noted between the rib gene and invasive GBS (p = 0.0329), whereas alp2 was related to the colonizing strains (p = 0.0495). Conclusions: The investigated GBS isolates originating from infections in newborns and healthy neonates represented serotype III in more than half of the cases and differed from one another in terms of resistance to macrolides, ST type affiliation and the presence of genes encoding surface proteins from the Alp family. Further comparative genetic research on a larger number of strains is necessary for epidemiological investigation and vaccine development.

Hospital-Acquired Pneumonia in the Intensive Care Units of Polish Hospitals

We analyzed the epidemiological characteristics of pneumonia in intensive care units of Polish hospitals. Among 11,587 patients, there were 191 cases of hospital-acquired pneumonia (HAP). The incidence of HAP was 5.6%, and that of ventilator-associated pneumonia (VAP) was 17.9%. The overall mortality rate was 12.6%, and the mortality rate for patients who received artificial ventilation was 15.0%. The predominant organisms causing HAP and VAP were Pseudomonas aeruginosa and Escherichia coli, and 21.1% of Staphylococcus aureus isolates were resistant to methicillin.