Jan Tkadlec

Antibiotic profiling of Clostridium difficile ribotype 176 – A multidrug resistant relative to C. difficile ribotype 027

Antibiotic profiling of twenty Czech Clostridium difficile PCR-ribotype 176 isolates revealed a high level of resistance to erythromycin, ciprofloxacin and moxifloxacin (n = 20) and to rifampicin (n = 13). Accumulation of resistance mechanisms to multiple antibiotics highlight that PCR-ribotype 176 belong to problematic epidemic strains.

Characterization of Staphylococcus aureus strains isolated from Czech cystic fibrosis patients: high rate of ribosomal mutation conferring resistance to MLSB antibiotics as a result of long-term and low-dose azithromycin treatment

Staphylococcus aureus is one of the most frequent pathogens infecting the respiratory tract of patients with cystic fibrosis (CF). This study was the first to examine S. aureus isolates from CF patients in the Czech Republic. Among 100 S. aureus isolates from 92 of 107 observed patients, we found a high prevalence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics (56%). More than half of the resistant strains (29 of 56) carried a mutation in the MLS(B) target site. The emergence of MLS(B) resistance and mutations conferring resistance to MLS(B) antibiotics was associated with azithromycin treatment (p=0.000000184 and p=0.000681, respectively). Methicillin resistance was only detected in 3% of isolates and the rate of resistance to other antibiotics did not exceed 12%. The prevalence of small-colony variant (SCV) strains was relatively low (9%) and eight of nine isolates with the SCV phenotype were thymidine dependent. The study population of S. aureus was heterogeneous in structure and both the most prevalent community-associated and hospital-acquired clonal lineages were represented. Of the virulence genes, enterotoxin genes seg (n=52), sei (n=49), and sec (n=16) were the most frequently detected among the isolates. The PVL genes (lukS-PV and lukF-PV) have not been revealed in any of the isolates.

NAD(P)H-hydrate dehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation.

Background One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. Methods and Results We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. Conclusion We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

Cutaneous bacillary angiomatosis due to Bartonella quintana in a renal transplant recipient

Bacillary angiomatosis (BA) is a disorder of neovascular proliferation involving skin and other organs of immunosuppressed patients caused by Bartonella species. BA has been recognized in both immunocompetent and immunodeficient patients, mostly in human immunodeficiency virus (HIV)‐infected persons, much more rare in those with other immunodeficiencies, including organ transplantation. Diagnosis is based on serologic analysis, culture and molecular biology [detection of Bartonella species deoxyribonucleic acid (DNA) in tissue biopsy extracts by real‐time polymerase chain reaction (PCR)]. All immunosuppressed patients with BA should be treated with antibiotics because of potentially life‐threatening course of the disease. We report the first case of cutaneous bacillary angiomatosis due to Bartonella quintana in renal transplant recipient. This presentation demonstrates that BA should be considered a differential diagnosis in immunocompromised patients presenting with fever and cutaneous angioma‐like lesions.

Characterisation of methicillin-susceptible Staphylococcus pseudintermedius isolates from canine infections and determination of virulence factors using multiplex PCR

Staphylococcus pseudintermedius is a genuine opportunistic pathogen of the skin, especially in canids. However, characterisation of virulence, antimicrobial resistance and genotypic variability in methicillin-susceptible S. pseudintermedius isolates has not been fully explored. In this study, coagulase-positive staphylococcal isolates collected from dogs of various breeds and ages suffering from dermatitis (n = 70), pyoderma (n = 7), and otitis (n = 7), from districts of Prague (Czech Republic) and surrounding areas, were characterised using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and repetitive sequence-based PCR fingerprinting. Susceptibility to antimicrobial agents was determined, virulence factor genes for leukocidin (lukSF-I), exfoliatins (exi, expB, and siet), enterotoxin C (seccanine) and enterotoxin-related genes (se-int and sel) were detected using multiplex PCR and the genotypes of S. pseudintermedius isolates were determined using SmaI macrorestriction analysis. The majority of the staphylococcal isolates (n = 84) were identified as S. pseudintermedius (n = 79) and all of them were susceptible to methicillin/oxacillin (MSSP). About half of the strains (n = 41) were resistant to macrolide-lincosamide-streptogramin B antimicrobial agents and resistance was mediated in all but one of the strains by the erm(B) gene. The genes for lukSF-I, siet, se-int, and sel were detected in the majority of the MSSP strains (96.2%, 100%, 100%, and 73.4%, respectively). Investigated canine S. pseudintermedius isolates were highly heterogeneous, which prevented the correlation of any specific lineage to a particular infection, dog breed, or region of origin.

Importance of Multifaceted Approaches in Infection Control: A Practical Experience from an Outbreak Investigation.

Background This study presents the results of a multidisciplinary, nosocomial MRSA outbreak investigation in an 8-bed medical intensive care unit (ICU). The identification of seven MRSA positive patients in the beginning of 2014 led to the closure of the ward for several weeks. A multidisciplinary, retrospective investigation was initiated in order to identify the reason and the source for the outbreak, describe MRSA transmission in the department and identify limitations in infection control. Methods The investigation comprised an epidemiological description of MRSA cases from 2012 to 2014 and a characterization of MRSA isolates, including phage-, spa- and PFGE-typing. Additionally, MRSA screening was performed from the hospital staff and the environment. To identify the reason for the outbreak, work-related, psychological and behavioral factors were investigated by impartial audits and staff interviews. Results Thirty-one MRSA cases were registered during the study period, and 36 isolates were investigated. Molecular typing determined the outbreak strain (phage type 54/812, PFGE type A4, spa type t003) and identified the probable index case. Nasal carriage in one employee and a high environmental contamination with the outbreak strain was documented. Important gaps in nursing procedures and general management were identified. Elevated stress levels and communication problems preceded the outbreak. Compliance with hand hygiene and isolation procedures was evaluated as appropriate. Conclusion This study demonstrates the complexity of controlling hospital-associated infections. The combined use of different typing methods is beneficial for outbreak investigations. Psychological, behavioral and other work-related factors have an important impact on the spread of nosocomial pathogens. These factors should be addressed and integrated in routine infection control practice.

A simple and cost-effective cover-glass test for the differentiation between staphylococci and micrococci in clinical laboratory.

A cover-glass placed on a heavily inoculated culture plate clearly differentiates facultatively anaerobic staphylococci growing underneath the cover-glass after overnight incubation from nongrowing aerobic micrococci. Even if there are some exceptions, all medically significant staphylococci can grow in the test. Thus, the test provides a cost-effective and highly specific tool for separation of both genera which fundamentally differ in their pathogenicity.