Oto Melter

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

How and why to monitor Pseudomonas aeruginosa infections in the long term at a cystic fibrosis centre

BACKGROUND Pseudomonas aeruginosa is a major cystic fibrosis (CF) pathogen causing chronic respiratory infections and posing a risk for cross-infection between patients with CF. AIM To propose an algorithm for long-term surveillance of P. aeruginosa and assess its suitability for monitoring the epidemiological situation at a CF centre with approximately 300 patients. METHODS Over a nine-year period, over 300 P. aeruginosa isolates from 131 infected patients were tested by multi-locus sequence typing (MLST) and/or random amplified polymorphic DNA (RAPD) assay. FINDINGS MLST analysis led to the identification of 97 different sequence types which were distributed among 17 RAPD-generated (pseudo)clusters. This indicates that the easy-to-perform RAPD assay is only suitable for intra-individual, not interindividual, strain analyses. No epidemic strains were observed. Longitudinal analysis revealed that 110 of the 131 patients were infected with the same strain over the observation period, whereas 21 patients had a strain replacement or a new infection. Chronic infection was found in 99 of the 131 patients, and the remaining 32 patients met the criteria for intermittent infection (as defined by the Leeds criteria). Eighteen of the 32 patients (56%) with intermittent infection were infected with the same strain for up to nine years. CONCLUSION The strain type only changed in 16% of 131 patients with chronic or intermittent infection. As many as 56% of patients considered to have intermittent infection were actually chronically infected with the same strain for many years.

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.

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.

96 Staphylococcus aureus in Czech cystic fibrosis patients – prospective study

Altogether, 242 respiratory isolates of S. aureus from 105 patients with CF were collected in 2011–2012. The isolates were typed by PFGE and screened for susceptibility to antibiotics and presence of resistance and virulence genes. Small colony variants (SCV) were identified and tested for auxotrophy. Results: Total of 142/58 (isolates/patients) were resistant to MLSb antibiotics (Macrolides-Lincosamides-Streptogramin B), 25/13 were resistant to aminoglycosides and 6/5 were MRSA. MLSb resistance was associated with genes from erm family or msrA gene. Isolates resistant to aminoglycosides harboured aadC, aphA3 or aac-aphD gene. mecA gene was detected in all of 6 MRSA isolates. Most frequent virulence genes detected in the isolates (n = 242) were genes for enterotoxins seg (n = 137), sei (n = 128), sec (n = 38), sea (n = 19), sej (n = 15) and seh (n = 18). Other virulence genes were not found or found sporadically. Isolates with SCV phenotype (n = 19), all thymidine auxotrophs, were isolated in 9 patients. Isolates were separated in to the 39 different pulsotypes by PFGE with two dominant pulsotypes of 61 isolates from 27 patient (26%) and 28 isolates from 10 patients (12%) respectively.

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.