Stefania Giedrys-Kalemba

Association of Recurrent Furunculosis with Panton-Valentine Leukocidin and the Genetic Background of Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a major cause of skin and soft tissue infections, such as furuncles, carbuncles, and abscesses, but it also frequently colonizes the human skin and mucosa without causing clinical symptoms. Panton-Valentine leukocidin (PVL) is a pore-forming toxin that has been associated with soft tissue infections and necrotizing pneumonia. We have compared the genotypes, virulence gene repertoires, and phage patterns of 74 furunculosis isolates with those of 108 control strains from healthy nasal carriers. The large majority of furunculosis strains were methicillin sensitive. Clonal cluster (CC) 121 (CC121) and CC22 accounted for 70% of the furunculosis strains but for only 8% of the nasal isolates. The PVL-encoding genes luk-PV were detected in 85% of furunculosis strains, while their prevalence among colonizing S. aureus strains was below 1%. luk-PV genes were distributed over several lineages (CCs 5, 8, 22, 30, and 121 and sequence type 59). Even within the same lineages, luk-PV-positive phages characterized furunculosis strains, while their luk-PV-negative variants were frequent among nasal strains. The very tight epidemiological linkage between luk-PV and furunculosis, which could be separated from the genetic background of the S. aureus strain as well as from the gene makeup of the luk-PV-transducing phage, lends support to the notion of an important role for PVL in human furunculosis. These results make a case for the determination of luk-PV in recurrent soft tissue infections with methicillin-sensitive as well as methicillin-resistant S. aureus.

Antibody responses in furunculosis patients vaccinated with autologous formalin-killed Staphylococcus aureus

Autologous vaccines (short: autovaccines) have been used since the beginning of the 20th century to treat chronic staphylococcal infections, but their mechanisms of action are still obscure. This prospective pilot study involved four patients with furunculosis who were vaccinated with autologous formalin-killed Staphylococcus aureus cells. Vaccines were individually prepared from the infecting S. aureus strain and repeatedly injected subcutaneously in increasing doses over several months. We characterized the virulence gene repertoire and spa genotype of the infecting and colonising S. aureus strains. Serum antibody responses to secreted and surface-bound bacterial antigens were determined by two-dimensional immunoblotting and flow-cytometry based assays (Luminex®). All patients reported clinical improvement. Molecular characterization showed that all strains isolated from one patient over time belonged to the same S. aureus clone. Already before treatment, there was robust antibody binding to a broad range of staphylococcal antigens. Autovaccination moderately boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected. Similarly, vaccination moderately enhanced the antibody response against some staphylococcal surface proteins, e.g. ClfA, ClfB, SdrD and SdrE. In summary, autovaccination only slightly boosted the pre-existing serum antibody response, predominantly to bacterial surface antigens.

Candidaemia in polish hospitals - a multicentre survey

Significant changes in the frequency of candidaemia and the distribution of causative species have been noted worldwide in the last two decades. In this study, we present the results of the first multicentre survey of fungaemia in Polish hospitals. A total of 302 candidaemia episodes in 294 patients were identified in 20 hospitals during a 2‐year period. The highest number of infections was found in intensive care (30.8%) and surgical (29.5%) units, followed by haematological (15.9%), ‘others’ (19.2%) and neonatological (4.6%) units. Candida albicans was isolated from 50.96% of episodes; its prevalence was higher in intensive care unit and neonatology (61.22% and 73.33%, respectively), and significantly lower in haematology (22%; P < 0.001). The frequency of C. krusei and C. tropicalis was significantly higher (24% and 18%) in haematology (P < 0.02); whereas, the distribution of C. glabrata (14.1%) and C. parapsilosis (13.1%) did not possess statistically significant differences between compared departments. Obtained data indicates that species distribution of Candida blood isolates in Polish hospitals reflects worldwide trends, particularly a decrease in the prevalence of infections due to C. albicans.

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.

Comparative Analysis of Superantigen Genes in Staphylococcus xylosus and Staphylococcus aureus Isolates Collected from a Single Mammary Quarter of Cows with Mastitis

The purpose of this study was to analyze and compare genes encoding superantigens (SAgs) in Staphylococcus xylosus and Staphylococcus aureus isolates collected simultaneously from milk of the same cows with clinical mastitis. Genes encoding staphylococcal enterotoxins and enterotoxin-like proteins (sea-selu), toxic shock syndrome toxin 1 (tst-1) and exfoliative toxins (eta and etd) were investigated. It was found that among 30 isolates of S. xylosus, 16 (53.3%) harbored from 1 to 10 SAg genes. In total, in 16 SAg positive S. xylosus, 11 different enterotoxin genes were detected: sec, sed, seg, seh, sei, selm, seln, selo, selp, ser, selu and one etd gene encoding exfoliative toxin D. The most prevalent genes were ser, selu, and selo. Among all the positive isolates of S. xylosus, a total of 14 different SAg gene combinations were detected. One combination was repeated in 3 isolates, whereas the rest were detected only once. However, in the case of S. aureus all the 30 isolates harbored the same combination of SAg genes: seg, sei, selm, seln, selo and on the basis of PFGE analysis all belonged to the same clonal type. Also noteworthy was the observation that SAg genes detected in S. aureus have also been found in S. xylosus. The findings of this study further extend previous observations that SAg genes are present not only in S. aureus but also in coagulase-negative staphylococci, including S. xylosus. Therefore, taking into account that the SAg genes are encoded on mobile genetic elements it is possible that these genes can be transferred between different species of coexisting staphylococci.

Cytokine response in patients with chronic infections caused by Staphylococcus aureus strains and diversification of their Agr system classes

This study aimed to describe the levels of circulating cytokine levels produced by Th lymphocytes (IFN-γ, IL-4, IL-10, IL-17A), as well as the levels of cytokines produced by monocytes/macrophages (TNF-α, IL-1β, IL-12), in patients with chronic infections caused by Staphylococcus aureus strains, particularly in the context of the diversification of their Agr system classes. The studies were conducted on adult patients, including 50 patients with chronic suppurative dermatitis, 40 patients with chronic infections of the upper respiratory tract and 25 healthy individuals (control group). Blood serum cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA). S. aureus was detected in cultures of suppurative dermal exudates or of pharyngeal smears. Classes of Agr systems in the S. aureus strains were identified using polymerase chain reaction (PCR). In both groups of patients, on average, levels of IFN-γ were doubled, while levels of IL-17A were increased by 2.5-fold, which, however, was not accompanied by increased levels of TNF-α or IL-12. The data indicate that the development of S. aureus infection among the studied patients was linked to an impoverished cytokine response of monocytes/macrophages, while that induced by the pathogen lymphocytes Th17/Th1 may be responsible for promotion of the chronic inflammatory response. In parallel, no quantitative or qualitative differences were disclosed between cytokine responses manifested by subgroups of patients infected with S. aureus strains belonging to class IV Agr, as compared to patients infected with strains of classes I to III Agr. Nevertheless, in the patients, strains belonging to class IV Agr prevailed, which points to the preferential relationship between the class and the pathogenicity of S. aureus.

Serodiagnosis of Borreliosis: Indirect Immunofluorescence Assay, Enzyme-Linked Immunosorbent Assay and Immunoblotting

Lyme disease is an infectious, multi-system, tick-borne disease caused by genospecies of Borrelia burgdorferi bacteria sensu lato, characterized by remarkable heterogeneity. In this situation choosing an optimal antigen array for diagnostic tests seems problematic. The serological tests for borrelia routinely done in laboratories often produce ambiguous results, which makes a proper diagnosis rather complicated and thus delays the implementation of an appropriate treatment regimen. Thirty-seven outpatients and eight inpatients with suspected borreliosis diagnosis hospitalized at the Clinics of the Pomeranian Medical University (Szczecin, Poland), participated in the study. In order to detect the antibodies against Borrelia sensu lato three kinds of serological tests were used: indirect immunofluorescence assay (IIFA), enzyme-linked immunosorbent assay (ELISA), and immunoblot. The IIFA and immunoblot tests conducted on 45 patients (100%) produced positive results for both the IgM and IgG antibody types. In the case of ELISA, positive or borderline results were observed in only 24 patients (53.3%). The immunoblot test for IgM most frequently detected antibodies against the outer surface protein C (OspC) antigen (p25), and, in the case of IgG, against the recombinant variable surface antigen (VlsE). The IIFA screening test used for diagnosing Lyme borreliosis produced the highest percentage of positive results, which were then confirmed by immunoblot, but not by ELISA. Therefore using only ELISA as a screening test or for diagnosing Lyme borreliosis seems debatable.

Molecular Studies of an Impetigo Bullosa Epidemic in Full-Term Infants

Background: Carriers ofStaphylococcus aureus strains can be the source of epidemic infection for patients. Objectives: A molecular epidemiological analysis of an impetigo bullosa outbreak in a neonatal ward was performed in order to determine a potential source of the infection and possible routes of subsequent spreading of the epidemic strain. Methods: The genetic relatedness of S. aureus strains isolated from 6 neonates with epidermal lesions and from 21 staff members was verified by the pulsed field gel electrophoresis (PFGE) method. Additionally, detection of eta and etb genes of S. aureus strains using PCR was performed. Results: None of the infected newborns’ mothers was a carrier. Seven strains, 6 isolated from the newborns and 1 taken from a midwife, showed the same restriction pattern, i.e. type A. In the other 20 health care workers colonized with S. aureus, 3 genetic types could be distinguished, i.e. B (2), C (7) and D (2), as well as 9 strains with unique PFGE patterns. The eta gene detected in 7 strains belonged to the genetic type A; there was no etb gene in any of the 27 S. aureus isolates. Conclusions: The presence of the same genetic type A of S. aureus in the infected newborns is a factor which indicates that the impetigo bullosa was a hospital infection. A probable source of the infection was a midwife who was colonized with the same S. aureus type. She was present at the birth of the first infected newborn. Today, molecular methods are essential for prompt recognition of an epidemic and implementation of appropriate infection control strategies.

Antibacterial activity of rosemary, caraway and fennel essential oils

Summary Introduction: Recently, interest in essential oils used in natural medicine, has been increasing. Essential oils are still being tested for their potential uses as an alternative remedies for the treatment of many infectious diseases. Objective: The aim of the study was to evaluate antibacterial properties of commercial essential oils (rosemary, caraway and fennel) to reduce the number of Staphylococcus aureus and Escherichia coli. Methods: The antibacterial activity of essential oils was investigated by agar dilution method. Results: The result of experiments showed that essential oils contained in microbiological media significantly reduced the number of S. aureus and E. coli cells. The best antibacterial properties possessed caraway oil: 1 mg/g for S. aureus and 10 mg/g for E. coli, weaker rosemary (5 mg/g) and fennel (20 mg/g) oils. Conclusion: Results confirmed the inhibiting effect of commercial essential oils on S. aureus and E. coli and provide a scientific ground for future research.

Superantigen gene profiles, genetic relatedness and biological activity of exosecretions of Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis.

This study evaluated the superantigen gene profiles, genetic relatedness and biological activity of exosecretions of 50 Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis. Genomic relatedness of S. aureus was determined by pulsed field gel electrophoresis analysis of macro‐restricted chromosomes. The presence of genes encoding superantigens was confirmed by multiplex PCR. To study the biological activity of S. aureus exosecretions, the supernatants from bacterial liquid cultures were classified into three groups: those with leukotoxin‐like properties, those with superantigen‐like properties and those with no particular activity on leukocytes cultured in vitro. It was shown that all analyzed bacterial isolates belonged to the same clonal type and harbored the same combination of superantigen genes, namely sed, selj and ser. However, 22% of all isolates produced factors with superantigen‐like and 48% of them with leukotoxin‐like activities. Finally, although there were no detectable genetic differences between the analyzed bacterial isolates, the virulence factors secreted by them differed considerably.