Whole genome sequencing and molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from patients with bacteraemia in Slovenia.

PURPOSE: Data on the molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from patients with bacteraemia in Slovenia are lacking. The aim of this study was to phenotypically and genotypically investigate 82 MRSA strains isolated from patients with bloodstream infections in central Slovenia between 2019 and 2022. METHODS: Whole-genome sequencing of selected strains was performed to characterize the strains based on sequence typing, antimicrobial resistance, toxin, and virulence factors genes. RESULTS: Most MRSA carried SCCmec II (63.4%), followed by SCCmec IV (34.1%) and SCCmec V (2.5%). A high proportion of strains belonging to the ST225 lineage (45.1%) was observed, followed by ST97 (18.3%), ST2883 (15.9%), ST22 (9.8%), ST5 (3.7%), and the ST1, ST398 and ST45 lineages (2.4% each). Sixteen different spa types were identified, predominantly ST225-t003 (31.7%), ST97-t359 (15.9%), and ST2883-t4336 (14.6%). None of the strains carried Panton-Valentine leukocidin, exfoliative toxins, or toxic shock toxin. All MRSA strains were susceptible to linezolid, rifampicin, vancomycin, teicoplanin, and trimethoprim-sulfamethoxazole. MRSA strains were resistant to erythromycin, clindamycin, tetracycline and gentamicin, with a frequency of 74.4%, 74.4%, 8.5%, and 1.2%, respectively. CONCLUSION: This study demonstrates that bacteraemia in central Slovenia is caused by diverse MRSA lineages. Identification of newly emerged lineages should be followed in the future to detect changes in the molecular epidemiology of MRSA in our country.

Characterization of Biomarker Levels in Crimean-Congo Hemorrhagic Fever and Hantavirus Fever with Renal Syndrome.

Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are important viral hemorrhagic fevers (VHF), especially in the Balkan region. Infections with Dobrava or Puumala orthohantavirus and Crimean-Congo hemorrhagic fever orthonairovirus can vary from a mild, nonspecific febrile illness, to a severe disease with a fatal outcome. The pathogenesis of both diseases is poorly understood, but it has been suggested that a host's immune mechanism might influence the pathogenesis of the diseases and survival. The aim of our study is to characterize cytokine response in patients with VHF in association with the disease progression and viral load. Forty soluble mediators of the immune response, coagulation, and endothelial dysfunction were measured in acute serum samples in 100 HFRS patients and 70 CCHF patients. HFRS and CCHF patients had significantly increased levels of IL-6, IL-12p70, IP-10, INF-γ, TNF-α, GM-CSF, MCP-3, and MIP-1b in comparison to the control group. Interestingly, HFRS patients had higher concentrations of serum MIP-1α, MIP-1ß, which promote activation of macrophages and NK cells. HFRS patients had increased concentrations of IFN-γ and TNF-α, while CCHF patients had significantly higher concentrations of IFN-α and IL-8. In both, CCHF and HFRS patients' viral load significantly correlated with IP-10. Patients with fatal outcome had significantly elevated concentrations of IL-6, IFN-α2 and MIP-1α, while GRO-α, chemokine related to activation of neutrophils and basophils, was downregulated. Our study provided a comprehensive characterization of biomarkers released in the acute stages of CCHF and HFRS.

Are patients with erythema migrans who have leukopenia and/or thrombocytopenia coinfected with Anaplasma phagocytophilum or tick-borne encephalitis virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.

Gene expression profile suggests that pigs (Sus scrofa) are susceptible to Anaplasma phagocytophilum but control infection.

BACKGROUND: Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species. RESULTS: For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level. CONCLUSIONS: These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.

The sequences of groESL operon of Anaplasma phagocytophilum among human patients in Slovenia.

Anaplasma phagocytophilum is an emerging tick-borne pathogen. Great genetic diversity of A. phagocytophilum has been described in animals and ticks. The present study is focused on the genetic variability of the groESL operon of A. phagocytophilum in human patients in Slovenia. During 1996­2008, there were 66 serologically confirmed patients with human granulocytic anaplasmosis. Of these, 46 were tested with a screening PCR for a small part of the 16S rRNA gene of A. phagocytophilum and 28 (60.9%) were positive. Positive samples were additionally tested with a PCR targeting the groESL operon and a larger fragment of the 16S rRNA gene. All amplicons were further sequenced and analyzed. The homology search and the alignment of the groESL sequences showed only one genetic variant. Sequence analysis of the 16S rRNA gene revealed 100% identity among amplicons. Slovenia is a small country with diverse climate, vegetation, and animal representatives. In previous studies in deer, dogs, and ticks, great diversity of the groESL operon was found. In contrast, in wild boar and in human patients from this study, only one genetic variant was detected. The results suggest that only one genetic variant might be pathogenic for humans or is competent enough to replicate in humans. To support this theory, other genetic markers and further studies need to be performed.

Anaplasmosis in dogs: the relation of haematological, biochemical and clinical alterations to antibody titre and PCR confirmed infection.

Laboratory and clinical parameters of 149 dogs, exposed to Anaplasma phagocytophilum (A. phagocytophilum), and 19 control dogs were evaluated and compared retrospectively. The aim of our study was to determine statistically significant differences of selected parameters between groups of patients, divided according to the immunofluorescence (IFA) titres, in attempt to improve current diagnostic and treatment criteria. Exposure to A. phagocytophilum was confirmed by IFA and infection by PCR. Based on the results, the dogs were divided into 8 groups (6 groups of seropositive dogs according to the antibody titre, 1 group of PCR positive dogs, and a control group). Selected parameters were compared between groups. Thrombocytopenia was confirmed to be the most prominent haematological change in IFA and/or PCR positive dogs. There were no statistically significant differences in clinical and haematological observations between groups of different IFA titre but clear overall differences between each IFA and PCR positive groups compared to the control group. Our results showed the necessity of introducing additional diagnostic procedures in clinical practice, since antibody titre and haematological parameters are not sufficient to confirm the clinical relevance of exposure to A. phagocytophilum in a particular patient.

Anaplasma phagocytophilum in ticks in Slovenia.

Ticks act as vectors of many pathogens of domestic animals and humans. Anaplasma phagocytophilum in Europe is transmitted by the ixodid tick vector Ixodes ricinus. A. phagocytophilum causes a disease with diverse clinical signs in various hosts. A great genetic diversity of the groESL operon of A. phagocytophilum has been found in ticks elsewhere. In Slovenia, the variety of the groESL operon was conducted only on deer samples. In this study, the prevalence of infected ticks was estimated and the diversity of A. phagocytophilum was evaluated. On 8 locations in Slovenia, 1924 and 5049 (6973) I. ricinus ticks were collected from vegetation in the years 2005 and 2006, respectively. All three feeding stages of the tick's life cycle were examined. The prevalence of ticks infected with A. phagocytophilum in the year 2005 and in the year 2006 was 0.31% and 0.63%, respectively, and it did not differ considerably between locations. The similarity among the sequences of groESL ranged from 95.6% to 99.8%. They clustered in two genetic lineages along with A. phagocytophilum from Slovenian deer. One sequence formed a separate cluster. According to our study, the prevalence of A. phagocytophilum in ticks is comparable to the findings in other studies in Europe, and it does not vary considerably between locations and tick stages. According to groESL operon analysis, two genetic lineages have been confirmed and one proposed. Further studies on other genes would be useful to obtain more information on genetic diversity of A. phagocytophilum in ticks in Slovenia.