Volga Punda-Polic

Rickettsia hoogstraalii sp. nov., isolated from hard- and soft-bodied ticks

A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gltA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia felis, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).

Outbreak in Croatia caused by a new carbapenem-resistant clone of Acinetobacter baumannii producing OXA-72 carbapenemase.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen that causes nosocomial infections and outbreaks, particularly in the intensive care unit (ICU) setting.1 Many outbreak strains belong to one of three worldwide lineages, known originally as European clones I, II and III. These correspond to sequence groups 2, 1 and 3, respectively, each of which includes a number of different genotypes defined by pulsed-field gel electrophoresis (PFGE).2 Only two previous reports have analysed carbapenem resistance inmultidrug-resistant isolates of A. baumannii from Croatia.3,4 In Split, the first carbapenem-resistant isolate of A. baumannii [meropenem minimum inhibitory concentration (MIC): 16 mg/mL] was isolated in 2002 at Split University Hospital. Between 2002 and 2008, >100 patient isolates were found to belong to the European clone 1 lineage, with most isolates carrying a blaOXA-107 gene associated with ISAba1.3 We now wish to report a new clone of multidrugresistant A. baumannii causing an outbreak in Split University Hospital following inward transfer of a patient. On 5 January 2009, a female aged 51 years was transferred to the intensive care unit (ICU) of Split University Hospital, Croatia, following brain surgery for glioblastoma at the General Hospital Mostar, Bosnia Herzegovina. According to the transfer letter, multidrug-resistant Acinetobacter sp. was isolated from a bronchial aspirate at Mostar General Hospital. No published data are available concerning the incidence, molecular basis and epidemiology of carbapenem-resistant isolates of Acinetobacter in Bosnia Herzegovina. On the day of transfer, blood cultures, cerebrospinal fluid and bronchial lavage were taken. Two days after transfer, carbapenemresistant A. baumannii was isolated from blood culture, and a day later from the bronchial lavage. Initial identification was made using the ATB 32GN and Vitek 2 systems (bioMérieux, Marcy l’Etoile, France), with A. baumannii confirmed by tRNA spacer fingerprinting and the presence of an OXA-51-type b-lactamase (specific to A. baumannii).2 Susceptibility to b-lactams (ceftazidime, cefepime, imipenem, meropenem), b-lactam/b-lactamase inhibitor combinations (ampicillin/sulbactam, piperacillin/tazobactam), aminoglycosides (amikacin, gentamicin) and fluoroquinolones (ciprofloxacin) was determined by disc-diffusion tests, and susceptibility to colistin by E-tests (AB Biodisk, Solna, Sweden). MICs were determined by broth microdilution according to Clinical and Laboratory Standards Institute recommendations. The A. baumannii isolates were resistant to all antimicrobials tested except colistin (MIC: 0.5 mg/mL) and ampicillin/sulbactam (MIC: 1 mg/mL). The patient was treated with intravenous ampicillin/sulbactam and colistin in contact isolation, but died two weeks following transfer. During the next 6 months (January to July 2009), 32 similar consecutive isolates were obtained from blood cultures, urine samples, catheter tip specimens, cerebrospinal fluid, throat and nasal swabs, and bronchial secretions collected from 23 different patients hospitalised in two ICUs and three different departments at Split University Hospital. PFGE following macrorestriction of genomic DNA with ApaI revealed that all isolates belonged to the European clone 2 lineage. All isolates also displayed the same multidrug resistance pattern (with no inhibition zone around imipenem or meropenem discs), but susceptibility to sulbactam and colistin (Table I). Bacterial DNA was extracted using a DNAze kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The presence of genes encoding class D carbapenemases was detected by multiplex polymerase chain reaction using primers specific for the OXA-23, OXA-40, OXA-51 and OXA-58 gene families.5 Each isolate possessed a blaOXA-51-like and a blaOXA-40-like gene. Sequencing (both strands) of the blaOXA-51-like amplicons from a randomly selected subset of isolates by a commercial laboratory (Macrogen, Seoul, South Korea) revealed the presence of the OXA-90 gene (a variant of OXA-66), whereas sequencing of the blaOXA-40-like amplicons revealed the presence of a gene encoding OXA-72.2 OXA-40-like carbapenemases were originally described in A. baumannii isolates from the Iberian peninsula, but have now been reported worldwide, with sequence divergences between the members of this family varying from one to five amino acids.6,7 OXA-72was first described in an Acinetobacter isolate fromThailand in 2004, and has since been reported in a single A. baumannii isolate from mainland China and as a major mechanism of carbapenem resistance in a Taiwanese hospital.8 This is the first report of OXA-72 in Southeast Europe. PFGE analysis revealed that this new imported clone belonged to the European clone 2 lineage, in contrast to carbapenem-resistant isolates of A. baumannii from the same hospital during the preceding six years, which belonged to the European clone 1 lineage and encoded only the unusual OXA-51-type enzyme OXA-107, associated with ISAba1.3 These observations reinforce the continued importance of careful epidemiological surveillance and enhanced control measures following international patient transfers. Wherever possible, hospitals should routinely isolate and screen all patients admitted from hospitals in other countries. This is particularly important when the source country is known to have a high prevalence of multidrug-resistant bacteria or when no antimicrobial surveillance data are available. It is mandatory to rapidly identify the source and/or reservoir of colonisation/infection with multidrug-resistant A. baumannii in order to prevent further dissemination inside a hospital.

Evidence of an autochthonous Toscana virus strain in Croatia.

BACKGROUND Phleboviruses are large and widespread group of viruses that are transmitted by arthropods and they have been reported to circulate in endemic regions of Mediterranean Basin, including Croatia. OBJECTIVES To investigate the role of Toscana virus, as a cause of the aseptic meningitis, in summer months in Croatia. STUDY DESIGN Samples from 30 patients with aseptic meningitis were retrospectively tested by serology and RT-PCR for TOSV. RESULTS TOSV RNA was detected in 2/30 and TOSV IgM antibodies were found in 4/30 of patients. Phylogenetic analysis of partial L and S segments suggests that TOSV from Croatia represents an autochthonous strain. CONCLUSIONS The study has confirmed the role of TOSV as an agent that causes aseptic meningitis in Croatia, therefore it should be considered by physicians when encountering meningitis or febrile illness among indigenous population or travellers during the summer months.

Antimicrobial susceptibility of Gram-negative and Gram-positive bacteria collected from countries in Eastern Europe: results from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) 2004-2010

The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) commenced in 2004 to longitudinally monitor global changes in bacterial susceptibility to a suite of antimicrobial agents. The current study examined the activity of tigecycline and comparators against isolates collected across Eastern Europe between 2004 and 2010. Minimum inhibitory concentrations were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methodologies. Antimicrobial susceptibility was determined using CLSI interpretive criteria, and tigecycline susceptibility was established using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. This study included 10 295 Gram-negative and 4611 Gram-positive isolates from 42 centres. Extended-spectrum β-lactamases (ESBLs) were reported among 15.3% of Escherichia coli and 39.3% of Klebsiella pneumoniae isolates; the highest rates were observed in Turkey (30.9%) and Bulgaria (53.8%), respectively. Imipenem-non-susceptible K. pneumoniae were identified only in Turkey. ESBL-positive E. coli were highly susceptible to imipenem (95.1%), meropenem (98.0%) and tigecycline (98.5%). Most antimicrobials showed poor activity against Acinetobacter baumannii and Pseudomonas aeruginosa. Vancomycin resistance was noted among 0.9% of Enterococcus faecalis and 11.7% of Enterococcus faecium isolates. High rates of susceptibility were reported for linezolid (99.7%) and tigecycline (100%) against E. faecium. One-quarter of Staphylococcus aureus isolates were meticillin-resistant S. aureus (MRSA), with the highest rate in Romania (51.5%); all MRSA were susceptible to linezolid, tigecycline and vancomycin. Antimicrobial resistance is high in much of Eastern Europe, with considerable variation seen among countries. Tigecycline and the carbapenems retain excellent activity against many pathogens from Eastern Europe; linezolid and vancomycin are active against most Gram-positive pathogens.

Antimicrobial Effects of Wine: Separating the Role of Polyphenols, pH, Ethanol, and Other Wine Components

While the antimicrobial effectiveness of wine is well documented, relative contributions of the wine components to its antimicrobial activity is controversial. To separate the role of wine phenolics, ethanol, and pH from other wine constituents, the antimicrobial effects of intact wine were compared to that of phenols-stripped wine, dealcoholized wine, ethanol, and low pH applied separately and in combination, against 2 common foodborne pathogens, Salmonella enterica serovar Enteritidis and Escherichia coli. All samples were biochemically characterized with respect to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, and pH. Antioxidative activity of the samples corresponded to their total phenolics content. Except for respective controls, pH and ethanol content were similar in all samples. The order of antibacterial activity of the samples was: intact wine > phenols-stripped wine > dealcoholized wine > combination of ethanol and low pH > low pH > ethanol. Separate application of ethanol or low pH showed negligible antibacterial activity while their combination showed synergistic effect. Antibacterial activity of the samples could not be related to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, or pH. Our study indicates that antimicrobial activity of complex solutions such as intact wine cannot be exclusively attributed to its phenolic or nonphenolic constituents, nor can the antimicrobial activity of wine be predicted on the basis of its particular components.

High prevalence and molecular characterization of extended-spectrum β-lactamase-producing Proteus mirabilis strains in southern Croatia.

The aim of this study was to determine the prevalence and antibiotic resistance rates of extended-spectrum β-lactamase (ESBL)-producing Proteus mirabilis strains isolated from inpatients at the Split University Hospital (southern Croatia) during a survey performed between 2005 and 2008. A total of 2152 consecutive isolates of P. mirabilis were isolated. The prevalence was 0.5 % in 2005 and increased significantly to 20.9 % by 2008. Strains were most frequently isolated from urine (36.5 %) and bronchial aspirates and wound swabs (11.3 %). ESBL-producing P. mirabilis isolates showed very high resistance rates to the majority of non-β-lactam antibiotics and were susceptible to a β-lactam/β-lactamase inhibitor and carbapenems. The isolates were genotyped and their ESBLs were molecularly characterized. Strains originating from the intensive care unit and the surgery and neurosurgery wards were clonally related. All P. mirabilis isolates produced the TEM-52 type of ESBL. To the best of our knowledge, our work detailed here and summarized in an earlier communication is the first report of such isolates from southern Croatia. Increased monitoring and screening for ESBL production in this species at our hospital is mandatory.

A case of Mediterranean spotted fever associated with severe respiratory distress syndrome.

Mediterranean spotted fever (MSF) is usually a mild endemic rickettsial disease occurring in southern Croatia. We have reported the clinical and epidemiological characteristics of an acute MSF case associated with severe respiratory distress syndrome and hemodynamical instability. The patient recovered completely after antimicrobial treatment. Indirect immunofluorescence assay (FOCUS Diagnostics Inc.) was performed to detect IgM and IgG antibodies to Rickettsia conorii. A significant increase of both IgM and IgG antibody titres found in paired acute- and convalescent-phase serum confirmed the diagnosis of acute MSF.

P1338 Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in a university hospital, Split, Croatia

Acinetobacter baumannii is an opportunistic pathogen that is frequently involved in outbreaks of infection, occurring mostly in intensive care units. Carbapenem resistance is now being reported increasingly in A. baumannii isolates in association with the production of carbapenem-hydrolysing class D beta-lactamases or oxacillinases that have now emerged worldwide. The aim of the present study was to analyse and compare genotypes of clinical isolates of carbapenem resistant Acinetobacter baumannii collected from three different Intensive Care Units in University Hospital Split, Croatia. During 2004, twenty-two non- repetitive A. baumannii isolates with an unusual resistance profile were obtained from patients hospitalised at three different Intensive Care Units (two adults ICU and one children ICU) inside University Hospital Split. All collected isolates of A. baumannii displayed intermediate (MICs>8 mg/L) or resistant (MICs>16 mg/L) profile to imipenem and/or meropenem. Minimum inhibitory concentrations were also determined for ceftazidime, cefepime, ceftriaxone, amikacin, gentamicin, ciprofloxacin and piperacillin-tazobactam by broth microdilution according to CLSI (formerly NCCLS) recommendation. All isolates were multidrug-resistant exhibiting high resistance to tested antimicrobials. The isolates of A. baumanii were genetically characterized using pulsed-field gel electrophoresis (PFGE). Strain typing was performed by macrorestriction analysis of chromosomal DNA by use of PFGE (Apa I enzyme, in a CHEF DR III drive module). Results. We report the clonal dissemination of pulsotype A between two different adult intensive care units in University Hospital Split, belonging to the same pulsed-field gel electrophoresis (PFGE) profile, probably by hospital staff during medical procedures. The strain characterised as pulsotype B was the only strain isolated from children intensive care unit without expanding inside the hospital. The infection control team of the hospital implemented restriction of carbapenem usage and strict antiseptic techniques, which included the rigorous use of alcohol-clorhexidine solutions before and between patient and equipment contact and before leaving the units. Consequently, incidence and spread of multidrug-resistant A. baumannii nosocomial infections suggest the necessity of a surveillance program and enforcing adequate control measures in different hospital settings.