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We report on the molecular evidence that Dermacentor reticulatus ticks in Croatia are infected with Rickettsia helvetica (10%) or Rickettsia slovaca (2%) or co-infected with both species (1%). These findings expand the knowledge of the geographic distribution of R. helvetica and D. reticulatus ticks. [ABSTRACT FROM AUTHOR]

Objective: The goal was to investigate the number and origin of typhoid fever cases in Split-Dalmatia County, Croatia, during the period of 1970-2005.Methods: Data from the medical records of typhoid fever cases treated at the University Hospital during the 35-year period were analyzed. Cases were defined as those in which Salmonella typhi was isolated from blood and/or stool. All isolates were tested for susceptibility to antimicrobial agents.Results: A total of 45 cases were identified. Forty-four patients originated from Bosnia and Herzegovina, and all of their isolates were susceptible to all antimicrobial agents tested. One patient from Pakistan was infected with multidrug-resistant S. typhi. No autochthonous case of typhoid fever was identified.Conclusion: All cases of typhoid fever were acquired in countries where typhoid fever is endemic, and their importation to Croatia was the result of war-associated population movements. Increased awareness of health care workers regarding persons who originated from or have traveled to a typhoid-endemic area is needed. To avoid treatment failure, particular attention should be given to persons coming from regions with a known presence of multidrug-resistant S. typhi. [ABSTRACT FROM AUTHOR]

Examines the serological response of a patient treated with prednisone to Leishmania infantum. Presentation of a case study on a patient with leishmaniasis; Symptoms of the disease; Daily accounts of the development of the patient.

We report a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). This strategy avoids issues related to weak substrate binding and ligand relocalization. The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4), the most important drug-metabolizing enzyme. Allosteric activation and inhibition of CYP3A4 by pharmaceuticals is an important mechanism of drug interactions. We performed HDX-MS analysis on several CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic) or do not show activation while still blocking the allosteric site (antagonistic). This allowed us to better define the position of the allosteric site, the protein structural dynamics associated with allosteric activation, and the presence of coexisting conformers.

Because of its exceptional substrate promiscuity, human P450 3A4 (CYP3A4) is arguably the most important drug-metabolizing enzyme. CYP3A4 also has the particularity of binding multiple ligands simultaneously, which is associated with heterotropic or homotropic, positive or negative, cooperativity or allostery. Solving the kinetics of such complex systems remains challenging, and so is identifying the binding pockets involved. Progesterone (PRG) is a known allosteric activator of CYP3A4-catalyzed 7-benzyloxy-4-trifluoromethylcoumarin (BFC) debenzylation. We report herein the use of bioconjugation as a successful strategy to identify this PRG allosteric site. A progesterone analogue (PGM) was covalently attached, separately at several locations, near a peripheral binding pocket previously proposed to be an allosteric site. Studies of BFC debenzylation in the presence of free PRG revealed that two of the bioconjugates successfully positioned the covalently attached PGM moiety in a way that mimics the allosteric activation observed with free PRG. Interestingly, the PGM bioconjugate with the better fit yielded a higher permanent activation of the enzyme.

Human cytochrome P450 3A4 (CYP3A4) is an important drug metabolizing enzyme involved in a number of drug-drug and food-drug interactions. As such, much effort has been devoted into investigating its mechanism of interaction with ligands. CYP3A4 has one of the highest levels of substrate promiscuity for an enzyme, and can even bind multiple ligands simultaneously. The location and orientation of these ligands depend on the chemical structure and stoichiometry, and are generally poorly understood. In the case of the steroid testosterone, up to three copies of the molecule can associate with the enzyme at once, likely two in the active site and one at a postulated allosteric site. Recently, we demonstrated that steroid bioconjugation at the allosteric site results in an increase in activity of CYP3A4 toward testosterone and 7-benzyloxy-4-trifluoromethylcoumarin oxidation. Here, using the established bioconjugation methodology, we show how steroid bioconjugation at the allosteric site affects the heme spin state, the binding affinity (K S ) of CYP3A4 for testosterone, as well as the enzyme coupling efficiency., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of the majority of drugs. As such, it is implicated in many adverse drug-drug and food-drug interactions, and is of significant interest to the pharmaceutical industry. This enzyme is known to simultaneously bind multiple ligands and display atypical enzyme kinetics, suggestive of allostery and cooperativity. As well, evidence of a postulated peripheral allosteric binding site has provoked debate around its significance and location. We report the use of bioconjugation to study the significance of substrate binding at the proposed allosteric site and its effect on CYP3A4 activity. CYP3A4 mutants were created and covalently modified with various small molecules including progesterone. The labeled mutants displayed enhanced kinetic stability and improved activity in testosterone and 7-benzyloxy-(4-trifluoromethyl)coumarin oxidation assays. Our work applies a new strategy to study cytochrome P450 allostery and supports the hypothesis that substrate binding at the postulated allosteric site of CYP3A4 may induce functional cooperativity.

Aim: To review the current state of knowledge concerning rickettsiae and rickettsioses in Croatia and to discuss their implications for travellers., Methods: The PubMed database was searched from 1991 to 2015 by combining the words "rickettsia," "rickettsiosis", "travellers" and "Croatia"., Results: Since 1969, Croatia appears to be free of epidemic typhus (ET) caused by Rickettsia prowazekii and the last case of Brill-Zinsser disease was recorded in 2008. Mediterranean spotted fever (MSF) caused by Rickettsia conorii is the most frequent human rickettsial infection in Croatia, followed by murine typhus caused by Rickettsia typhi. Human cases of MSF and murine typhus have been predominantly observed along the eastern Adriatic coast from Zadar to Dubrovnik and between Zadar and Split, respectively. Rickettsia akari, etiologic agent of rickettsialpox, was isolated from blood of a patient diagnosed with MSF in Zadar, but no cases of rickettsialpox were reported. Several species of pathogenic (Rickettsia slovaca, Rickettsia aeschlimannii, Ricketsia helvetica, and Ricketsia raoultii) and species of undetermined pathogenicity (Ricketsia hoogstraalii sp. nov.) rickettsiae were identified in ticks collected in different ecological regions of Croatia. A search of the literature revealed no evidence of rickettsial infection in travellers visiting Croatia. Three imported cases of Rickettsia africae were observed in travellers returning from South Africa., Conclusion: Rickettsiae and rickettsial diseases continue to be present in Croatia. As they can be acquired while travelling, physicians should consider rickettsial infection in the differential diagnosis of patients returning from Croatia and presenting with febrile illness., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio- and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drug-metabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme.

P450 enzymes (P450s) are well known for their ability to oxidize unactivated CH bonds with high regio- and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The meropenem yearly Susceptibility Test Information Collection (MYSTIC) programme is a global, longitudinal resistance surveillance network that monitors the activity of meropenem and compares its activity with other broadspectrum antimicrobial agents. We now report the antimicrobial efficacy of meropenem compared to other broad-spectrum agents within the selective Gram-negative pathogen groups from two Croatian Hospitals investigated between 2002-2007. A total of 1510 Gram-negative pathogens were tested and the minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI.There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in both medical centers. High resistance rates of K. pneumoniae to ceftazidime (18%), cefepime (17%) and gentamicin (39%) are raising concern. Acinetobacter baumannii turned out to be the most resistant Gram-negative bacteria with 81% resistant to ceftazidime, 73% to cefepime, 69% to gentamicin and 71% to ciprofloxacin. Almost 20% of Pseudomonas aeruginosa strains were resistant to imipenem, 13% to meropenem, 69% to gentamicin and 38% to ciprofloxacin.The prevalence of extended-spectrum beta-lactamases (ESBLs) in E. coli was 10% and in K. pneumoniae 49%. PCR and sequencing of the amplicons revealed the presence of SHV-5 in nine E. coli strains and additional tem-1 beta-lactamase five strains. Five K. pneumoniae strains were positive for bla(SHV-5 )gene. Eight ESBL positive Enterobacter spp. strains were found to produce tem and CtX-m beta-lactamases. Plasmid-mediated AmpC beta-lactamases were not found among K. pneumoniae, E. coli and Enterobacter spp. Three A. baumannii strains from Zagreb University Center were identified by multiplex PCR as OXA-58 like producers. Six A. baumannii strains from Split University Center were found to possess an ISAba1 insertion sequence upstream of bla(OXA-51 )gene. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gram-negative bacteria. These data indicate that despite continued use of meropenem, carbapenem resistance is not increasing among species tested, except for A. Baumannii, in the two study hospitals and suggest that clinicians can still administer carbapenems as a reliable and effective choice in managing serious nosocomial infections.

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.

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)).

Carbapenem-resistant isolates of Acinetobacter baumannii from intensive care units at Split University Hospital, Split, Croatia, were studied. Most (100 of 106) had ISAba1 inserted upstream of a bla(OXA-107) gene, encoding an unusual OXA-51-type oxacillinase. Pulsed-field gel electrophoresis revealed that the isolates formed three clusters belonging to the sequence group 2 (European clone 1) lineage.

A retrospective serological survey was carried out by the indirect immunofluorescence assay (IFA) and complement fixation (CF) test in a sample of 425 healthy residents (240 females and 185 males) to investigate whether Lymphocytic choriomeningitis virus (LCMV) circulates in the rural area of the northern Croatian island of Vir, which is known to be endemic for murine typhus. The overall prevalence of LCMV antibodies detected by IFA was found to be 36% (155 out of 425) and 13% (54 out of 425) by CF. No significant difference in the LCMV seroprevalence was observed with respect to age and gender by any of the methods used. The results show that inhabitants in the area studied are clearly being exposed to LCMV and the infection of the population occurs in the first 10 years of life. Equal prevalence of IgG and especially of short-lived CF antibodies in the elderly in comparison to other age groups and children can be explained by reinfections and boosting of antibodies through permanent contact with the virus. To the best of our knowledge this study reports the first evidence of endemic LCMV in Croatia and one of the highest LCMV human prevalence reported worldwide to date. The epidemiological association between LCMV and other zoonoses which can be expected in this community as well as the etiology of summer influenza-like illness along the coastal area of Croatia has to be explored further., ((c) 2006 Wiley-Liss, Inc.)

The prevalence of Escherichia coli and Klebsiella pneumoniae that produce extended-spectrum b-lactamases (ESBL) was investigated in patients of a university hospital in Split, Croatia. Patients were grouped according to age (pediatric vs. adult), antibiotic type, and hospital ward. From Jan. 2001 to Dec. 2002, the susceptibility of E. coli and K. pneumoniae isolates to antimicrobials was tested. ESBL production was assayed using the double-disk synergy test. ESBL-producing E. coli and K. pneumoniae were detected in all sites of infection sampled. The percentages of ESBL-positive isolates were higher in the pediatric wards than in the adult wards. The antibiotics most commonly prescribed to patients in all hospital wards belonged to the third-generation cephalosporin group. Among ESBL producers, E. coli isolates were more resistant to aminoglycosides, but less resistant to ciprofloxacin and cotrimoxazole. Resistance of E. coli and K. pneumoniae to ciprofloxacin was exclusively found in isolates from adult patients. None of the isolates, regardless of ESBL production, was resistant to carbapenemes. In addition, the prevalence and antimicrobial resistance of ESBL-producing E. coli and K. pneumoniae isolates differed between pediatric and adult patients.

A total of 197 ticks belonging to four species (Haemaphysalis punctata, Hyalomma marginatum, Rhipicephalus bursa and Dermacentor marginatus) collected in October 2000 from domestic animals in southern Croatia were examined for the presence of rickettsiae by molecular techniques. Specific sequences of the rickettsiae were detected in 25 (12.7%) of ticks tested. The prevalence of infection in D. marginatus and H. marginatum ticks was 36.8 and 64.7%, respectively. None of the ticks belonging to the species H. punctata or Rh. bursa were infected. Sequence analysis of amplified products revealed that D. marginatus ticks are infected with Rickettsia slovaca, whereas H. marginatum are infected with R. aeschlimannii. The results of this study extend the knowledge of the geographic distribution of SFG rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in ticks in southern Croatia.

The prevalence of rickettsial antibodies in north-western part of Bosnia and Herzegovina was studied. Among 231 sera tested by complement fixation (CF) positive were: 61.5% for Rickettsia typhi, 4.3% for R. prowazekii, 1.7% for R. conorii and 19.0% for Coxiella burnetii. Of 183 sera tested by indirect immunofluorescence assay (IFA) 37.7% reacted with R. typhi, 1.6% with R. conorii and 22.4% with C. burnetii. The results show that at least R. typhi and C. burnetii are highly endemic in this area.

Addition of interferon (IFN) to nucleoside analog therapy for herpetic keratitis has been shown to significantly increase the efficacy of therapy compared to nucleoside alone. We have analysed several nucleoside analogs and recombinant IFN-alpha 2 to determine which combinations have increased anti-herpes simplex virus type 1 (HSV) activity. Synergistic anti-HSV activity between IFN-alpha 2 and the acyclic guanosine analogs, acyclovir (ACV) and ganciclovir (DHPG), was demonstrated in cytopathic effect reduction assay in human corneal cell cultures as well as in Vero cells. In this assay system IFN-alpha 2 alone had little detectable antiviral activity at titers of greater than or equal to 2,000 IU/ml, however, treatment of cells with about 100 IU/ml of IFN-alpha 2 for 24 hrs prior to infection decreased the ED50 of ACV approximately 2- to 3-fold and of DHPG approximately 5- to 6-fold in Vero cells. Combinations of IFN-alpha 2 with bromovinyldeoxyuridine (BVdU) in Vero cells or human corneal stromal cells did not increase the antiviral activity of BVdU. Combinations of IFN-alpha 2 with trifluorothymidine (TFT) also did not increase the effective antiviral activity of this nucleoside and resulted in decreased uptake of TFT from the medium. These studies document that combinations of acyclic nucleoside analogs, ACV and DHPG, with IFN-alpha 2 resulted in synergistic anti-HSV activities in both Vero and human corneal stromal cells, while the pyrimidine analogs, TFT and BVdU, were not synergistic with IFN-alpha 2. IFN-alpha 2 treatment of cells induced modifications of nucleoside (e.g., thymidine and TFT), but not nucleobase (e.g., ACV) uptake. These studies suggest that selective inhibition of nucleoside versus nucleobase uptake may contribute to the mechanism of IFN/nucleobase synergy in the inhibition of HSV replication.