Vassilios Sandalakis

Proteome studies of bacterial antibiotic resistance mechanisms.

Ever since antibiotics were used to help humanity battle infectious diseases, microorganisms straight away fought back. Antibiotic resistance mechanisms indeed provide microbes with possibilities to by-pass and survive the action of antibiotic drugs. Several methods have been employed to identify these microbial resistance mechanisms in an ongoing effort to reduce the steadily increasing number of treatment failures due to multi-drug-resistant microbes. Proteomics has evolved to an important tool for this area of research. Following rapid advances in whole genome sequencing, proteomic technologies have been widely used to investigate microbial gene expression. This review highlights the contribution of proteomics in identifying microbial drug resistance mechanisms. It summarizes different proteomic studies on bacteria resistant to different antibiotic drugs. The review further includes an overview of the methodologies used, as well as lists key proteins identified, thus providing the reader not only a summary of research already done, but also directions for future research. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

TICK-BORNE BACTERIA IN MOUFLONS AND THEIR ECTOPARASITES IN CYPRUS

The Cypriot mouflon (Ovis orientalis ophion), a once almost extirpated species of wild sheep, is under strict surveillance because it can be threatened by likely transmission of pathogenic bacteria, such as Anaplasma spp., Rickettsia spp., and Coxiella burnetii, primarily from domestic ungulates. We collected 77 blood samples from Cypriot mouflons and 663 of their ectoparasites (Rhipicephalus turanicus, Rhipicephalus sanguineus, Rhipicephalus bursa, Hyalomma anatolicum excavatum, Hyalomma marginatum, Haemaphysalis punctata, Haemaphysalis sulcata, and Ixodes gibossus) and tested them by polymerase chain reaction and sequencing. Twenty-three mouflon blood samples (30%) were positive for C. burnetii, 23 (30%) for Rickettsia spp., and 8 (10%) for Anaplasma ovis. Of 109 pools of ectoparasites, 32.1% were positive for C. burnetii, 28.4% for Rickettsia spp., and 10.9% for A. ovis; 11.9% were positive for both C. burnetii and Rickettsia spp., 6.4% for both Rickettsia spp. and A. ovis, and 2.8% for all three pathogens. This is the first survey that records the presence of tick-borne pathogens, both in the Cypriot mouflon and in ticks parasitizing it.

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics.

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12 836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.

Use of MALDI-TOF mass spectrometry in the battle against bacterial infectious diseases: recent achievements and future perspectives.

ABSTRACT Introduction: Advancements in microbial identification occur increasingly faster as more laboratories explore, refine and extend the use of mass spectrometry in the field of microbiology. Areas covered: This review covers the latest knowledge found in the literature for quick identification of various classes of bacterial pathogens known to cause human infection by the use of MALDI-TOF MS technology. Except for identification of bacterial strains, more researchers try to ‘battle time’ in favor of the patient. These novel approaches to identify bacteria directly from clinical samples and even determine antibiotic resistance are extensively revised and discussed. Expert commentary: Mass spectrometry is the future of bacterial identification and creates a new era in modern microbiology. Its incorporation in routine practice seems to be not too far, providing a valuable alternative, especially in terms of time, to conventional techniques. If the technology further advances, quick bacterial identification and probable identification of common antibiotic resistance might guide patient decision-making regarding bacterial infectious diseases in the near future.

DNA Gyrase and Topoisomerase IV Mutations in an In Vitro Fluoroquinolone-Resistant Coxiella burnetii Strain

The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a vacuole with lysosomal characteristics. Quinolones have been used as an alternative therapy for Q fever. In this study, quinolone-resistance-determining regions of the genes coding for DNA gyrase and topoisomerase IV were analyzed by DNA sequencing from an in vitro fluoroquinolone-resistant C. burnetii strain (Q212). Sequencing and aligning of DNA gyrase encoding genes (gyrA and gyrB) and topoisomerase IV genes (parC and parE) revealed one gyrA mutation leading to the amino acid substitution Asp87Gly (Escherichia coli numbering), two gyrB mutations leading to the amino acid substitutions Ser431Pro and Met518Ile, and three parC mutations leading to the amino acid substitutions Asp69Asn, Thr80Ile, and Gly104Ser. The corresponding alignment of the C. burnetii Q212 reference strain, the in vitro developed fluoroquinolone-resistant C. burnetii Q212 strain, and E. coli resulted in the identification of several other naturally occurring mutations within and outside the quinolone-resistance-determining regions of C. burnetii providing indications of possible natural resistance to fluoroquinolones. The present study adds additional potential mutations in the DNA topoisomerases that may be involved in fluoroquinolone resistance in C. burnetii due to their previous characterization in other bacterial species.

Phylogentic analysis of Anaplasma ovis strains isolated from sheep and goats using groEL and mps4 genes.

Evidence of Anaplasma spp. in goats and sheep in Cyprus has been demonstrated by previous research. Herein, further research was performed for the identification of the exact Anaplasma spp. resulting in the identification of Anaplasma ovis strains in all samples examined. We used a bioinformatics as well as a molecular approach (study of groEl and mps4 genes) in order to verify the validity of the results. All samples depicted the presence of A. ovis regardless of the host (goat or sheep).

Identification of a novel uncultured Rickettsia species strain (Rickettsia species strain Tselenti) in Cyprus.

In a previous study conducted in Cyprus, various spotted fever group Rickettsia species were detected and identified in ticks by molecular analysis. Among them, a partially characterized Rickettsia species was detected in Hyalomma anatolicum excavatum and Rhipicephalus turanicus ticks. We report characterization of this rickettsial strain by using polymerase chain reaction sequencing analysis of partial citrate synthase A, outer membrane protein A, outer membrane protein B, and 17-kD protein genes. We propose a provisional name Rickettsia sp. strain Tselenti for this strain until it is isolated and further characterized.

Environmental investigation for the presence of Vibrio species following a case of severe gastroenteritis in a touristic island

Global changes have caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves, and corals. Following a possible human case of infection due to V. cholerae in the island of Kos (eastern Aegean, Greece), environmental samples were collected and tested for the presence of Vibrio species. Using chromogenic agar and MALDI-TOF MS, V. parahaemolyticus, V. vulnificus V. furnisii, V. alginolyticus, and V. fluvialis were isolated and/or identified. The presence of V. cholerae was established by PCR-sequencing analysis only. Following the susceptibility testing of the Vibrio isolates, only one, V. furnisii, showed intermediate resistance to ciprofloxacin. The rest of the isolates were susceptible to all antibiotics tested. The presence of Vibrio species in aquatic samples reveals potential dangers due to exposure to contaminated seawaters.

Distribution of the ompA-types among ruminant and swine pneumonic strains of Pasteurella multocida exhibiting various cap-locus and toxA patterns

Pasteurella multocida is an important pathogen in food-producing animals and numerous virulence genes have been identified in an attempt to elucidate the pathogenesis of pasteurellosis. Currently, some of these genes including the capsule biosynthesis genes, the toxA and the OMPs-encoding genes have been suggested as epidemiological markers. However, the number of studies concerning ruminant isolates is limited, while, no attempt has ever been made to investigate the existence of ompA sequence diversity among P. multocida isolates. The aim of the present study was the comparative analysis of 144 P. multocida pneumonic isolates obtained from sheep, goats, cattle and pigs by determining the distribution of the ompA-types in conjunction with the cap-locus and toxA patterns. The ompA genotypes of the isolates were determined using both a PCR-RFLP method and DNA sequence analysis. The most prevalent capsule biosynthesis gene among the isolates was capA (86.1%); a noticeable, however, rate of capD-positive isolates (38.6%) was found among the ovine isolates that had been associated primarily with the capsule type A in the past. Moreover, an unexpectedly high percentage of toxA-positive pneumonic isolates was noticed among small ruminants (93.2% and 85.7% in sheep and goats, respectively), indicating an important epidemiological role of toxigenic P. multocida for these species. Despite their great heterogeneity, certain ompA-genotypes were associated with specific host species, showing evidence of a host preference. The OmpA-based PCR-RFLP method developed proved to be a valuable tool in typing P. multocida strains.

Tetracycline resistance genes in Pasteurella multocida isolates from bovine, ovine, caprine and swine pneumonic lungs originated from different Greek prefecturesa

The aim of the present work was to analyze the antibiotic resistance of Pasteurella multocida isolates from sheep, goats, cattle and pigs originated from eight different Greek prefectures, to determine the class(es) of the tet genes, to investigate any possible association with plasmids or/and chromosomes and to study the geographical distribution of the resistant isolates. Eighteen out to one hundred isolates were resistant to tetracycline with a minimal inhibitory concentration (MIC) higher than 128 μg/ml. The tetH gene was found in thirteen (72.2%) and the tetB gene was found in four isolates (22.2%). After treatment with the restriction enzymes EcoRV, EcoRI, PstI and BspHI, the tetB gene showed a new polymorphism. Partial sequencing of the tetH and tetB genes revealed five polymorphisms in the tetB gene and two polymorphisms in the tetH gene; phylogenetic and molecular evolutionary analyses were also conducted. Both tetB and tetH genes revealed patterns clearly differentiated from the rest of Pasteurella species meaning that the genes could be transferred from a non Pasteurella species. The results presented provide further knowledge and evidence of the role played by commensals as of resistance determinants.