Pieter Deschaght

Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU).

Acinetobacter genomic species (gen. sp.) 3 and gen. sp. 13TU are increasingly recognized as clinically important taxa within the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. To define the taxonomic position of these genomic species, we investigated 80 strains representing the known diversity of the ACB complex. All strains were characterized by AFLP analysis, amplified rDNA restriction analysis and nutritional or physiological testing, while selected strains were studied by 16S rRNA and rpoB gene sequence analysis, multilocus sequence analysis and whole-genome comparison. Results supported the genomic distinctness and monophyly of the individual species of the ACB complex. Despite the high phenotypic similarity among these species, some degree of differentiation between them could be made on the basis of growth at different temperatures and of assimilation of malonate, l-tartrate levulinate or citraconate. Considering the medical relevance of gen. sp. 3 and gen. sp. 13TU, we propose the formal names Acinetobacter pittii sp. nov. and Acinetobacter nosocomialis sp. nov. for these taxa, respectively. The type strain of A. pittii sp. nov. is LMG 1035(T) (=CIP 70.29(T)) and that of A. nosocomialis sp. nov. is LMG 10619(T) (=CCM 7791(T)).

Pseudomonas aeruginosa population structure revisited.

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS +/exoU − genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set.

Gardnerella vaginalis comprises three distinct genotypes of which only two produce sialidase

OBJECTIVE Sialidase and the presence of Gardnerella vaginalis have been proposed as biomarkers for bacterial vaginosis. Sialidase has been associated with adverse pregnancy outcome. We genotyped G vaginalis isolates, assessed the presence and diversity of sialidase-encoding genes, and determined the production of sialidase. STUDY DESIGN One hundred thirty-four G vaginalis isolates were genotyped by random amplified polymorphic deoxyribonucleic acid (RAPD) and a selection of 29 isolates with amplified ribosomal deoxyribonucleic acid restriction analysis (ARDRA). A G vaginalis sialidase quantitative polymerase chain reaction was developed, and the sialidase production was assessed with the filter spot test. RESULTS Three G vaginalis genotypes could be distinguished by both RAPD and ARDRA. Only 2 genotypes encoded and produced sialidase. CONCLUSION Three genotypes exist among G vaginalis isolates, and there is a clear link between genotype and sialidase production. A possible link between sialidase production and (symptomatic) bacterial vaginosis and biofilm production can be hypothesized.

PCR and the detection of Pseudomonas aeruginosa in respiratory samples of CF patients. A literature review.

Previous studies proved the importance of rapid antibacterial intervention in case of Pseudomonas aeruginosa detection in respiratory samples of cystic fibrosis patients. To improve the early detection of P. aeruginosa, several culture, PCR and serology based approaches have been compared. Because an increasing number of routine microbiology laboratories have access to real-time PCR (qPCR), we reviewed the specificity and sensitivity of published PCR formats. The importance of choice of DNA-extraction methods and PCR formats and of the validation of their specificity and sensitivity with clinical samples is stressed.

OXA-23-producing Acinetobacter species from horses: a public health hazard?

Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; Department of Bacteriology and Immunology, CODA-CERVA-VAR, Groeselenberg 99, 1180 Brussels, Belgium; Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; Laboratory of Bacterial Genetics, National Institute of Public Health, Srobarova 48, 100 42 Prague, Czech Republic; Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine & Health Sciences, Ghent University, De Pintenlaan 185, Ghent, Belgium

The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniae and comparison with four other species specific PCR assays

BackgroundStreptococcus pneumoniae is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a challenge.MethodsThis report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of S. pneumoniae, and its performance compared to other genotypic and phenotypic tests.ResultsThe new PCR assay designed in this study, proved to be specific at 57°C for S. pneumoniae, not amplifying S. pseudopneumoniae or any other streptococcal strain or any strains from other upper airway pathogenic species. PCR assays (psaA, LytA, ply, spn9802-PCR) were previously described for the specific amplification of S. pneumoniae, but psaA-PCR was the only one found not to cross-react with S. pseudopneumoniae.ConclusionSpne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify S. pseudopneumoniae as S. pneumoniae. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.

Genotype based evaluation of Pseudomonas aeruginosa eradication treatment success in cystic fibrosis patients

BACKGROUND Longitudinal data regarding the genotypes of Pseudomonas aeruginosa isolates after eradication treatment are limited. We followed cystic fibrosis patients after a first ever isolation of P. aeruginosa and evaluated the P. aeruginosa-free time period after eradication therapy. METHODS Between January 2003 and December 2008 respiratory samples were cultured prospectively from 41 patients with a first ever P. aeruginosa isolate. Twenty five patients had at least one subsequent isolate. Treatment efficacy was assessed based on the time to a second isolation and on comparison of the RAPD genotypes of the P. aeruginosa isolates. RESULTS Eleven patients became chronically colonized during the study period. For ten of these the second isolate had the same genotype as the first isolate. Moreover, these patients had a significantly shorter P. aeruginosa-free time interval between the first ever and the second isolate compared to the 14 not chronically colonized patients (median 0 months versus 7.5 months, p<0.05). CONCLUSION Our results indicate that the presence of a genotypically identical subsequent P. aeruginosa isolate and/or a short P. aeruginosa-free time interval after treatment are ominous signs and might be useful additional tools to predict impending chronic colonization. Current routine bacteriological methods for the detection of P. aeruginosa may lack the sensitivity to discriminate between true eradication and low bacterial persistence.

Isolates belonging to CDC group II-i belong predominantly to Sphingobacterium mizutaii Yabuuchi et al. 1983: emended descriptions of S. mizutaii and of the genus Sphingobacterium

Two clinical strains, NF 296 and NF 931, present in our collection, were identified biochemically as members of CDC group II-i. Determination of the 16S rRNA gene sequence revealed highest similarity with strains of Sphingobacterium mizutaii. Because these strains produced indole, whereas S. mizutaii has been described as indole-negative, we also investigated the type strain and a reference strain of S. mizutaii, LMG 8340(T) (=CCUG 15907(T)) and LMG 8341 (=CCUG 15908), and found both strains also to be positive for indole production. These data warrant inclusion of some of the CDC group II-i strains into S. mizutaii and emended descriptions of Sphingobacterium mizutaii as indole-production-positive and of the genus Sphingobacterium as variable for indole production.

Edwardsiella tarda sepsis in a live-stranded sperm whale (Physeter macrocephalus).

Whale strandings remain poorly understood, although bacterial infections have been suggested to contribute. We isolated Edwardsiella tarda from the blood of a stranded sperm whale. The pathogen was identified with MALDI-TOF MS, confirmed by 16S rRNA gene sequencing and quantified in blood by qPCR. We report the first case of sepsis in a sperm whale. The zoonotic potential of E. tarda and the possible role of bacterial infections in the enigmatic strandings of cetaceans are discussed.

Molecular Epidemiology and Clinical Impact of Acinetobacter calcoaceticus-baumannii Complex in a Belgian Burn Wound Center.

Multidrug resistant Acinetobacter baumannii and its closely related species A. pittii and A. nosocomialis, all members of the Acinetobacter calcoaceticus-baumannii (Acb) complex, are a major cause of hospital acquired infection. In the burn wound center of the Queen Astrid military hospital in Brussels, 48 patients were colonized or infected with Acb complex over a 52-month period. We report the molecular epidemiology of these organisms, their clinical impact and infection control measures taken. A representative set of 157 Acb complex isolates was analyzed using repetitive sequence-based PCR (rep-PCR) (DiversiLab) and a multiplex PCR targeting OXA-51-like and OXA-23-like genes. We identified 31 rep-PCR genotypes (strains). Representatives of each rep-type were identified to species by rpoB sequence analysis: 13 types to A. baumannii, 10 to A. pittii, and 3 to A. nosocomialis. It was assumed that isolates that belonged to the same rep-type also belonged to the same species. Thus, 83.4% of all isolates were identified to A. baumannii, 9.6% to A. pittii and 4.5% to A. nosocomialis. We observed 12 extensively drug resistant Acb strains (10 A. baumannii and 2 A. nosocomialis), all carbapenem-non-susceptible/colistin-susceptible and imported into the burn wound center through patients injured in North Africa. The two most prevalent rep-types 12 and 13 harbored an OXA-23-like gene. Multilocus sequence typing allocated them to clonal complex 1 corresponding to EU (international) clone I. Both strains caused consecutive outbreaks, interspersed with periods of apparent eradication. Patients infected with carbapenem resistant A. baumannii were successfully treated with colistin/rifampicin. Extensive infection control measures were required to eradicate the organisms. Acinetobacter infection and colonization was not associated with increased attributable mortality.