Browsing by Author "Lastovica, Albert J."

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    Characterisation of campylobacter concisus strains from South Africa using amplified fragment length polymorphism (AFLP) profiling and a genomospecies-specific polymerase chain reaction (PCR) assay: Identification of novel genomospecies and correlation with clinical data
    (Academic Journals, 2013) On, S.L.W.; Siemer, B. L.; Brandt, S. M.; Chung, P.; Lastovica, Albert J.
    Amplified Fragment Length Polymorphism (AFLP) profiling was used to evaluate the distribution of phenotypically indistinguishable, but genetically distinct, among Campylobacter concisus strains from South Africa. A Polymerase Chain Reaction (PCR) assay described for identifying strains belonging to Genomospecies 1 and 2 was applied in this study. Forty-seven C. concisus strains were studied in total, of which 42 were of South African origin. Forty of the South African isolates were assigned to the major existing genomospecies typified by the type strain of oral origin (GS1), and reference strains from bloody diarrhoea (GS2). Eighteen South African isolates were distributed in the GS1 cluster including two oral strains. Twenty-two faecal South African isolates clustered with reference GS2 strains. Two novel genomospecies (GS 5 and 6) were inferred by their AFLP profile characteristics. Use of an existing PCR assay first described for identification of GS1 and GS2 strains generally indicated that the tool was accurate, although the novel genomospecies described here yielded an amplicon in the GS2 assay. No consistent clinical pattern among the diarrhoea South African strains could be discerned. The study extends the known genetic diversity among C. concisus, elucidates the presence of multiple genomospecies in South Africa, and confirms for the first time an association of GS1 with diarrhoea as well as the utility (with caveats) of a PCR assay for identifying GS1 and GS2 strains.
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    A designed experiments approach to optimizing MALDI-TOF MS spectrum processing parameters enhances detection of antibiotic resistance in Campylobacter jejuni
    (Frontiers Media, 2016) Penny, Christian; Lastovica, Albert J.
    MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.
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    Molecular characterization of the 16S rRNA Gene of helicobacter fennelliae isolated from stools and blood cultures from paediatric patients in South Africa
    (Hindawi, 2011) Smuts, H.E.M; Lastovica, Albert J.
    Forty strains of H. fennelliae collected from paediatric blood and stool samples over an 18 year period at a children's hospital in Cape Town, South Africa, were amplified by PCR of the 16S rRNA. Two distinct genotypes of H. fennelliae were identified based on the phylogenetic analysis. This was confirmed by sequencing a portion of the beta subunit of the RNA polymerase (rpoB) gene. All isolates from South Africa clustered with a proposed novel Helicobacter strain (accession number AF237612) isolated in Australia, while three H. fennelliae type strains from the northern hemisphere, NCTC 11612, LMG 7546 and CCUG 18820, formed a separate branch. A large (355bp) highly conserved intervening sequence (IVS) in the 16S rRNA was found in all isolates. Predicted secondary structures of the IVS from the 16S rRNA and 23S rRNA were characterised by a primary stem structure formed by base pairing of the 3 and 5 ends and internal loops and stems. This phylogenetic analysis is the largest undertaken of H. fennelliae. The South African H. fennelliae isolates are closely related to an Australian isolate previously reported to be a possible novel species of Helicobacter. This study suggests that the latter is strain of H. fennelliae.
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    Multilocus sequence typing methods for the emerging Campylobacter species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus
    (Frontiers Media, 2012) Miller, William G.; Lastovica, Albert J.
    Multilocus sequence typing (MLST) systems have been reported previously for multiple food - and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.