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Rayner R.E.,Kelvin Institute | Savill J.,Public Health Microbiology Laboratory | Hafner L.M.,Queensland University of Technology | Huygens F.,Kelvin Institute
Future Microbiology | Year: 2015

Streptococcus pneumoniae is a potentially deadly human pathogen associated with high morbidity, mortality and global economic burden. The universally used bacterial genotyping methods are multilocus sequence typing and pulsed field gel electrophoresis. However, another highly discriminatory, rapid and less expensive genotyping technique, multilocus variable number of tandem repeat analysis (MLVA), has been developed. Unfortunately, no universal MLVA protocol exists, and some MLVA protocols do not amplify certain loci for all pneumococcal serotypes, leaving genotyping profiles incomplete. A number of other genotyping or characterization methods have been developed and will be discussed. This review examines the various protocols for genotyping S. pneumoniae and highlights the current direction technology and research is heading to understand this bacterium. © 2015 Future Medicine Ltd.

Rayner R.E.,Queensland University of Technology | Savill J.,Public Health Microbiology Laboratory | Hafner L.M.,Queensland University of Technology | Huygens F.,Queensland University of Technology
PLoS ONE | Year: 2015

Background: Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The 'gold standard' genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST. Methods: Four different MLVA protocols, including a modified method, were applied to 317 isolates of serotyped invasive S . pneumoniae isolated from sterile body sites of Queensland children under 15 years from 2007-2012. MLST was applied to 202 isolates for comparison. Results: The modified MLVA4 is significantly more discriminatory than the 'gold standard' MLST method. MLVA4 has similar discrimination compared to other MLVA techniques in this study). The failure to amplify particular loci in previous MLVA methods were minimised in MLVA4. Failure to amplify BOX-13 and Spneu19 were found to be serotype specific. Conclusion: We havemodified a highly discriminatory MLVA technique for genotyping Queensland invasive S. pneumoniae. MLVA4 has the ability to enhance our understanding of the pneumococcal epidemiology and the changing genetics of the pneumococcus in localised and short-term studies. © 2015 Rayner et al.

Bachmann N.L.,University of Queensland | Petty N.K.,University of Queensland | Petty N.K.,University of Technology, Sydney | Ben Zakour N.L.,University of Queensland | And 3 more authors.
BMC Genomics | Year: 2014

Background: Salmonella enterica subsp. enterica serovar Virchow has been recognized as a significant health burden in Asia, Australia and Europe. In addition to its global distribution, S. Virchow is clinically significant due to the frequency at which it causes invasive infections and its association with outbreaks arising from food-borne transmission. Here, we examine the genome of an invasive isolate of S. Virchow SVQ1 (phage type 8) from an outbreak in southeast Queensland, Australia. In addition to identifying new potential genotyping targets that could be used for discriminating between S. Virchow strains in outbreak scenarios, we also aimed to carry out a comprehensive comparative analysis of the S. Virchow genomes.Results: Genome comparisons between S. Virchow SVQ1 and S. Virchow SL491, a previously published strain, identified a high degree of genomic similarity between the two strains with fewer than 200 single nucleotide differences. Clustered Regularly Interspaced Palindromic Repeats (CRISPR) regions were identified as a highly variable region that could be used to discriminate between S. Virchow isolates. We amplified and sequenced the CRISPR regions of fifteen S. Virchow isolates collected from seven different outbreaks across Australia. We observed three allelic types of the CRISPR region from these isolates based on the presence/absence of the spacers and were able to discriminate S. Virchow phage type 8 isolates originating from different outbreaks. A comparison with 27 published Salmonella genomes found that the S. Virchow SVQ1 genome encodes 11 previously described Salmonella Pathogenicity Islands (SPI), as well as additional genomic islands including a remnant integrative conjugative element that is distinct from SPI-7. In addition, the S. Virchow genome possesses a novel prophage that encodes the Type III secretion system effector protein SopE, a key Salmonella virulence factor. The prophage shares very little similarity to the SopE prophages found in other Salmonella serovars suggesting an independent acquisition of sopE.Conclusions: The availability of this genome will serve as a genome template and facilitate further studies on understanding the virulence and global distribution of the S. Virchow serovar, as well as the development of genotyping methods for outbreak investigations. © 2014 Bachmann et al.; licensee BioMed Central Ltd.

Usta E.,Public Health Microbiology Laboratory | Erolu C.,Ondokuz Mayis University | Yanik K.,Ondokuz Mayis University | Karada A.,Ondokuz Mayis University | And 2 more authors.
Mikrobiyoloji Bulteni | Year: 2015

Stenotrophomonas maltophilia is an opportunistic emergent pathogen causing hospital-acquired infections. It is resistant to majority of the broad spectrum antibiotics due to several mechanisms which significantly limit the treatment options. Although the relationship between integrons, mobile genetic elements which play role in transferring resistance genes, and the antibiotic resistance in different gram- negative bacteria have been investigated, the data are limited in Turkey especially for S.maltophilia. The aims of this study were to detect the presence of different classes of integrons and plasmids in clinical isolates of S.maltophilia and to investigate the antibiotic resistance profiles of those isolates. One hundred S.maltophilia strains isolated from various clinical samples (32 sputum, 25 tracheal aspirates, 9 urine and blood, 7 exudates and catheters, 4 sterile body fluids and wounds, 2 CSF, 1 conjunctiva) in our microbiology laboratory during January 2011 -September 2012, were included in the study. The isolates were identified by VITEK2 Compact (BioMerieux, France) or Phoenix 100 (BD, USA) automatized systems, and the susceptibilities of the strains to levofloxacin, chloramphenicol, ceftazidime and trimethoprim/sulfamethoxazol (SXT) were evaluated via broth microdilution method according to the CLSI recommendations. Class 1 (intl-1), class 2 (intl-2), class 3 (intl-3) integron gene cassettes and inte- gron 5'-3' conserved gene regions (intl-5'-3'CS) were investigated by polymerase chain reaction (PCR) using specific primers in all of the strains. Nucleotide sequence analysis of PCR products was performed in case of positive result, and the presence and size of plasmids were further investigated. The susceptibility rates of S.maltophilia strains to ceftazidime, chloramphenicol, SXT and levofloxacin were found as 24%, 66%, 93% and 95%, respectively, while MIC50 and MIC90 values were 64-128 Mg/ml, 8-16 μg/ ml, 1/19-2/38 μg/ml and 1-2 μg/ml, respectively. In PCR amplification with intl-1, intl-2 and intl-3 primers, 12%, 2% and 10% of the isolates yielded expectative bands, respectively. DNA sequence analysis of the amplified products revealed five isolates to harbour intl-1 gene, while intl class 2 and class 3 genes were not detected in any of the strains. Furthermore in PCR amplification with intl-5'CS and 3'CS primers, 20% of the strains yielded expected bands. Sequence analysis of these amplicons revealed the presence of quaternary ammonium compound resistance protein genes (qacL) in two, aminoglycoside adenyltransferase gene (aadA) in one and integron-associated recombination site (attll) genes in five strains. Additionally, the presence of plasmids have been detected in 9 (9%) of the strains, however all of them was integron-negative. The sizes of plasmids were 2340, 1350, 2760, 18600, 20000, 3570-2540, 2510 and 5000-2540 base pairs, respectively. When the antibiotic susceptibility patterns of strains were compared with the presence of intl gene regions, no statistically significant relationship was observed (p> 0.05). In conclusion, the demonstration of integron class 1 genes and plasmids among clinical S.maltophilia strains is regarded as a warning data to indicate the potential for spread of those resistant strains in our hospital.

Li X.,U.S. Center for Disease Control and Prevention | Huang B.,Public Health Microbiology Laboratory | Eglezos S.,EML Consulting Services QLD | Graham T.,Public Health Microbiology Laboratory | And 2 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2013

Listeria monocytogenes is an important worldwide foodborne pathogen. For listeriosis outbreak investigations, an optimal multiple-locus variable-number-tandem-repeat analysis typing panel was identified from 4 previously reported schemes. The optimal combination of loci consisted of 9 loci (LMV6, LMV1, LMV2, Lm11, Lm10, LMV7, Lm32, LM-TR6, and Lm23), which produced the same level of differentiation ability Simpson index of 0.9914 as that of all 14 loci combined in the previous 4 reports. This panel provided higher differentiation ability than the individual of the 4 previously reported schemes, suggesting it would be useful for future surveillance and outbreak investigations. © 2013.

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