Studies on Prn Variation in the Mouse Model and Comparison with Epidemiological Data

The virulence factor pertactin (Prn) is a component of pertussis vaccines and one of the most polymorphic Bordetella pertussis antigens. After the introduction of vaccination shifts in predominant Prn types were observed and strains with the Prn vaccine type (Prn1) were replaced by strains carrying non-vaccine types (Prn2 and Prn3), suggesting vaccine-driven selection. The aim of this study was to elucidate the shifts observed in Prn variants. We show that, although Prn2 and Prn3 circulated in similar frequencies in the 1970s and 1980s, in the 1990s Prn2 strains expanded and Prn3 strains disappeared, suggesting that in vaccinated populations Prn2 strains are fitter than Prn3 strains. We established a role for Prn in the mouse model by showing that a Prn knock-out (Prn-ko) mutation reduced colonization in trachea and lungs. Restoration of the mutation resulted in a significant increase in colonization compared to the knock-out mutant. The ability of clinical isolates with different Prn variants to colonize the mouse lung was compared. Although these isolates were also polymorphic at other loci, only variation in the promoter for pertussis toxin (ptxP) and Prn were found to contribute significantly to differences in colonization. Analysis of a subset of strains with the same ptxP allele revealed that the ability to colonize mice decreased in the order Prn1>Prn2 and Prn3. Our results are consistent with the predominance of Prn1 strains in unvaccinated populations. Our results show that ability to colonize mice is practically the same for Prn2 and Prn3. Therefore other factors may have contributed to the predominance of Prn2 in vaccinated populations. The mouse model may be useful to assess and predict changes in the B. pertussis population due to vaccination

PFGE diversity within the methicillin-resistant Staphylococcus aureus clonal lineage ST398

<p>Abstract</p> <p>Background</p> <p>Livestock has recently been identified as a new reservoir of methicillin-resistant <it>Staphylococcus aureus </it>(MRSA). Most isolates belong to ST398 and are non-typeable with PFGE using <it>Sma</it>I, making it difficult to study transmission and outbreaks. Therefore, a new PFGE using <it>Cfr</it>9I, a neoschizomer of <it>Sma</it>I was optimized and evaluated to investigate ST398 isolates.</p> <p>Results</p> <p>After optimizing and evaluating the <it>Cfr</it>9I PFGE, clear and reproducible banding patterns were obtained from all previously non-typeable MRSA (NT_{<it>Sma</it>I}-MRSA) isolates. The PFGE patterns of ST398 isolates showed more diversity than with <it>spa</it>-typing and/or MLST. The PFGE results showed diversity within and between the two most prevalent <it>spa</it>-types of NT_{<it>Sma</it>I}-MRSA (t011 and t108). No match was found, when comparing banding patterns of the NT_{<it>Sma</it>I}-MRSA with 700 different PFGE types, obtained with <it>Sma</it>I digestion, in our database of more than 4000 strains. Furthermore, possible transmission among veterinarians and their family members was investigated and an outbreak of ST398 MRSA in a residential care facility was confirmed with the <it>Cfr</it>9I PFGE.</p> <p>Conclusions</p> <p>The adjusted PFGE can be used as a method for selecting important and distinct ST398 isolates for further research. The adjustments in the PFGE protocol using <it>Cfr</it>9I are easy to implement to study the ST398 clonal lineage in laboratories which already have a PFGE facility.</p

Towards standardisation:comparison of five whole genome sequencing (WGS) analysis pipelines for detection of epidemiologically linked tuberculosis cases

BackgroundWhole genome sequencing (WGS) is a reliable tool for studying tuberculosis (TB) transmission. WGS data are usually processed by custom-built analysis pipelines with little standardisation between them.AimTo compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases.MethodsFrom the Netherlands, 535 Mycobacterium tuberculosis complex (MTBC) strains from 2016 were included. Epidemiological information obtained from municipal health services was available for all mycobacterial interspersed repeat unit-variable number of tandem repeat (MIRU-VNTR) clustered cases. WGS data was analysed using five different pipelines: one core genome multilocus sequence typing (cgMLST) approach and four single nucleotide polymorphism (SNP)-based pipelines developed in Oxford, United Kingdom; Borstel, Germany; Bilthoven, the Netherlands and Copenhagen, Denmark. WGS clusters were defined using a maximum pairwise distance of 12 SNPs/alleles.ResultsThe cgMLST approach and Oxford pipeline clustered all epidemiologically linked cases, however, in the other three SNP-based pipelines one epidemiological link was missed due to insufficient coverage. In general, the genetic distances varied between pipelines, reflecting different clustering rates: the cgMLST approach clustered 92 cases, followed by 84, 83, 83 and 82 cases in the SNP-based pipelines from Copenhagen, Oxford, Borstel and Bilthoven respectively.ConclusionConcordance in ruling out epidemiological links was high between pipelines, which is an important step in the international validation of WGS data analysis. To increase accuracy in identifying TB transmission clusters, standardisation of crucial WGS criteria and creation of a reference database of representative MTBC sequences would be advisable

Multiple-Locus Variable Number Tandem Repeat Analysis of Staphylococcus Aureus: Comparison with Pulsed-Field Gel Electrophoresis and spa-Typing

(MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology

Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening

Findings. To facilitate automation, a novel DNA extraction method for MRSA was adopted. The MRSA specific chromosome-SCCmec PCR was adapted, additional primers were added, and the performance was validated. From various laboratories in The Netherlands we received a total of 86 MRSA clinical isolates, that were negative in commercially available tests. We identified 14 MRSA strains with new variant chromosome-SCCmec junctions by sequence analysis. These MRSA strains appeared to carry SCCmec sequences with a high degree of homology to SCC regions of S. epidermidis and S. haemolyticus. All were included for detection in chromosome-SCCmec based PCR. Background: Efficient management of Methicillin Resistant Staphylococcus aureus (MRSA) in the hospital is needed to prevent dissemination. It is important that MRSA can be rapidly identified, and effective infection control measures can be initiated. Equally important is a rapid MRSA negative report, especially for patients in isolation. For negative screening we implemented fully automated high through-put molecular screening for MRSA. Conclusions: Fourteen variant chromosome-SCCmec junctions in MRSA, that are not detected in commercially available MRSA detection kits were added to our PCR to detect all currently known variant SCC-mec types of MRSA