Identifying useful info from MySQL row-based binary logs
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To overcome some of the deficiencies with current molecular typing schema for Campylobacter spp. We investigated the distribution of 68 gene targets in 58 Campylobacter 5001 binary strains, one Campylobacter lari strain, and two Campylobacter coli strains for this purpose. Gene targets were selected on the basis of distribution in multiple genomes or plasmids, and known or putative status as an epidemicity factor.
We identified 18 gene targets that conferred the same level of discrimination as the 68 initially examined. We conclude that P-BIT is a useful approach for subtyping, offering advantages of speed, cost, and potential for strain risk ranking unavailable from current molecular typing schema for Campylobacter spp.
Campylobacter 5001 binary, particularly C. The sheer scale of infection makes concerted epidemiological studies difficult, as does the extremely wide distribution of the organism, found in all major avian and mammalian food animals, 5001 binary products, and indeed environments.
Moreover, 5001 binary Campylobacter spp. The poor discrimination of phenotypic typing methods led to intense developments in molecular epidemiological tools for more accurate data. Although a wide range of genotypic methods have been described 47two methods are now more commonly used by laboratories worldwide. The availability of standardized protocols for macrorestriction profiling with pulsed-field gel electrophoresis PFGE and multilocus sequence typing MLST have facilitated major contributions to our understanding of the epidemiology of these bacteria.
Nonetheless, issues remain, notably relating to the speed, cost, and ease of data analysis from these methods. Furthermore, although MLST has proven useful in evaluating the original host of a given 5001 binary, no current methods provide information on the relative risk to human health from individual strains.
Various studies, including those identifying stable clones found in humans and various animals as well as strain types only in a particular animal host 513384861and whole-genome microarray-based comparisons revealing a correlation between genome content and stress survival 46 indicate that not all 5001 binary are of equal risk to humans. In this study, we designed a range of specific PCR assays and investigated the distribution of 68 genes associated with epidemicity factors in C.
Between 5 and 10 colonies were transferred to a 1. DNA was diluted in sterile Milli-Q water as required. Risk-based binary typing of isolates. We chose predominantly those genes implicated as markers of epidemicity that were overrepresented in genotypes associated with human illness 14141617202226 - 3135 5001 binary 3743 5001 binary, 444649 5001 binary, 505253596064656769 - 71 or associated with virulence factors that were not components of the core genome 333460to evaluate a 5001 binary for subtyping that could 5001 binary be related to the risk to human health from individual strains.
These genes were generally involved in cell surface, mobility, and toxin production. Primers were designed using Primer3 58 via Geneious Pro 10 ; available from http: Primers were designed to produce PCR products with sizes and melting temperatures that will allow for easy multiplexing in the future. In addition, one primer set Cj designed by Price and colleagues 56 was also included in the risk-based binary typing system.
The target genes and primer sequences are listed in Table 2. Interstrain relationships were assessed by numerical analysis of the P-BIT data using the simple matching coefficient and 5001 binary clustering.
The Penner serotyping system was 5001 binary to determine the heat-stable serotypes of the isolates using the passive hemagglutination technique and antisera produced in house according to 5001 binary method of Penner and Hennessy Samples were digested using SmaI and KpnI.
Novel alleles and sequence types ST were submitted for allele and ST and clonal complex CC designations when appropriate.
Sixty-eight PCRs were designed for 67 prospective risk-based binary typing genes. Two primer sets for 5001 binary were designed using the sequence from positions 81 to accession no. Both sequences aligned only with C. Four targets cheWcftpglB -g, and p19 were positive for all C. One target csrA was positive for all C. The remaining 49 targets were detected in at least 1, and at most 57, of the 58 C.
The risk-based binary typing system produced 47 types from 61 isolates with a diversity index 62 of A subset of 18 targets was selected to yield the same discriminatory potential as the entire scheme.
Their selection was based upon several criteria, including discriminatory potential among the strain set examined, position in the genome, and known or predicted function. Numerical analysis of P-BIT data. A dendrogram of the cluster analysis Fig. Cluster 5001 binary comprised the two C. Cluster 2 contained 13 C. Cluster 3 contained 13 isolates assigned to CC 45 and in which each of these types clustered closely with isolates unassigned 5001 binary a CC and one isolate SVS for which MLST data was not available.
Cluster 4 contained six isolates, three of which were unassigned to a CC and one each of CC, and Cluster 5 was represented by a single isolate of CC Cluster 6 comprised three 5001 binary unassigned to any CC.
Cluster analysis of P-BIT 5001 binary based on 68 gene targets using the simple matching coefficient and Ward's clustering. Cluster analysis of P-BIT data based on the suggested minimal set of 18 gene targets using the simple matching coefficient and 5001 binary clustering.
A minimum spanning tree was prepared for the target P-BIT subset using the binary coefficient, maximum neighbor distance of 2 changes, and minimum size of 2 types. The tree is displayed in Fig.
Six clusters were revealed. Cluster 3 comprised isolates of CC 48, 52, and C. Cluster 4 contained isolates of CC and one unassigned isolate. Clusters 6 and 7 contained only isolates that had not been assigned a CC.
Minimum spanning tree based on 18 P-BIT targets. The tree was created using BioNumerics 5. The MST is presented with logarithmic branch lengths; complexes are shaded based on a maximum neighbor distance of 5001 binary changes 5001 binary minimum of 2 types. Thick lines between nodes indicate one change, thinner lines two changes, dark dotted lines three to four changes, and gray 5001 binary lines five or more changes.
Mix 1 contains ST 5001 binaryand mix 2 contains ST 21 and The wide range of sources and wide variation in detection rates between target genes in this study make investigation of source 5001 binary problematic. However, we do not know how many of the human isolates were derived from diarrheal cases that arose from consumption of contaminated poultry but assume the proportion is not insignificant In our laboratory, Penner serotyping is only available for C.
Three isolates were untypeable by Penner serotyping, 5001 binary the remaining 55 isolates had 22 Penner serotypes and a diversity index of Combining SmaI and KpnI produced 54 types and a diversity index of The 59 isolates analyzed by 5001 binary produced 32 sequence types and a diversity index of Typing 5001 binary for microorganisms may be assessed by a number of criteria to establish their fitness for purpose 68 ; R. We found KpnI-based PFGE typing to be the most discriminatory of the methods used in our study, notwithstanding that not all strains prove typeable with this enzyme Although the more widespread use of standardized methodologies for PFGE-based typing of bacterial pathogens has proven valuable for epidemiological studies 211181932 5001 binary, determining and using normalization parameters to enable a meaningful comparison of PFGE macrorestriction profiles can be challenging.
Furthermore, the PFGE apparatus is of moderate cost. The increased availability of DNA sequencing facilities has made MLST a popular 5001 binary and offers excellent portability, enabling researchers to readily compare their data with results obtained worldwide.
The MLST approach also offers phylogenetically meaningful data that have proven invaluable for population genetic 641 and source attribution 1342 studies. However, while technological advances continue to reduce the cost of performing the analysis, MLST still requires substantive capital outlay for implementation and running costs can be prohibitive for small routine laboratories.
Our P-BIT approach 5001 binary only the most basic molecular biology laboratory equipment for implementation approximately one-tenth the cost of PFGE equipment, for example and no more than e-mail for the exchange of data between laboratories. The PCRs used have been designed to enable multiplexing, to further reduce running costs.
5001 binary have identified a core set of 18 gene targets that conferred the same discriminatory potential in this study as the complete range of 68 targets employed. We believe these features, together with its high discriminatory power and complete typeability, render P-BIT a worthy tool for laboratories engaged in epidemiological 5001 binary of C.
The distinctiveness of profiles obtained from the few C. The P-BIT approach to 5001 binary yields a simple binary code albeit one based on whole-genomic 5001 binary that is not suited for analysis with algorithms aimed at predicting phylogenetic relationships.
The bases for determining strain relatedness with MLST and P-BIT are 5001 binary very different, with the former evaluating change between housekeeping gene sequences and the latter determining, by PCR, the presence of diverse, 5001 binary distributed genes that may be subject to selective pressure.
Nevertheless, some congruence between these methods was seen when comparing the interstrain relationships inferred. With the population genetic structure of C. As with P-BIT, the data are not aimed at determining strain phylogeny unless banding patterns 5001 binary indistinguishable, in which case strains are assumed to be related.
It has become common 5001 binary to confirm strain relationships inferred with one restriction enzyme with the use of a second Despite these caveats, we compared the clustering of C. One feature we had in mind when developing this approach was the possibility of quantifying the risk to human health from individual strains through generation of a 5001 binary binary code 5001 binary to 5001 binary factors. The selection of initial marker genes in this study was on a metagenomic basis but focused on genes that conferred, or were implicated in, some aspect of strain virulence.
We recognize that our P-BIT system uses PCR primers that are designed to amplify specific sequence orientations of the target genes and that 5001 binary may be present in particular strains with different flanking regions that would therefore not be 5001 binary. Thus, P-BIT data provides 5001 binary but not absolute data regarding the presence or absence of a gene.
Nevertheless, for the establishment of a simple, inexpensive, and effective typing method, we feel this is an acceptable 5001 binary. For genes carried on plasmids, there is a risk in long-term studies that strains will be cured of 5001 binary extrachromosomal DNA and thereby lose a specific marker. This appears to be 5001 binary case 5001 binary one strain examined here, 5001 binaryin which the plasmid-borne tet O 5001 binary conferring tetracycline resistance was not detected.
Our example of this strain was a kind gift from colleagues, and we have no 5001 binary on the number of passages it may have experienced before it was received. 5001 binary, the isolate's first description was in in another laboratory, and it is likely to 5001 binary been subcultured many times before we received it, allowing ample opportunity for the plasmid to be cured.
We examined a heated lysate of to ascertain if 5001 binary result may have been due to the DNA preparation method used being 5001 binary for genomic DNA extraction, but still obtained no tet O-derived amplicon data not shown. In the context of the P-BIT system, we do not consider the inclusion of tet O inappropriate since it represents an important virulence marker found in two of 5001 binary strains examined here: Further studies of the relationship between P-BIT profile, stress survival, and strain type frequency in human populations are also warranted.
We conclude that P-BIT is a useful approach for subtyping, offering advantages of speed, cost, and 5001 binary for strain risk ranking unavailable elsewhere. We hope the wider scientific community will be encouraged to investigate further its usage in national and 5001 binary surveillance studies and outbreak investigations.
We acknowledge Aruni Premaratane and Stephanie Brandt for technical assistance and Beverley Horn for statistical assistance. We also thank the anonymous reviewers for constructive comments on the manuscript.