Genomes of Vibrio cholerae O1 Serotype Ogawa Associated with Current Cholera Activity in Pakistan.

Here, this report presents two genomes of Vibrio cholerae O1 serotype Ogawa, recovered from cholera cases in Australia linked to travel to Pakistan in 2022. Their multidrug-resistant genotype represents the current activity of cholera within the seventh pandemic. One of the genome sequences was assembled using both short- and long-read sequences.

Genome Sequences of Enteroinvasive Escherichia coli Sequence Type 6, 99, and 311 Strains Acquired in Asia Pacific.

Laboratory diagnosis of enteroinvasive Escherichia coli (EIEC) remains difficult and limits the availability of EIEC genomes to the research community. We report the draft genome sequences of three EIEC strains which represent three distinct sequence types and serotypes circulating in Asia Pacific and causing enterocolitis in humans.

Novel multiplex PCR assay for identification and subtyping of enteroinvasive Escherichia coli and differentiation from Shigella based on target genes selected by comparative genomics.

PURPOSE: Both Shigella and enteroinvasive Escherichia coli (EIEC) can cause enterocolitis, but they have a distinct epidemiology and public health relevance. Current culture-independent testing (CIT) methods to identify Shigella in faecal samples rely on the ipaH gene as the target, which is also found in EIEC genomes. The aim of this study was to design an assay that can identify EIEC in cultures from CIT ipaH-positive samples. METHODOLOGY: Shigella and EIEC genomes were screened to find unique regions present in EIEC genomes using a comparative genomics approach and differentiating genetic loci that are suitable PCR targets were identified. The primers for these loci were designed and tested in 6501 and 104 genomes of Shigella and EIEC, respectively. RESULTS: An assay with two sets of multiplex PCR reactions that differentiates Shigella and EIEC based on the presence/absence of at least two out of six loci was developed and evaluated. The majority of Shigella genomes lacked all six loci, while at least two loci were present in most EIEC genomes. This assay successfully differentiated clinical EIEC from Shigella with a limit of detection of 105 cells ml-1. The sensitivity and specificity were over 95 and 99%, respectively. The assay can further subtype EIEC genomes into their genetic lineages. CONCLUSION: This new highly specific assay can assist in the identification of EIEC in ipaH PCR-positive samples and augment the public health laboratory surveillance of EIEC and shigellosis.

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance.

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing. Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing. Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing. In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

Draft genome comparison of representatives of the three dominant genotype groups of dairy Bacillus licheniformis strains.

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.

Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions.

Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products.

Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.

Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.