- Volume 7, Issue 3, 2021

In 2018, a cluster of two cases of cryptococcosis occurred at the Queen Elizabeth University Hospital (QEUH) in Glasgow, Scotland (UK). It was postulated that these cases may have been linked to pigeon droppings found on the hospital site, given there have been previous reports of Cryptococcus neoformans associated with pigeon guano. Although some samples of pigeon guano taken from the site yielded culturable yeast from genera related to Cryptococcus, they have since been classified as Naganishia or Papiliotrema spp., and no isolates of C. neoformans were recovered from either the guano or subsequent widespread air sampling. In an attempt to further elucidate any possible shared source of the clinical isolates, we used whole-genome sequencing and phylogenetic analysis to examine the relationship of the two Cryptococcus isolates from the QEUH cases, along with two isolates from sporadic cases treated at a different Glasgow hospital earlier in 2018. Our work demonstrated that these four clinical isolates were not clonally related; while all isolates were from the VNI global lineage and of the same mating type (MATα), the genotypes of the two QEUH isolates were separated by 1885 base changes and belonged to different sub-lineages, recently described as the intercontinental sub-clades VNIa-93 and VNIa-5. In contrast, one of the two sporadic 2018 clinical isolates was determined to belong to the VNIb sub-lineage and the other classified as a VNIV/VNI hybrid. Our work demonstrated that the two 2018 QEUH isolates and the two prior C. neoformans clinical isolates were all genetically distinct. It was not possible to determine whether the QEUH genotypes stemmed from independent sources or from the same source, i.e. pigeons carrying different genotypes, but it should be noted that whilst members of allied genera within the Tremellomycetes were isolated from the hospital environment, there were no environmental isolations of C. neoformans.

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.

Whole genome sequencing (WGS) enables complete characterization of bacterial pathogenic isolates at single nucleotide resolution, making it the ultimate tool for routine surveillance and outbreak investigation. The lack of standardization, and the variation regarding bioinformatics workflows and parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing. The workflow supports three commonly used bioinformatics approaches for the detection of genes and alleles: alignment with blast+, kmer-based read mapping with KMA, and direct read mapping with SRST2. A collection of 131 STEC isolates collected from food and human sources, extensively characterized with conventional molecular methods, was used as a validation dataset. Using a validation strategy specifically adopted to WGS, we demonstrated high performance with repeatability, reproducibility, accuracy, precision, sensitivity and specificity above 95 % for the majority of all assays. The WGS workflow is publicly available as a ‘push-button’ pipeline at https://galaxy.sciensano.be. Our validation strategy and accompanying reference dataset consisting of both conventional and WGS data can be used for characterizing the performance of various bioinformatics workflows and assays, facilitating interoperability between laboratories with different WGS and bioinformatics set-ups.

Mycobacterium tuberculosis is a known human pathogen that causes the airborne infectious disease tuberculosis (TB). Every year TB infects millions of people worldwide. The emergence of multi-drug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis strains against the first- and second-line anti-TB drugs has created an urgent need for the development and implementation of new drug strategies. In this study, the complete genomes of 174 strains of M. tuberculosis are analysed to understand the evolution of molecular drug target (MDT) genes. Phylogenomic placements of M. tuberculosis strains depicted close association and temporal clustering. Selection pressure analysis by deducing the ratio of non-synonymous to synonymous substitution rates (dN/dS) in 51 MDT genes of the 174 M . tuberculosis strains led to categorizing these genes into diversifying (D, dN/dS>0.70), moderately diversifying (MD, dN/dS=0.35–0.70) and stabilized (S, dN/dS<0.35) genes. The genes rpsL, gidB, pncA and ahpC were identified as diversifying, and Rv0488, kasA, ndh, ethR, ethA, embR and ddn were identified as stabilized genes. Furthermore, sequence similarity networks were drawn that supported these divisions. In the multiple sequence alignments of diversifying and stabilized proteins, previously reported resistance mutations were checked to predict sensitive and resistant strains of M. tuberculosis . Finally, to delineate the potential of stabilized or least diversified genes/proteins as anti-TB drug targets, protein–protein interactions of MDT proteins with human proteins were analysed. We predict that kasA (dN/dS=0.29), a stabilized gene that encodes the most host-interacting protein, KasA, should serve as a potential drug target for the treatment of TB.

Species belonging to the family Lactobacillaceae are found in highly diverse environments and play an important role in fermented foods and probiotic products. Many of these species have been individually reported to harbour plasmids that encode important genes. In this study, we performed comparative genomic analysis of publicly available data for 512 plasmids from 282 strains represented by 51 species of this family and correlated the genomic features of plasmids with the ecological niches in which these species are found. Two-thirds of the species had at least one plasmid-harbouring strain. Plasmid abundance and GC content were significantly lower in vertebrate-adapted species as compared to nomadic and free-living species. Hierarchical clustering highlighted the distinct nature of plasmids from the nomadic and free-living species than those from the vertebrate-adapted species. EggNOG-assisted functional annotation revealed that genes associated with transposition, conjugation, DNA repair and recombination, exopolysaccharide production, metal ion transport, toxin–antitoxin system, and stress tolerance were significantly enriched on the plasmids of the nomadic and in some cases nomadic and free-living species. On the other hand, genes related to anaerobic metabolism, ABC transporters and the major facilitator superfamily were overrepresented on the plasmids of the vertebrate-adapted species. These genomic signatures correlate with the comparatively nutrient-depleted, stressful and dynamic environments of nomadic and free-living species and nutrient-rich and anaerobic environments of vertebrate-adapted species. Thus, these results indicate the contribution of the plasmids in the adaptation of lactobacilli to their respective habitats. This study also underlines the potential application of these plasmids in improving the technological and probiotic properties of lactic acid bacteria.

The mobilome plays a crucial role in bacterial adaptation and is therefore a starting point to understand and establish the gene flow occurring in the process of bacterial evolution. This is even more so if we consider that the mobilome of environmental bacteria can be the reservoir of genes that may later appear in the clinic. Recently, new genera have been proposed in the family Mycobacteriaceae , including the genus Mycolicibacterium , which encompasses dozens of species of agricultural, biotechnological, clinical and ecological importance, being ubiquitous in several environments. The current scenario in the Mycobacteriaceae mobilome has some bias because most of the characterized mycobacteriophages were isolated using a single host strain, and the few plasmids reported mainly relate to the genus Mycobacterium . To fill in the gaps in these issues, we performed a systematic in silico study of these mobile elements based on 242 available genomes of the genus Mycolicibacterium . The analyses identified 156 putative plasmids (19 conjugative, 45 mobilizable and 92 non-mobilizable) and 566 prophages in 86 and 229 genomes, respectively. Moreover, a contig was characterized by resembling an actinomycete integrative and conjugative element (AICE). Within this diversity of mobile genetic elements, there is a pool of genes associated with several canonical functions, in addition to adaptive traits, such as virulence and resistance to antibiotics and metals (mercury and arsenic). The type-VII secretion system was a common feature in the predicted plasmids, being associated with genes encoding virulent proteins (EsxA, EsxB, PE and PPE). In addition to the characterization of plasmids and prophages of the family Mycobacteriaceae , this study showed an abundance of these genetic elements in a dozen species of the genus Mycolicibacterium .

Research into the lower urinary tract (LUT) microbiota has primarily focused on its relationship to LUT symptoms (LUTS), taking snapshots of these communities in individuals with and without LUTS. While certain bacterial taxa have been associated with LUTS, or the lack thereof, the temporal dynamics of this community were largely unknown. Recently, we conducted a longitudinal study and found that vaginal intercourse resulted in a shift in species richness and diversity within the LUT microbiota. This is particularly relevant as frequent vaginal intercourse is a major risk factor for urinary tract infection (UTI) in premenopausal women (Aydin et al. Int Urogynecol J 2015;26:795–804). To further investigate the relationship between vaginal intercourse and LUT microbiota, here we present the results of a 3 week study in which daily urogenital specimens were collected from a female participant and her male sexual partner. Consistent with our previous findings, the LUT microbiota changed after vaginal intercourse, most notably a high abundance of Streptococcus mitis was observed post-coitus. We isolated and sequenced S. mitis from both sexual partners finding that: (i) the S. mitis isolates from the female partner’s urogenital tract were genomically similar throughout the duration of the study, and (ii) they were related to one isolate from the male partner’s oral cavity collected at the end of the study, suggesting transmission between the two individuals. We hypothesize that blooms in S. mitis after vaginal intercourse may play a role in coitus-related UTI. We found that a S. mitis isolate, in contrast to a Lactobacillus jensenii isolate displaced after vaginal intercourse, cannot inhibit the growth of uropathogenic Escherichia coli . Thus, this bloom in S. mitis may provide a window of opportunity for a uropathogen to colonize the LUT.

The vaginal microbiome plays an important role in human health and species of vaginal bacteria have been associated with reproductive disease. Strain-level variation is also thought to be important, but the diversity, structure and evolutionary history of vaginal strains is not as well characterized. We developed and validated an approach to measure strain variation from metagenomic data based on SNPs within the core genomes for six species of vaginal bacteria: Gardnerella vaginalis , Lactobacillus crispatus , Lactobacillus iners , Lactobacillus jensenii , Lactobacillus gasseri and Atopobium vaginae . Despite inhabiting the same environment, strain diversity and structure varies across species. All species except L. iners are characterized by multiple distinct groups of strains. Even so, strain diversity is lower in the Lactobacillus species, consistent with a more recent colonization of the human vaginal microbiome. Both strain diversity and the frequency of multi-strain samples is related to species-level diversity of the microbiome in which they occur, suggesting similar ecological factors influencing diversity within the vaginal niche. We conclude that the structure of strain-level variation provides both the motivation and means of testing whether strain-level differences contribute to the function and health consequences of the vaginal microbiome.

Staphylococcus aureus chronic airway infection in patients with cystic fibrosis (CF) allows this pathogen to adapt over time in response to different selection pressures. We have previously shown that the main sequence types related to community-acquired methicillin-resistant S. aureus (MRSA) infections in Argentina – ST5 and ST30 – are also frequently isolated from the sputum of patients with CF, but in these patients they usually display multi-drug antimicrobial resistance. In this study, we sequenced the genomes of MRSA from four paediatric CF patients with the goal of identifying mutations among sequential isolates, especially those possibly related to antimicrobial resistance and virulence, which might contribute to the adaptation of the pathogen in the airways of patients with CF. Our results revealed genetic differences in sequential MRSA strains isolated from patients with CF in both their core and accessory genomes. Although the genetic adaptation of S. aureus was distinct in different hosts, we detected independent mutations in thyA, htrA, rpsJ and gyrA – which are known to have crucial roles in S. aureus virulence and antimicrobial resistance – in isolates recovered from multiple patients. Moreover, we identified allelic variants that were detected in all of the isolates recovered after a certain time point; these non-synonymous mutations were in genes associated with antimicrobial resistance, virulence, iron scavenging and oxidative stress resistance. In conclusion, our results provide evidence of genetic variability among sequential MRSA isolates that could be implicated in the adaptation of these strains during chronic CF airway infection.

Background. Infections caused by carbapenem-resistant Acinetobacter baumannii (CR-Ab) have become increasingly prevalent in clinical settings and often result in significant morbidity and mortality due to their multidrug resistance (MDR). Here we present an integrated whole-genome sequencing (WGS) response to a persistent CR-Ab outbreak in a Brisbane hospital between 2016–2018.

Methods. A. baumannii, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa isolates were sequenced using the Illumina platform primarily to establish isolate relationships based on core-genome SNPs, MLST and antimicrobial resistance gene profiles. Representative isolates were selected for PacBio sequencing. Environmental metagenomic sequencing with Illumina was used to detect persistence of the outbreak strain in the hospital.

Results. In response to a suspected polymicrobial outbreak between May to August of 2016, 28 CR-Ab (and 21 other MDR Gram-negative bacilli) were collected from Intensive Care Unit and Burns Unit patients and sent for WGS with a 7 day turn-around time in clinical reporting. All CR-Ab were sequence type (ST)1050 (Pasteur ST2) and within 10 SNPs apart, indicative of an ongoing outbreak, and distinct from historical CR-Ab isolates from the same hospital. Possible transmission routes between patients were identified on the basis of CR-Ab and K. pneumoniae SNP profiles. Continued WGS surveillance between 2016 to 2018 enabled suspected outbreak cases to be refuted, but a resurgence of the outbreak CR-Ab mid-2018 in the Burns Unit prompted additional screening. Environmental metagenomic sequencing identified the hospital plumbing as a potential source. Replacement of the plumbing and routine drain maintenance resulted in rapid resolution of the secondary outbreak and significant risk reduction with no discernable transmission in the Burns Unit since.

Conclusion. We implemented a comprehensive WGS and metagenomics investigation that resolved a persistent CR-Ab outbreak in a critical care setting.

The Liverpool epidemic strain (LES) is an important transmissible clonal lineage of Pseudomonas aeruginosa that chronically infects the lungs of people with cystic fibrosis (CF). Previous studies have focused on the genomics of the LES in a limited number of isolates, mostly from one CF centre in the UK, and from studies highlighting identification of the LES in Canada. Here we significantly extend the current LES genome database by genome sequencing 91 isolates from multiple CF centres across the UK, and we describe the comparative genomics of this large collection of LES isolates from the UK and Canada. Phylogenetic analysis revealed that the 145 LES genomes analysed formed a distinct clonal lineage when compared with the wider P. aeruginosa population. Notably, the isolates formed two clades: one associated with isolates from Canada, and the other associated with UK isolates. Further analysis of the UK LES isolates revealed clustering by clinic geography. Where isolates clustered closely together, the association was often supported by clinical data linking isolates or patients. When compared with the earliest known isolate, LESB58 (from 1988), many UK LES isolates shared common loss-of-function mutations, such as in genes gltR and fleR. Other loss-of-function mutations identified in previous studies as common adaptations during CF chronic lung infections were also identified in multiple LES isolates. Analysis of the LES accessory genome (including genomic islands and prophages) revealed variations in the carriage of large genomic regions, with some evidence for shared genomic island/prophage complement according to clinic location. Our study reveals divergence and adaptation during the spread of the LES, within the UK and between continents.

Sequence similarity of pathogen genomes can infer the relatedness between isolates as the fewer genetic differences identified between pairs of isolates, the less time since divergence from a common ancestor. Clustering based on hierarchical single linkage clustering of pairwise SNP distances has been employed to detect and investigate outbreaks. Here, we evaluated the evidence-base for the interpretation of phylogenetic clusters of Shiga toxin-producing Escherichia coli (STEC) O157:H7. Whole genome sequences of 1193 isolates of STEC O157:H7 submitted to Public Health England between July 2015 and December 2016 were mapped to the Sakai reference strain. Hierarchical single linkage clustering was performed on the pairwise SNP difference between all isolates at descending distance thresholds. Cases with known epidemiological links fell within 5-SNP single linkage clusters. Five-SNP single linkage community clusters where an epidemiological link was not identified were more likely to be temporally and/or geographically related than sporadic cases. Ten-SNP single linkage clusters occurred infrequently and were challenging to investigate as cases were few, and temporally and/or geographically dispersed. A single linkage cluster threshold of 5-SNPs has utility for the detection of outbreaks linked to both persistent and point sources. Deeper phylogenetic analysis revealed that the distinction between domestic UK and imported isolates could be inferred at the sub-lineage level. Cases associated with domestically acquired infection that fall within clusters that are predominantly travel associated are likely to be caused by contaminated imported food.

Acinetobacter baumannii is a successful opportunistic pathogen that can compete for iron under iron-limiting conditions. Here, large novel transposons that carry genes for synthesis and transport of the fimsbactin siderophores present in some A. baumannii strains were examined. Tn6171, originally found in the A. baumannii global clone 1 (GC1) lineage 2 isolate D36, includes tns genes encoding proteins related to the TnsA, TnsB, TnsC transposition proteins (50–59 % identity), TnsD targeting protein (43 % identity) and TnsE (31 % identity) of Tn7, and is found in the chromosome downstream of the glmS gene, the preferred location for Tn7, flanked by a 5 bp target site duplication. Tn6171 is bounded by 29 bp inverted repeats and, like Tn7, includes additional TnsB binding sites at each end. Tn6171 or minor variants were detected in the equivalent location in complete or draft genomes of several further A. baumannii isolates belonging to GC1 [sequence type (ST) 1, ST81, ST94, ST328, ST623, ST717], GC2 (ST2) and ST10. However, in some of these isolates the surrounding glmS region was clearly derived from a different A. baumannii lineage, indicating that the transposon may have been acquired by replacement of a segment of the chromosome. A recombination-free phylogeny revealed that there were several transposon acquisition events in GC1. The GC1 isolates were mainly lineage 2, but a potential third lineage was also detected. A related transposon, designated Tn6552, was detected in ATCC 17978 (ST437) and other ST437 isolates. However, the Tn6552 tnsD targeting gene was interrupted by an ISAba12, and Tn6552 is not downstream of glmS.