Emergence of a multidrug-resistant and virulent Streptococcus pneumoniae lineage mediates serotype replacement after PCV13: an international whole-genome sequencing study.

BACKGROUND: Serotype 24F is one of the emerging pneumococcal serotypes after the introduction of pneumococcal conjugate vaccine (PCV). We aimed to identify lineages driving the increase of serotype 24F in France and place these findings into a global context. METHODS: Whole-genome sequencing was performed on a collection of serotype 24F pneumococci from asymptomatic colonisation (n=229) and invasive disease (n=190) isolates among individuals younger than 18 years in France, from 2003 to 2018. To provide a global context, we included an additional collection of 24F isolates in the Global Pneumococcal Sequencing (GPS) project database for analysis. A Global Pneumococcal Sequence Cluster (GPSC) and a clonal complex (CC) were assigned to each genome. Phylogenetic, evolutionary, and spatiotemporal analysis were conducted using the same 24F collection and supplemented with a global collection of genomes belonging to the lineage of interest from the GPS project database (n=25 590). FINDINGS: Serotype 24F was identified in numerous countries mainly due to the clonal spread of three lineages: GPSC10 (CC230), GPSC16 (CC156), and GPSC206 (CC7701). GPSC10 was the only multidrug-resistant lineage. GPSC10 drove the increase in 24F in France and had high invasive disease potential. The international dataset of GPSC10 (n=888) revealed that this lineage expressed 16 other serotypes, with only six included in 13-valent PCV (PCV13). All serotype 24F isolates were clustered in a single clade within the GPSC10 phylogeny and long-range transmissions were detected from Europe to other continents. Spatiotemporal analysis showed GPSC10-24F took 3-5 years to spread across France and a rapid change of serotype composition from PCV13 serotype 19A to 24F during the introduction of PCV13 was observed in neighbouring country Spain. INTERPRETATION: Our work reveals that GPSC10 alone is a challenge for serotype-based vaccine strategy. More systematic investigation to identify lineages like GPSC10 will better inform and improve next-generation preventive strategies against pneumococcal diseases. FUNDING: Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

Genomic adaptations of Campylobacter jejuni to long-term human colonization.

BACKGROUND: Campylobacter is a genus of bacteria that has been isolated from the gastrointestinal tract of humans and animals, and the environments they inhabit around the world. Campylobacter adapt to new environments by changes in their gene content and expression, but little is known about how they adapt to long-term human colonization. In this study, the genomes of 31 isolates from a New Zealand patient and 22 isolates from a United Kingdom patient belonging to Campylobacter jejuni sequence type 45 (ST45) were compared with 209 ST45 genomes from other sources to identify the mechanisms by which Campylobacter adapts to long-term human colonization. In addition, the New Zealand patient had their microbiota investigated using 16S rRNA metabarcoding, and their level of inflammation and immunosuppression analyzed using biochemical tests, to determine how Campylobacter adapts to a changing gastrointestinal tract. RESULTS: There was some evidence that long-term colonization led to genome degradation, but more evidence that Campylobacter adapted through the accumulation of non-synonymous single nucleotide polymorphisms (SNPs) and frameshifts in genes involved in cell motility, signal transduction and the major outer membrane protein (MOMP). The New Zealand patient also displayed considerable variation in their microbiome, inflammation and immunosuppression over five months, and the Campylobacter collected from this patient could be divided into two subpopulations, the proportion of which correlated with the amount of gastrointestinal inflammation. CONCLUSIONS: This study demonstrates how genomics, phylogenetics, 16S rRNA metabarcoding and biochemical markers can provide insight into how Campylobacter adapts to changing environments within human hosts. This study also demonstrates that long-term human colonization selects for changes in Campylobacter genes involved in cell motility, signal transduction and the MOMP; and that genetically distinct subpopulations of Campylobacter evolve to adapt to the changing gastrointestinal environment.

Genomic and phenotypic comparison of two Salmonella Typhimurium strains responsible for consecutive salmonellosis outbreaks in New Zealand.

Salmonella enterica serovar Typhimurium DT160 was the predominant cause of notified human salmonellosis cases in New Zealand from 2000 to 2010, before it was superseded by another S. Typhimurium strain, DT56 variant (DT56v). Whole genome sequencing and phenotypic testing were used to compare 109 DT160 isolates with eight DT56v isolates from New Zealand animal and human sources. Phylogenetic analysis provided evidence that DT160 and DT56v strains were distantly related with an estimated date of common ancestor between 1769 and 1821. The strains replicated at different rates but had similar antimicrobial susceptibility profiles. Both strains were resistant to the phage expressed from the chromosome of the other strain, which may have contributed to the emergence of DT56v. DT160 contained the pSLT virulence plasmid, and the sseJ and sseK2 genes that may have contributed to the higher reported prevalence compared to DT56v. A linear pBSSB1-family plasmid was also found in one of the DT56v isolates, but there was no evidence that this plasmid affected bacterial replication or antimicrobial susceptibility. One of the DT56v isolates was also sequenced using long-read technology and found to contain an uncommon chromosome arrangement for a Typhimurium isolate. This study demonstrates how comparative genomics and phenotypic testing can help identify strain-specific elements and factors that may have influenced the emergence and supersession of bacterial strains of public health importance.

Genomic Surveillance of a Globally Circulating Distinct Group W Clonal Complex 11 Meningococcal Variant, New Zealand, 2013-2018.

Genomic surveillance is an essential part of effective disease control, enabling identification of emerging and expanding strains and monitoring of subsequent interventions. Whole-genome sequencing was used to analyze the genomic diversity of all Neisseria meningitidis isolates submitted to the New Zealand Meningococcal Reference Laboratory during 2013-2018. Of the 347 isolates submitted for whole-genome sequencing, we identified 68 sequence types belonging to 18 clonal complexes (CC). The predominant CC was CC41/44; next in predominance was CC11. Comparison of the 45 New Zealand group W CC11 isolates with worldwide representatives of group W CC11 isolates revealed that the original UK strain, the 2013 UK strain, and a distinctive variant (the 2015 strain) were causing invasive group W meningococcal disease in New Zealand. The 2015 strain also demonstrated increased resistance to penicillin and has been circulating in Canada and several countries in Europe, highlighting that close monitoring is needed to prevent future outbreaks around the world.

Global Distribution of Invasive Serotype 35D Streptococcus pneumoniae Isolates following Introduction of 13-Valent Pneumococcal Conjugate Vaccine.

A newly recognized pneumococcal serotype, 35D, which differs from the 35B polysaccharide in structure and serology by not binding to factor serum 35a, was recently reported. The genetic basis for this distinctive serology is due to the presence of an inactivating mutation in wciG, which encodes an O-acetyltransferase responsible for O-acetylation of a galactofuranose. Here, we assessed the genomic data of a worldwide pneumococcal collection to identify serotype 35D isolates and understand their geographical distribution, genetic background, and invasiveness potential. Of 21,980 pneumococcal isolates, 444 were originally typed as serotype 35B by PneumoCaT. Analysis of the wciG gene revealed 23 isolates from carriage (n = 4) and disease (n = 19) with partial or complete loss-of-function mutations, including mutations resulting in premature stop codons (n = 22) and an in-frame mutation (n = 1). These were selected for further analysis. The putative 35D isolates were geographically widespread, and 65.2% (15/23) of them was recovered after the introduction of pneumococcal conjugate vaccine 13 (PCV13). Compared with serotype 35B isolates, putative serotype 35D isolates have higher invasive disease potentials based on odds ratios (OR) (11.58; 95% confidence interval[CI], 1.42 to 94.19 versus 0.61; 95% CI, 0.40 to 0.92) and a higher prevalence of macrolide resistance mediated by mefA (26.1% versus 7.6%; P = 0.009). Using the Quellung reaction, 50% (10/20) of viable isolates were identified as serotype 35D, 25% (5/20) as serotype 35B, and 25% (5/20) as a mixture of 35B/35D. The discrepancy between phenotype and genotype requires further investigation. These findings illustrated a global distribution of an invasive serotype, 35D, among young children post-PCV13 introduction and underlined the invasive potential conferred by the loss of O-acetylation in the pneumococcal capsule.

Long-term Colonization by Campylobacter jejuni Within a Human Host: Evolution, Antimicrobial Resistance, and Adaptation.

Background: Campylobacteriosis is inflammation of the gastrointestinal tract as a result of Campylobacter infection. Most campylobacteriosis cases are acute and self-limiting, with Campylobacter excretion ceasing a few weeks after symptoms cease. We identified a patient with fecal specimens positive for Campylobacter jejuni (ST45) intermittently during a 10-year period. Methods: Sixteen Campylobacter isolates were collected from the patient during 2006-2016. The isolates' genomes were sequenced to determine their relatedness, and their antimicrobial susceptibility patterns and motility were measured to determine the effects of antibiotic therapy and long-term excretion on the Campylobacter population. Results: Phylogenetic analyses estimated that the isolates shared a date of common ancestor between 1998 and 2006, coinciding with the onset of symptoms for the patient. Genomic analysis identified selection for changes in motility, and antimicrobial susceptibility testing suggested that the Campylobacter population developed resistance to several antibiotics coinciding with periods of antibiotic therapy. Conclusions: The patient was consistently colonized with organisms from a Campylobacter population that adapted to the internal environment of the patient. Genomic and phylogenetic analyses can give insight into a patient's infection history and the effect of antimicrobial treatment on Campylobacter populations in this unusual situation of long-term colonization of an individual.

Genomic Analysis of Salmonella enterica Serovar Typhimurium DT160 Associated with a 14-Year Outbreak, New Zealand, 1998-2012.

During 1998-2012, an extended outbreak of Salmonella enterica serovar Typhimurium definitive type 160 (DT160) affected >3,000 humans and killed wild birds in New Zealand. However, the relationship between DT160 within these 2 host groups and the origin of the outbreak are unknown. Whole-genome sequencing was used to compare 109 Salmonella Typhimurium DT160 isolates from sources throughout New Zealand. We provide evidence that DT160 was introduced into New Zealand around 1997 and rapidly propagated throughout the country, becoming more genetically diverse over time. The genetic heterogeneity was evenly distributed across multiple predicted functional protein groups, and we found no evidence of host group differentiation between isolates collected from human, poultry, bovid, and wild bird sources, indicating ongoing transmission between these host groups. Our findings demonstrate how a comparative genomic approach can be used to gain insight into outbreaks, disease transmission, and the evolution of a multihost pathogen after a probable point-source introduction.

M-Protein Analysis of Streptococcus pyogenes Isolates Associated with Acute Rheumatic Fever in New Zealand.

We applied an emm cluster typing system to group A Streptococcus strains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenic emm types but found a high proportion of emm types previously associated with pyoderma, further suggesting a role for skin infection in ARF.

Increasing incidence of invasive group A streptococcus disease in New Zealand, 2002-2012: a national population-based study.

OBJECTIVES: To analyse the incidence, demographics and molecular epidemiology of invasive group A streptococcal (GAS) disease in New Zealand between 2002 and 2012. METHODS: Using laboratory-based surveillance data, invasive GAS isolates were identified from the Institute of Environmental Science and Research, New Zealand. Hospitalization and mortality data were obtained from the New Zealand Ministry of Health. Molecular typing was performed by sequence analysis of the emm gene. RESULTS: The incidence of invasive GAS infections increased from 3.9 per 100,000 population in 2002 to 7.9 per 100,000 population (P < 0.001) in 2012. The incidence was highest in the over 75-year age group, and in Pacific peoples. There was temporal variation in emm types associated with invasive GAS disease, with emm1 being the overall predominant emm type. The diversity of emm types varied significantly according to ethnicity. Overall, 59% of GAS isolates were theoretically covered by an experimental M-protein vaccine. CONCLUSIONS: Our study provides valuable data on the epidemiology of invasive GAS disease in New Zealand, and represents one of the few studies to assess such longitudinal data across an entire nation. The increase in invasive GAS disease is concerning, and reasons for this should be explored further.

Survey of the bp/tee genes from clinical group A streptococcus isolates in New Zealand - implications for vaccine development.

Group A streptococcus (GAS) is responsible for a wide range of diseases ranging from superficial infections, such as pharyngitis and impetigo, to life-threatening diseases, such as toxic shock syndrome and acute rheumatic fever (ARF). GAS pili are hair-like extensions protruding from the cell surface and consist of highly immunogenic structural proteins: the backbone pilin (BP) and one or two accessory pilins (AP1 and AP2). The protease-resistant BP builds the pilus shaft and has been recognized as the T-antigen, which forms the basis of a major serological typing scheme that is often used as a supplement to M typing. A previous sequence analysis of the bp gene (tee gene) in 39 GAS isolates revealed 15 different bp/tee types. In this study, we sequenced the bp/tee gene from 100 GAS isolates obtained from patients with pharyngitis, ARF or invasive disease in New Zealand. We found 20 new bp/tee alleles and four new bp/tee types/subtypes. No association between bp/tee type and clinical outcome was observed. We confirmed earlier reports that the emm type and tee type are associated strongly, but we also found exceptions, where multiple tee types could be found in certain M/emm type strains, such as M/emm89. We also reported, for the first time, the existence of a chimeric bp/tee allele, which was assigned into a new subclade (bp/tee3.1). A strong sequence conservation of the bp/tee gene was observed within the individual bp/tee types/subtypes (>97 % sequence identity), as well as between historical and contemporary New Zealand and international GAS strains. This temporal and geographical sequence stability provided further evidence for the potential use of the BP/T-antigen as a vaccine target.

Same-day subtyping of Campylobacter jejuni and C. coli isolates by use of multiplex ligation-dependent probe amplification-binary typing.

Campylobacteriosis is the most commonly reported form of human bacterial gastroenteritis in the world. Sound identification of infectious sources requires subtyping, but the most widely used methods have turnaround times measured in days and require specialist equipment and skills. A multiplex ligation-dependent probe amplification-binary typing (MBiT) assay was developed for subtyping Campylobacter jejuni and Campylobacter coli. It was tested on 245 isolates, including recent isolates from Belgium and New Zealand, and compared to multilocus sequence typing (MLST). When used in an outbreak setting, MBiT identified the predominant genotype and possible additional cases days before pulsed-field gel electrophoresis (PFGE) results were available. MBiT was more discriminatory than MLST and, being a single assay with results produced within 6 h, was more rapid and cost-effective than both MLST and PFGE. In addition, MBiT requires only basic molecular biology equipment and skills.

Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery.

The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by both quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.

Whole-genome comparison of two Campylobacter jejuni isolates of the same sequence type reveals multiple loci of different ancestral lineage.

Campylobacter jejuni ST-474 is the most important human enteric pathogen in New Zealand, and yet this genotype is rarely found elsewhere in the world. Insight into the evolution of this organism was gained by a whole genome comparison of two ST-474, flaA SVR-14 isolates and other available C. jejuni isolates and genomes. The two isolates were collected from different sources, human (H22082) and retail poultry (P110b), at the same time and from the same geographical location. Solexa sequencing of each isolate resulted in ~1.659 Mb (H22082) and ~1.656 Mb (P110b) of assembled sequences within 28 (H22082) and 29 (P110b) contigs. We analysed 1502 genes for which we had sequences within both ST-474 isolates and within at least one of 11 C. jejuni reference genomes. Although 94.5% of genes were identical between the two ST-474 isolates, we identified 83 genes that differed by at least one nucleotide, including 55 genes with non-synonymous substitutions. These covered 101 kb and contained 672 point differences. We inferred that 22 (3.3%) of these differences were due to mutation and 650 (96.7%) were imported via recombination. Our analysis estimated 38 recombinant breakpoints within these 83 genes, which correspond to recombination events affecting at least 19 loci regions and gives a tract length estimate of ~2 kb. This includes a ~12 kb region displaying non-homologous recombination in one of the ST-474 genomes, with the insertion of two genes, including ykgC, a putative oxidoreductase, and a conserved hypothetical protein of unknown function. Furthermore, our analysis indicates that the source of this recombined DNA is more likely to have come from C. jejuni strains that are more closely related to ST-474. This suggests that the rates of recombination and mutation are similar in order of magnitude, but that recombination has been much more important for generating divergence between the two ST-474 isolates.

Species distribution of Burkholderia cepacia complex isolates in cystic fibrosis and non-cystic fibrosis patients in New Zealand.

Burkholderia cepacia complex (Bcc) isolates from 39 CF patients and 25 non-CF patients in New Zealand were speciated and characterised using the multilocus sequence typing (MLST) scheme for Bcc. B. multivorans predominated in CF patients (31/39, 79.5%) and in non-CF patients (7/25, 28%). Sequence types (ST) with an international distribution were identified (27/64, 42.2%) among the New Zealand Bcc isolates. MLST revealed a high level of diversity among Bcc isolates in CF patients indicating a lack of person-to-person transmission. Non-CF patients showed less diversity in MLST types, however, individuals with shared STs were geographically and chronologically separated. The use of MLST analysis allows continued surveillance of isolates with the potential to identify outbreaks. The identification of internationally distributed strains may provide an indicator of the relative transmissibility and infectivity of these strains and warrants further investigation.

Multilocus sequence typing of Campylobacter jejuni, and the correlation between clonal complex and pulsed-field gel electrophoresis macrorestriction profile.

The characterization of Campylobacter jejuni has been significantly improved by the use of multilocus sequence typing (MLST), which allows the relationship between isolates to be determined. The sequence types (STs) of 261 isolates of C. jejuni from New Zealand were determined. Isolates were obtained from a range of sources including chicken meat, cattle, pigs, duck, sheep, water and human infections. Thirty-two new alleles and 44 new STs were identified. Comparison of the MLST data and pulsed-field gel electrophoresis macrorestriction profiles showed that the macrorestriction profiles were good predictors of the clonal complex (CC) but not ST. All the major CCs identified elsewhere in the world were found in New Zealand as well as the association of certain CCs with particular animal niches. The majority of new STs identified were from river water isolates.

Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites.

Erythromycin-resistant isolates of Streptococcus pneumoniae from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for mef(A), but sequencing revealed that the ST9 clone possessed the mef(A) sequence commonly associated with Streptococcus pyogenes. All M phenotype isolates with this mef(A) sequence also had sequences consistent with the presence of the Tn1207.1 genetic element inserted in the celB gene. In contrast, isolates with the mef(E) sequence normally associated with S. pneumoniae contained sequences consistent with the presence of the mega insertion element. All MLS(B) isolates carried erm(B), and two isolates carried both erm(B) and mef(E). Fourteen of the 15 MLS(B) isolates were tetracycline resistant and contained tet(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained tet(M). MICs for isolates with the mef(A) sequence were significantly higher than MICs for isolates with the mef(E) sequence (P < 0.001). Thus, the ST9 clone of S. pneumoniae is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance.