Abstract
Despite recent gains in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains an enigma. Aiming to understand if S. Typhi in the gallbladder are vital for transmission and/or adapted for long-term colonisation we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneous sequences from organisms isolated from the blood of acute patients.S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes but reflective of the general population. However, gallbladder isolates had a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p=2.8×10−9), respectively. This variation was associated with a higher prevalence of nonsense mutations in the gallbladder isolates in the predominant genotype. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Particularly, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to a 134Kb deletion. Long-term gallbladder carriage of S. Typhi results in atypically long branch lengths that can distinguish between carriage and acute infection. Our data strongly suggests typhoid carriers are unlikely to play a principal role in disease transmission in endemic settings, but that the hostile environment of the human gallbladder may generate new antigenic variants through immune selection.
Background
Typhoid fever, a potentially life-threatening systemic infection caused predominantly by the bacterium Salmonella enterica serovar Typhi (S. Typhi), remains a significant public health problem in resource-poor settings including parts of Asia and Africa 1. The disease is contracted via ingestion of contaminated food or water or through contact with individuals excreting the organism 2. The majority of typhoid patients fully recover with appropriate treatment; however, some individuals can become asymptomatic carriers and shed infectious bacteria in their faeces for an ill-defined period of time. Asymptomatic carriage of S. Typhi has been recognized as a public health threat for more than a century, with infamous typhoid carriers like Mary Mallon, a cook in New York, and Mr N, a milker in England, identified in the early part of the 20th century 3,4.
Typhoid carriage can be differentiated into three categories depending on the duration of shedding: convalescent (three weeks to three months), temporary (three to twelve months), and chronic (more than one year) 5. In endemic regions, an estimated 2-5 percent of acute typhoid patients become chronic carriers, meaning that they continue to intermittently shed the bacteria indefinitely after apparent clinical resolution 3,5. Consequently, chronic carriers are widely believed to be an ecological niche that facilitates the transmission and persistence of typhoid in human populations 6. S. Typhi is a human-restricted pathogen, meaning that the disease may be theoretically eliminated locally by reducing transmission through targeted treatment, improved sanitation, and mass vaccination. Consequently, understanding the role of chronic carriers in disease transmission, and detecting them prospectively, may accelerate disease elimination.
Despite substantive gains in understanding the biology of typhoid, we have generated limited new insights into typhoid carriage in recent decades. Data from murine models of Salmonella carriage and human clinical investigations have determined that the gallbladder is a key permissive niche for long-term bacterial persistence 7–13. Various epidemiological investigations have shown that gallstones and gallbladder damage may facilitate typhoid carriage 9,13–17, and that Salmonella preferentially attach to, and form biofilms on, cholesterol-rich gallstones 7,11,13,18,19. S. Typhi carriage isolates have been previously genetically compared with isolates from acute infection, with the aim of identifying signatures associated with carriage 20–23. However, these studies were unable to infer how carriage isolates directly relate to those causing acute disease.
It is apparent we need a better understanding of the role of the typhoid carrier and associated organisms to generate new approaches to the management of such individuals in endemic locations. Although it is widely accepted that that S. Typhi carriage play a key role in the transmission of typhoid in endemic settings it is unknown if carriage organisms are somehow adapted for long-term colonisation. Aiming to address this question, we performed whole genome sequencing and detailed genetic analyses on S. Typhi isolated from the gallbladders of typhoid carriers in Kathmandu. We compared these data to the sequences of contemporaneous organisms isolated from the blood of acutely infected patients in the same community over the same time period. Our data provides new insight into the role of typhoid carriage in disease transmission, showing that whilst carriage isolates are reflective of the general S. Typhi population circulating in the community, gallbladder carriage subjects organisms to immune pressures, which induces genetic variation and genomic degradation.
Results
The phylogenetic relationships between acute and gallbladder S. Typhi isolates
Between June 2007 and October 2010, we conducted a Salmonella carriage study in Kathmandu13. Patients undergoing cholecystectomy for acute or chronic cholecystitis were enrolled; bile and stool samples from 1,377 individuals were collected and subjected to microbiological examination. Twenty-four S. Typhi were isolated from bile samples taken from these patients and designated as gallbladder isolates. Ninety-six S. Typhi isolates recovered from patients with acute typhoid fever living in the same population over the same time period were used for comparison 24 (denoted as acute isolates) (Table S1). A phylogenetic analysis of these 120 S. Typhi isolates demonstrated that subclade 4.3.1 (H58) was the dominant genotype, constituting 62.5% (15/24) of all gallbladder isolates and 65.6% (63/96) of all acute isolates. The second most common genotype was 3.3.0 (H1), accounting for 12.5% (3/24) and 14.6% (14/96) of all gallbladder and acute isolates, respectively.
We identified a significant degree of genetic diversity within this collection of acute and carriage organisms, with multiple less-common genotypes co-circulating, included various clades (4.1, 3.1 and 2.2), subclades (3.2.2, 3.0.1, 2.2.2 and 2.2.1), and organisms within primary cluster 2 (Figure 1). The less common genotypes from the gallbladder fell within subclade 3.2.2 (8.3%; 2/24), 2.2.2 (4.2%; 1/24), clade 2.2 (8.3%; 2/24) and primary cluster 2 (4.2%; 1/24). Overall, gallbladder isolates were not significantly associated with subclade 4.3.1 in comparison with other genotypes (15/24 versus 9/24, p=0.083; Chi-squared test). These initial observations indicate that S. Typhi carriage was not restricted to any particular S. Typhi genotype; instead, the genotype distribution among gallbladder isolates generally reflected a genetic structure similar to that of the acute S. Typhi infections circulating in the community.
Antimicrobial susceptibility
We speculated resistance to key antimicrobials may facilitate the development of carriage. However, we found that the S. Typhi gallbladder isolates did not carry any obviously acquired AMR genes. However, chromosomal mutations associated with reduced susceptibility against fluoroquinolones were common. These fluoroquinolone resistance-associated mutations within the gallbladder organisms were more commonly observed in subclade 4.3.1 than in non-subclade 4.3.1 (73% (11/15) versus 11% (1/9), p=0.01; Chi-squared test). In comparing the respective gyrA mutation profiles between the acute and gallbladder isolates within subclade 4.3.1, we found that 76.2% (48/63) and 60% (9/15) carried the S83F mutation respectively, 7.9% (5/63) and 13.3% (2/15) carried the S83Y mutation, and 15.9% (10/63) and 26.7% (4/15) had no gyrA mutation. Consequently, there was no significant difference (p=0.327; Chi-squared test) in the presence of fluoroquinolone resistance-associated mutations between acute and gallbladder isolates within subclade 4.3.1.
Phylogenetic signatures of long-term Salmonella Typhi carriage
Despite the acute and gallbladder S. Typhi isolates generally clustering within the same genotypes across the phylogeny, we observed that a substantial proportion of the gallbladder isolates had higher genetic variability, which could be distinguished by long terminal branches (Figure 1). The median pairwise SNP distance of gallbladder isolates within subclade 4.3.1 was 21 SNPs (IQR: 12-24), which was significantly greater than that of the corresponding acute isolates (13 SNPs (IQR: 8-19 SNPs) (p=2.8×10−9, Wilcoxon rank-sum test) (Figure S2). Similarly, the median pairwise SNP distance of gallbladder isolates within subclade 3.3.0 (20 SNPs, IQR: 13-22 SNPs) was higher than that of acute isolates (13 SNPs, IQR: 4-15 SNPs) (p=0.26, Wilcoxon rank-sum test).
We mapped the contemporary acute and gallbladder S. Typhi sequences onto the global S. Typhi phylogeny, which indicated that the majority of these Nepalese acute and gallbladder S. Typhi isolates fell within known domestic genotypes, with limited evidence of importation from other countries (Figure S1). This observation suggests that the long terminal branches associated with gallbladder isolates were unlikely to be driven by the importation of these organisms from alternative countries.
We next estimated and plotted the nearest phylogenetic distances (NPDs) between each taxon and its nearest neighbour on the subclade 4.3.1 tree versus the year of isolation, the age of the individual from whom the organism was isolated, and the gyrA mutation profile. We hypothesized that the annual distribution of NPDs of S. Typhi acute isolates would represent the phylogenetic diversity (mutation accumulation) occurring annually via acute disease transmission and would be comparable over multiple years. Alternatively, we considered that S. Typhi in the gallbladder may develop characteristic adaptive mutations facilitating long-term persistence, causing them to gradually become increasingly distinct from contemporaneous acute isolates, leading to greater phylogenetic distances relative to their neighbours. In addition, given that all acute subclade 4.3.1 isolates here exhibited a gyrA mutation, the gallbladder subclade 4.3.1 isolates without a gyrA mutation were more likely to have colonized the gallbladder prior to nalidixic acid resistance becoming commonplace.
Our analyses showed that the average (±SD) NPD per year of acute subclade 4.3.1 isolates was comparable; specifically, 0.00163 (± 0.00202) substitutions/site (∼3.6 (± 4.4) SNPs) in 2008; 0.00110 (± 0.00229) substitutions/site (∼2.4 (± 5) SNPs) in 2009, and 0.00144 (± 0.00238) substitutions/site (∼3.2 (± 5.2) SNPs) in 2010. The majority of the subclade 4.3.1 gallbladder isolates (8/10) for which NPDs fell within the annual NPD distribution of acute subclade 4.3.1 isolates were associated with comparable terminal branch lengths and had a gyrA S83F mutation. Based on these findings, we surmised that gallbladder colonization with these isolates was likely to have occurred relatively recently in these individuals. Notable exceptions were two gallbladder isolates (GB266 and GB387) that did not possess a gyrA mutation and were associated with long terminal branches but had low NPDs as they clustered closely within the main phylogeny (Figures 1 and 2). Further, our data showed that all subclade 4.3.1 gallbladder isolates exhibiting abnormally high NPDs were associated with long terminal branches, indicative of chronic carriage (Figure 2). In particular, two subclade 4.3.1 gallbladder isolates (GB76 and GB441) lacked gyrA mutations, two others (GB003 and GB044) had gyrA S83Y mutations, and the remaining one (GB169) exhibited gyrA mutation S83F. With respect to the age distribution, typhoid carriers were significantly older (median age 36 years, range: 20-67) than patients with acute illness (median age 16 years, range: 0-31) (p=3.8×10−8, Wilcoxon rank-sum test). The gallbladder isolates thought to have originated from chronic carriers (based on above data) were obtained from individuals between aged between 27 and 40 years, which was older than the majority of the sampled acute typhoid patients; however, there was no significant difference in age distribution between those estimated to be recent and chronic carriers (Figure 2).
The genetic traits of Salmonella Typhi gallbladder isolates
To identify potentially adaptive mutations associated with typhoid carriage, all nonsynonymous SNPs (NSs) occurring exclusively within the S. Typhi gallbladder genome sequences were identified and grouped by their predicted or known function. A corresponding analysis was performed for all NSs in the acute S. Typhi isolates. A total of 228 gallbladder-specific NSs (212 missense and 16 nonsense mutations) and 469 acute-specific NSs (437 missense and 32 nonsense mutations) were identified. In general, there was no significant difference (p=0.924; Chi-square test) in the proportion of nonsense mutations out of total specific NSs in the gallbladder versus the acute isolates across all genotypes. However, among subclade 4.3.1 isolates, the proportion of nonsense mutations out of total specific NSs was significantly higher for gallbladder isolates than for acute isolates (10/60 compared to 2/67, Fisher exact test, p=0.01). These data suggest that gene degradation resulting from nonsense mutations was more common in the subclade 4.3.1 gallbladder isolates compared to the subclade 4.3.1 acute isolates.
Inactivated genes in the gallbladder isolates included genes involved in the synthesis of peptidoglycan (pbpC), vitamin B12 receptor (btuB), general stress response regulator (rpoS), a laterally acquired protein in SPI-7 (STY4562), membrane transport protein (STY3932), central metabolism (STY0230, ggt), hypothetical proteins (STY0929 and STY4178), and osmotically inducible lipoprotein E precursor (osmE) (Table 1).
Overall, the gallbladder- and acute-specific NSs across all genotypes could be grouped into 78 functional categories. The highest prevalence of these NSs was found in genes encoding hypothetical proteins, membrane lipoproteins, unknown functions, transport/binding proteins, SPI-7, general regulatory functions, surface polysaccharides and antigens, carbohydrate degradation, and DNA replication/modification. The proportions of NSs in SPI-7, surface polysaccharides and antigens, pathogenicity, cell envelope, anaerobic respiration, fatty acid biosynthesis and transport/binding proteins were higher in gallbladder than acute isolates (Figure 3). Notably, the data showed that the proportion of NSs in the viaB operon (encoding the Vi antigen, target of the typhoid conjugate vaccine (TCV)) was significantly higher in gallbladder isolates compared to the acute isolates across all genotypes (9/228 compared to 7/469, Chi-squared test, p=0.04). Similar results were obtained when considering only S. Typhi isolates belonging to subclade 4.3.1, with gallbladder isolates having more specific NSs in the viaB operon than the acute isolates (5/60 compared to 1/67, Fisher’s exact test, p=0.09). Additionally, we identified two gallbladder isolates (GB428 and GB003) that had lost the Vi capsular polysaccharide due to the deletion of the entire SPI-7 region (134kb).
Positive selection associated with typhoid carriage
Finally, we investigated signatures of positive selection by identifying analogous genetic variation detected in different gallbladder isolates. Among the gallbladder specific NSs, a number of different mutations were present in the same gene or the same biological pathway in at least two phylogenetically unlinked gallbladder isolates. For example, within the viaB operon, there were two NSs at codon 137 and 462 in the tviE gene (isolates GB580 and GB026) and six NSs in codons 166, 504, 506, 508, 665 and 752 in the tviD gene (isolates GB005, GB026, GB076, GB125 and GB281, respectively). Both genes facilitate the polymerization and translocation of the Vi capsule 25. Convergent NSs were also observed in the rpoS gene (sigma factor sigma-38) of isolates GB125 (nonsense mutation at codon 247) and GB705 (NSs at codon 94 and 250), which may impact general stress response and nutrient starvation. A further example was NSs at codon 59 and 230 in the degS gene (serine protease) (isolates GB005 and GB169). DegS is a component of the DegS-DegU two-component regulation system involved in expression of several degradative enzymes for salt stress responses and growth-limiting conditions. Additionally, three isolates (GB005, GB026 and GB705) each possessed an NS (codons 335, 406 and 946, respectively) in STY1242 (ptsG - glucose-specific PTS system IIBC component). PtsG enzyme is a component of the glucose-specific phosphotransferase system and plays a role in phosphorylation and translocation of glucose across the bacterial membrane, and is induced in carbon-limited conditions 26. NSs in several other genes were observed in >2 carriage isolates, including STY0429 (SbcC - exonuclease), STY0661 (dmsC - molybdopterin containing oxidoreductase membrane anchor subunit), STY1447 (putative ribulose-5-phosphate 3-epimerase) and STY2760 (ratA - putative exported protein) (Table 2).
Evidence of selective pressure on lipopolysaccharide
With respect to convergent mutations within the same biological pathways, there were a number of gallbladder-specific NSs involved in LPS O-antigen synthesis and modification; for example, an NS in the rfc gene (regulator of O-antigen polymerization) in isolate GB441, an NS in STY2629 (LPS modification acyltransferase) in isolate GB335, two NSs in rfbE (CDP-tyvelose-2-epimerase) and rfaG genes (LPS core biosynthesis protein) in isolate GB281, and three NSs in the rfbK (phosphomannomutase), manB (phosphomannomutase), and rfaD genes (ADP-L-Glycero-D-mannoheptose-6-epimease) in isolate GB026. RfbK and manB are both related to GDP-mannose synthesis for the LPS, and rfaD is an enzyme that catalyzes the conversion of ADP-D-glycerol-D-mannoheptose to ADP-L-glycerol-D-mannoheptose, a precursor for the synthesis of inner-core LPS.
Discussion
As stakeholders consider introduction of a new TCV into their national immunization programmes, research into the role of chronic carriers in bacterial persistence and disease transmission in endemic settings is needed to forecast the longer-term impact of vaccination on the transmission dynamics of typhoid and inform appropriate public health measures. However, epidemiological investigations of typhoid carriage are challenging, given that this population is problematic to identify and follow. Currently, the environmental factors driving the evolution of S. Typhi within the gallbladder are poorly understood and little is known about the adaptive mechanisms that may promote long-term survival. Our study is the largest genomic investigation of S. Typhi gallbladder carriage in a typhoid endemic setting, which allowed us to provide unprecedented insight into the genetic and phylogenetic signatures associated with typhoid carriage, but also to utilize these data to infer the potential role of typhoid carriage in disease transmission.
S1 Table. Gallbladder and acute Salmonella Typhi isolates and their associated metadata
Our data demonstrated, contrary to previous suggestions 27, that carriage of typhoid in the gallbladder was not restricted to any particular genotype and was associated a diverse range of bacterial genotypes, which largely mirrored the genetic structure of the bacterial population causing acute disease in Nepal. Further, typhoid carriage was not confined to specific AMR phenotypes, signifying that carriage is not associated with treatment failure with specific antimicrobials interacting with corresponding AMR profiles. However, by comparing the pairwise SNP distances between gallbladder and acute isolates within the same genotype, we found that gallbladder isolates displayed significantly greater genetic diversity compared to acute isolates, which suggests that long-term exposure to the gallbladder environment results in different accumulated adaptive mutations over time than would be generated in acute isolates. Our phylogenetic reconstruction of S. Typhi revealed that a number of gallbladder isolates had atypically long terminal branches, signifying that chronic carriage isolates may have a distinct phylogenetic signature which could be potentially utilized for the identification of organisms arising from chronic carriers. Further investigating this phenomenon, we found that the annual distribution of NPDs of acute isolates, which likely reflects mutation accumulation in the natural environment, was highly comparable across years and could be exploited to disaggregate recent carriers from longer-term carriers. If carriers are relevant, then we would predict they would be proportionally more important in causing acute disease in immunised populations with reduced environmental transmission. Therefore, we can use the annual NPD distribution to assess the impact of typhoid vaccination on disease transmission dynamics in endemic areas.
The role of chronic carriage in disease transmission represents one of the most long-standing questions in typhoid fever. Though typhoid carriers have been widely considered as an important source of infection, their exact contribution to transmission in endemic areas is not well understood. Previous molecular epidemiological studies in endemic regions highlighted an abundance of long-cycle environmental transmission in these settings, with a wide diversity of co-circulating bacterial genotypes isolated from acute typhoid patients 28–31, suggesting that person-to-person transmission makes a minimal contribution to new typhoid cases in an endemic area. Here, few gallbladder isolates clustered in close proximity or were directly linked with acute isolates and had long terminal branches. These observations suggest that these organisms play a negligible role in causing onward acute infections. Notably, none of the pre-surgical stool cultures from these patients undergoing cholecystectomy were positive for S. Typhi. However, the infectivity and transmission fitness of gallbladder isolates must be investigated further, as we cannot rule out the possibility that gallbladder isolates can become a more important source of infection when environmental transmission is successfully reduced. Further, the fact that gallbladder isolates display greater genetic variation than acute isolates implies that the gallbladder may act as an important ecological niche for generating novel genotypes.
By identifying NS mutations occurring specifically in gallbladder isolates and classifying them into predicted functional classes for comparison with those of acute isolates, we found that gene degradation by nonsense mutation was significantly higher in gallbladder compared to acute isolates within subclade 4.3.1. The effects of gene inactivation on phenotype, fitness and adaptation of carriage isolates inside the gallbladder are currently unknown. Further investigation of this phenomenon is necessary, as gene inactivation has been shown to be an important molecular mechanism in human adaptation in the evolutionary history of S. Typhi 32,33.
We additionally found evidence for the enrichment of NSs in genes encoding the Vi polysaccharide capsule in gallbladder isolates. The Vi antigen is immunogenic and anti-Vi antibody gradually wanes in acute typhoid patients after recovery, but can be detected in plasma from chronic carriers 34,35. Data from sero-surveillance studies for chronic carriage have commonly reported elevated anti-Vi antibodies in healthy individuals, which could be associated with carriage or repeated infections 36,37. Immunofluorescent staining of biofilms produced by S. Typhi on the surface of human gallstones demonstrated an abundance of Vi capsule on the surface of the colonising bacteria, suggesting that S. Typhi constitutively expresses Vi during carriage 19. The increased frequency of nonsynonymous mutations in the viaB operon (tviB, tviD and tviE) of gallbladder isolates, combined with high anti-Vi antibody titres in plasma 38 suggest that S. Typhi residing in the gallbladder are under sustained immune pressure. The observation that two gallbladder isolates lacked genes encoding proteins for Vi capsule biosynthesis again suggests that these were subject to selective pressure and that the loss of Vi may be an adaptive mechanism for long-term survival. The generation of Vi-negative S. Typhi may also question the possibility of their proliferation following mass immunization with TCV.
Identifying genes under selection among gallbladder isolates is crucial for understanding the evolutionary forces and bacterial adaptation to the gallbladder environment during carriage. Signatures of positive selection were detected in a number of genes containing differing gallbladder-specific NS mutations in at least two phylogenetically unlinked gallbladder isolates. Many of these genes are associated with gene regulation under stress and virulence gene expression. For example, the global regulatory gene rpoS is responsible for general stress responses and nutrient starvation, and regulates biofilm formation, colonization of Peyer’s patches, persistence in the spleen and the synthesis of Vi 39–41. The degS gene is involved in salt stress responses and growth-limiting conditions; STY1242 (ptsG - glucose-specific PTS system IIBC component) is activated during carbon starvation. These observations suggest that S. Typhi is exposed to a range of differing stressors within the gallbladder. Furthermore, the genes responsible for LPS biosynthesis had additionally accumulated NS mutations. LPS is the major component of the outer membrane of Gram-negative bacteria and represents one of the main factors contributing to bile salt resistance 42,43. LPS is also a key structural component of the biofilm extracellular matrix forming on human gallstones 19. The disruption of genes involved in LPS biosynthesis of S. Typhimurium may have a negative influence on biofilm production and attachment 44. The enrichment of NS mutations in genes involved in LPS biosynthesis and modification will lead to structural changes in LPS, which we predict will enhance bile resistance and biofilm formation.
This study has its limitations. The number of gallbladder and acute isolates was relatively small and thus might affect the interpretation of the phylogenetic distances between some of the gallbladder isolates and their nearest neighbour. Specifically, our ability to infer associations with uncommon genotypes was limited. Additionally, the identified phylogenetic signature inferred to be associated with carriage was not observed for all gallbladder isolates, due to an underrepresentation in the acutely infected population. Additionally, it was impossible to determine the duration of carriage to confirm our findings, as most typhoid carriers from our study do not recall a history of typhoid 13. However, our data suggest that the potential duration of carriage within our gallbladder isolates was variable, which led to variable terminal branch lengths. Despite these limitations, our study is unique and opens up new possibilities for evaluating associations between gallbladder-specific genetic variation and phenotypic differences to better understand the biology of this infectious disease paradox.
Conclusions
We conclude that typhoid carriage is not associated with any specific genotype nor driven by AMR phenotypes. However, we show that long-term gallbladder carriage results in atypically long phylogenetic branch lengths that can be used to distinguish between carriage and acute infection. Additionally, we found evidence that typhoid carriers are unlikely to play a major role in disease transmission in endemic settings such as Kathmandu, and long-cycle transmission is the primary driver of disease transmission in highly endemic settings. Public health efforts should continue to focus on providing people with safe water and promoting safe food handling and the introduction of TCV to interrupt environmental transmission in endemic settings. It remains important to further investigate the epidemiology, genomics, biology and public health impacts of carriage in parallel to the deployment of these public health measures. The role of carriers may become increasingly important as we move toward eradication, especially as immune selection appears to play a critical role in gallbladder colonisation.
Methods
Sampling
Between June 2007 and October 2010, we conducted a Salmonella carriage study at Patan Hospital in Kathmandu13. In brief, patients undergoing cholecystectomy for acute or chronic cholecystitis were enrolled; bile and stool samples from these patients were subjected to microbiological examination. S. Typhi were isolated from bile samples taken from these patients (referred to as gallbladder isolates). Additionally, S. Typhi isolates recovered from patients with acute typhoid fever living in the same population recruited into a randomized controlled trial were used for a comparison 24 (referred to as acute isolates) (Table S1).
Bacterial isolation and antimicrobial susceptibility testing
Bile and stool were collected from all cholecystectomy patients for culture. Bile was inoculated into equal volumes of Selenite F broth and Peptone broth and incubated at 37°C overnight. Broth was subcultured onto MacConkey agar and Xylene Lysine Deoxycholate (XLD) agar. After overnight incubation at 37°C, the plates were examined for the growth of Gram-negative bacteria and colonies were identified by API20E (bioMerieux, France). S. Typhi were confirmed by slide agglutination using specific antisera (Murex Biotech, Biotech, England).
For the acute isolates, 5-10 ml of blood was taken from all patients with a clinical suspicion of typhoid fever and inoculated into media containing tryptone soya broth and sodium polyanethol sulphonate (up to 25mL). Blood culture bottles were incubated for up to seven days, with blind sub-cultures at 24 hours, 48 hours, and 7 days, or when the broth became cloudy on sheep blood, chocolate, and MacConkey agar. Presumptive Salmonella colonies were identified as above.
Antimicrobial susceptibility testing was performed by the modified Bauer-Kirby disc diffusion method with zone size interpretation based on CLSI guidelines 45. Etests® were used to determine MICs following the manufacturer’s recommendations (bioMérieux, France). Ciprofloxacin MICs were used to categorise S. Typhi isolates as susceptible (≤0.06 μg/mL), intermediate (0.12-0.5 μg/mL) and resistant (≥1 μg/mL) following CLSI guidelines 45.
Vi agglutination assay
Two gallbladder isolates of S. Typhi (GB003 and GB428) that lacked the Vi polysaccharide biosynthesis (viaB) operon were grown on LB agar plates supplemented with increasing concentrations (1mM, 85mM and 170mM) of NaCl. Vi agglutinations were performed on microscope slides by mixing 10μl of single colony suspensions with 50μl of Vi antisera (Murex Biotech, Biotech, England). Agglutination was recorded after gently agitating the slide for 1 minute. Two gallbladder isolates of S. Typhi (GB125 and GB169) containing the viaB operon were used as controls.
Whole genome sequencing and SNP analyses
Total genomic DNA from acute and gallbladder S. Typhi isolates was extracted using the Wizard Genomic DNA Extraction Kit (Promega, Wisconsin, USA) (Table S1). 50ng of genomic DNA was subjected to library preparation using the Nextera DNA library prep kit; whole genome sequencing (WGS) was performed on an Illumina MiSeq platform following the manufacturer’s recommendations to generate 250bp paired end reads.
Single nucleotide polymorphisms (SNPs) were called using previously described methods46. Briefly, all reads were mapped to the reference sequence of S. Typhi strain CT18 (Accession no: AL513382), plasmid pHCM1 (AL513383) and pHCM2 (AL513384) using SMALT (version 0.7.4). Candidate SNPs were called against the reference sequence using SAMtools and filtered with a minimal mapping quality of 30 and a quality ratio cut-off of 0.75. The allele at each locus in each isolate was determined by reference to the consensus base in that genome. This process was performed using samtools mpileup and by removing low confidence alleles with consensus base quality ≤20, read depth ≤5 or heterozygous base calls. SNPs in phage regions, repetitive sequences or recombinant regions were excluded, 47,48 which resulted in a final set of 2,186 chromosomal SNPs. SNPs were subsequently annotated using the parseSNPTable.py script in the RedDog pipeline (https://github.com/katholt/RedDog). From the identified SNPs in S. Typhi genomes, a subset of 68 were used to assign S. Typhi isolates to previously defined lineages according to the existing extended S. Typhi genotyping framework 49.
To identify the potential function of genes containing key SNPs, we investigated the known or predicted functions of the identified genes. We identified SNPs occurring exclusively in either acute or gallbladder isolates and genes containing these SNPs were grouped by their predicted or known function based on the S. Typhi functional classification scheme developed by the Sanger Institute (www.sanger.ac.uk) using the genome annotation of S. Typhi CT18 50.
The antimicrobial resistance (AMR) gene and plasmid contents of S. Typhi isolates were determined using a local assembly approach with ARIBA (Antimicrobial Resistance Identifier by Assembly) 51. Resfinder 52 and Plasmidfinder 53 were used as reference databases of antimicrobial resistance genes and plasmid replicons, respectively.
Phylogenetic analyses and pairwise SNP distance
A maximum likelihood phylogenetic tree was reconstructed from the SNP alignment of 120 S. Typhi isolates (an S. Paratyphi A isolate was included as an outgroup) using RAxML (version 8.2.8) with the generalized time-reversible model and a Gamma distribution to model the site-specific rate variation (GTR+Г). Support for the maximum likelihood (ML) tree was assessed via bootstrap analysis with 1,000 pseudoreplicates. Pairwise phylogenetic distances depicting the phylogenetic branch length separating each pair of taxa within subclade 4.3.1 (H58) were estimated using the function cophenetic in the ape package (v4.1) in R (v3.3.2). Phylogenetic distances between each taxon and its nearest neighbour on the phylogenetic tree of subclade 4.3.1 were plotted using ggplot2. To investigate the phylogenetic structure of acute and gallbladder S. Typhi isolates from Nepal in the global context, a second maximum likelihood tree was inferred from a separate alignment of 23438 SNPs identified from 120 Nepali S. Typhi along with 1820 globally representative S. Typhi described previously 54. A S. Paratyphi A isolate was included as an outgroup to root the tree. Support for this ML tree was assessed via 100 bootstrap replicates.
Pairwise genetic distances (the difference in the number of SNPs) within and between acute and gallbladder S. Typhi isolates were estimated from the SNP alignment using the ape (v4.1) and adegenet (v2.0.1) packages in R (v3.3.2). Pairwise SNP distances were extracted and plotted using the function pairDistPlot in the adegenet package. The Wilcoxon rank-sum test was used for testing the difference in the average pairwise SNP distances between groups.
Declarations
Ethics approval and consent to participate
This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the institutional ethical review boards of Patan Hospital, The Nepal Health Research Council and The Oxford University Tropical Research Ethics Committee (OXTREC, Reference number: 2108). All enrollees were required to provide written informed consent for the collection and storage of all samples and subsequent data analysis. In the case of those under 18 years of age, a parent or guardian was asked to provide written informed consent.
Consent for publication
Consent for publication was incorporated as a component of entrance into the study.
Availability of data and materials
The raw sequence data generated from this study are available in the European Nucleotide Archive (ENA) under the accession numbers described in Table S1.
Competing interests
The authors declare no competing interests.
Funding
This work was supported by a Wellcome senior research fellowship to Stephen Baker to (215515/Z/19/Z). DTP is funded as leadership fellow through the Oak Foundation. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Authors’ contributions
Conceptualization: SB
Formal analysis: PTD, NTVT, NTNT, MAR Provided samples: SD, AK, BB
Methodology: PTD, NTVT, NTNT, HNDT, SD, AK, MC, MAR
Writing original draft: DPT, MAR, SB Review and editing: DPT, MC, GD, MAR, SB
Read and approved final version of manuscript: PDT, NTVT, NTNT, HNDT, SD, AK, MC, BB, GD, MAR, SB
Acknowledgments
We wish to acknowledge all members of the enteric infections group at Oxford University Clinical Research Unit (OUCRU) in Vietnam and Nepal and the study team at Patan Hospital.
Footnotes
↵* Joint senior authors