Estimating the Burden of Paratyphoid A in Asia and Africa

Despite the increasing availability of typhoid vaccine in many regions, global estimates of mortality attributable to enteric fever appear stable. While both Salmonella enterica serovar Typhi (S. Typhi) and serovar Paratyphi (S. Paratyphi) cause enteric fever, limited data exist estimating the burden of S. Paratyphi, particularly in Asia and Africa.We performed a systematic review of both English and Chinese-language databases to estimate the regional burden of paratyphoid within Africa and Asia. Distinct from previous reviews of the topic, we have presented two separate measures of burden; both incidence and proportion of enteric fever attributable to paratyphoid. Included articles reported laboratory-confirmed Salmonella serovar classification, provided clear methods on sampling strategy, defined the age range of participants, and specified the time period of the study.A total of 64 full-text articles satisfied inclusion criteria and were included in the qualitative synthesis. Paratyphoid A was commonly identified as a cause of enteric fever throughout Asia. The highest incidence estimates in Asia came from China; four studies estimated incidence rates of over 150 cases/100,000 person-years. Paratyphoid A burden estimates from Africa were extremely limited and with the exception of Nigeria, few population or hospital-based studies from Africa reported significant Paratyphoid A burden.While significant gaps exist in the existing population-level estimates of paratyphoid burden in Asia and Africa, available data suggest that paratyphoid A is a significant cause of enteric fever in Asia. The high variability in documented incidence and proportion estimates of paratyphoid suggest considerable geospatial variability in the burden of paratyphoid fever. Additional efforts to monitor enteric fever at the population level will be necessary in order to accurately quantify the public health threat posed by S. Paratyphi A, and to improve the prevention and treatment of enteric fever.     

Differential Epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu,Nepal: A Matched Case Control Investigation in a Highly Endemic Enteric Fever Setting

Background

Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.

Methodology and principal findings

A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.

Conclusions and significance

Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.     

Evaluation of an Electricity-free,Culture-based Approach for Detecting Typhoidal Salmonella Bacteremia during Enteric Fever in a High Burden,Resource-limited Setting

Background

In many rural areas at risk for enteric fever, there are few data on Salmonella enterica serotypes Typhi (S. Typhi) and Paratyphi (S. Paratyphi) incidence, due to limited laboratory capacity for microbiologic culture. Here, we describe an approach that permits recovery of the causative agents of enteric fever in such settings. This approach involves the use of an electricity-free incubator based upon use of phase-change materials. We compared this against conventional blood culture for detection of typhoidal Salmonella.

Methodology/Principal Findings

Three hundred and four patients with undifferentiated fever attending the outpatient and emergency departments of a public hospital in the Kathmandu Valley of Nepal were recruited. Conventional blood culture was compared against an electricity-free culture approach. Blood from 66 (21.7%) patients tested positive for a Gram-negative bacterium by at least one of the two methods. Sixty-five (21.4%) patients tested blood culture positive for S. Typhi (30; 9.9%) or S. Paratyphi A (35; 11.5%). From the 65 individuals with culture-confirmed enteric fever, 55 (84.6%) were identified by the conventional blood culture and 60 (92.3%) were identified by the experimental method. Median time-to-positivity was 2 days for both procedures. The experimental approach was falsely positive due to probable skin contaminants in 2 of 239 individuals (0.8%). The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% (95% CI: 80.0%–97.0%) and 96.0% (92.7%–98.1%), respectively. After initial incubation, Salmonella isolates could be readily recovered from blood culture bottles maintained at room temperature for six months.

Conclusions/Significance

A simple culture approach based upon a phase-change incubator can be used to isolate agents of enteric fever. This approach could be used as a surveillance tool to assess incidence and drug resistance of the etiologic agents of enteric fever in settings without reliable local access to electricity or local diagnostic microbiology laboratories.     

East London Experience with Enteric Fever 2007-2012

Purpose

The clinical presentation and epidemiology for patients with enteric fever at two hospitals in East London during 2007–2012 is described with the aim to identify preventive opportunities and to reduce the cost of treatment.

Methods

A retrospective analysis of case notes from patients admitted with enteric fever during 2007 to 2012 with a microbiologically confirmed diagnosis was undertaken. Details on clinical presentation, travel history, demographic data, laboratory parameters, treatment, patient outcome and vaccination status were collected.

Results

Clinical case notes were available for 98/129 (76%) patients including 69 Salmonella enterica serovar Typhi (S. Typhi) and 29 Salmonella enterica serovar Paratyphi (S. Paratyphi). Thirty-four patients (35%) were discharged from emergency medicine without a diagnosis of enteric fever and then readmitted after positive blood cultures. Seventy-one of the 98 patients (72%) were UK residents who had travelled abroad, 23 (23%) were foreign visitors/new entrants to the UK and four (4%) had not travelled abroad. Enteric fever was not considered in the initial differential diagnosis for 48/98 (49%) cases. The median length of hospital stay was 7 days (range 0–57 days). The total cost of bed days for managing enteric fever was £454,000 in the two hospitals (mean £75,666/year). Median time to clinical resolution was five days (range 1–20). Seven of 98 (7%) patients were readmitted with relapsed or continued infection. Six of the 71 (8%) patients had received typhoid vaccination, 34 (48%) patients had not received vaccination, and for 31 cases (44%) vaccination status was unknown.

Conclusions

Further interventions regarding education and vaccination of travellers and recognition of the condition by emergency medicine clinicians in travellers to South Asia is required.     

Bloodstream Infection among Children Presenting to a General Hospital Outpatient Clinic in Urban Nepal

Background

There are limited data on the etiology and characteristics of bloodstream infections in children presenting in hospital outpatient settings in South Asia. Previous studies in Nepal have highlighted the importance of murine typhus as a cause of febrile illness in adults and enteric fever as a leading bacterial cause of fever among children admitted to hospital.

Methods

We prospectively studied a total of 1084 febrile children aged between 2 months and 14 years presenting to a general hospital outpatient department in Kathmandu Valley, Nepal, over two study periods (summer and winter). Blood from all patients was tested by conventional culture and by real-time PCR for Rickettsia typhi.

Results

Putative etiological agents for fever were identified in 164 (15%) patients. Salmonella enterica serovar Typhi (S. Typhi) was identified in 107 (10%), S. enterica serovar Paratyphi A (S. Paratyphi) in 30 (3%), Streptococcus pneumoniae in 6 (0.6%), S. enterica serovar Typhimurium in 2 (0.2%), Haemophilus influenzae type b in 1 (0.1%), and Escherichia coli in 1 (0.1%) patient. S. Typhi was the most common organism isolated from blood during both summer and winter. Twenty-two (2%) patients were PCR positive for R. typhi. No significant demographic, clinical and laboratory features distinguished culture positive enteric fever and murine typhus.

Conclusions

Salmonella infections are the leading cause of bloodstream infection among pediatric outpatients with fever in Kathmandu Valley. Extension of immunization programs against invasive bacterial disease to include the agents of enteric fever and pneumococcus could improve the health of children in Nepal.     

Multidrug-resistant Salmonella enterica serovar paratyphi A harbors IncHI1 plasmids similar to those found in serovar typhi

Salmonella enterica serovars Typhi and Paratyphi A cause systemic infections in humans which are referred to as enteric fever. Multidrug-resistant (MDR) serovar Typhi isolates emerged in the 1980s, and in recent years MDR serovar Paratyphi A infections have become established as a significant problem across Asia. MDR in serovar Typhi is almost invariably associated with IncHI1 plasmids, but the genetic basis of MDR in serovar Paratyphi A has remained predominantly undefined. The DNA sequence of an IncHI1 plasmid, pAKU_1, encoding MDR in a serovar Paratyphi A strain has been determined. Significantly, this plasmid shares a common IncHI1-associated DNA backbone with the serovar Typhi plasmid pHCM1 and an S. enterica serovar Typhimurium plasmid pR27. Plasmids pAKU_1 and pHCM1 share 14 antibiotic resistance genes encoded within similar mobile elements, which appear to form a 24-kb composite transposon that has transferred as a single unit into different positions into their IncHI1 backbones. Thus, these plasmids have acquired similar antibiotic resistance genes independently via the horizontal transfer of mobile DNA elements. Furthermore, two IncHI1 plasmids from a Vietnamese isolate of serovar Typhi were found to contain features of the backbone sequence of pAKU_1 rather than pHCM1, with the composite transposon inserted in the same location as in the pAKU_1 sequence. Our data show that these serovar Typhi and Paratyphi A IncHI1 plasmids share highly conserved core DNA and have acquired similar mobile elements encoding antibiotic resistance genes in past decades.     

Ultra-fast and sensitive detection of non-typhoidal Salmonella using microwave-accelerated metal-enhanced fluorescence ("MAMEF")

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1:1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids).     

25 Years after Vi Typhoid Vaccine Efficacy Study,Typhoid Affects Significant Number of Population in Nepal

Salmonella Typhi, first isolated in 1884, results in infection of the intestines and can end in death and disability. Due to serious adverse events post vaccination, whole cell killed vaccines have been replaced with new generation vaccines. The efficacy of Vi polysaccharide (ViPS) vaccine, a new generation, single-dose intramuscular typhoid vaccine was assessed in Nepal in 1987. However, despite the availability of ViPS vaccine for more than 25 years, Nepal has one of the highest incidence of typhoid fever. Therefore we collected information from hospitals in the Kathmandu Valley from over the past five years. There were 9901 enteric fever cases between January 2008 and July 2012. 1,881 of these were confirmed typhoid cases from five hospitals in the Kathmandu district. Approximately 70% of the cases involved children under 15 years old. 1281 cases were confirmed as S. Paratyphi. Vaccines should be prioritized for control of typhoid in conjunction with improved water and sanitation conditions in Nepal and in endemic countries of Asia and Africa.     

Genetic diversity of Salmonella Paratyphi A isolated from enteric fever patients in Bangladesh from 2008 to 2018

BackgroundThe proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh.Principal findingsWe conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms.SignificanceThis study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies.     

O:2-CRM197 Conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM₁₉₇, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM₁₉₇ as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.     

Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission

Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, human-restricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case-control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens.     

The Ecological Dynamics of Fecal Contamination and Salmonella Typhi and Salmonella Paratyphi A in Municipal Kathmandu Drinking Water

One of the UN sustainable development goals is to achieve universal access to safe and affordable drinking water by 2030. It is locations like Kathmandu, Nepal, a densely populated city in South Asia with endemic typhoid fever, where this goal is most pertinent. Aiming to understand the public health implications of water quality in Kathmandu we subjected weekly water samples from 10 sources for one year to a range of chemical and bacteriological analyses. We additionally aimed to detect the etiological agents of typhoid fever and longitudinally assess microbial diversity by 16S rRNA gene surveying. We found that the majority of water sources exhibited chemical and bacterial contamination exceeding WHO guidelines. Further analysis of the chemical and bacterial data indicated site-specific pollution, symptomatic of highly localized fecal contamination. Rainfall was found to be a key driver of this fecal contamination, correlating with nitrates and evidence of S. Typhi and S. Paratyphi A, for which DNA was detectable in 333 (77%) and 303 (70%) of 432 water samples, respectively. 16S rRNA gene surveying outlined a spectrum of fecal bacteria in the contaminated water, forming complex communities again displaying location-specific temporal signatures. Our data signify that the municipal water in Kathmandu is a predominant vehicle for the transmission of S. Typhi and S. Paratyphi A. This study represents the first extensive spatiotemporal investigation of water pollution in an endemic typhoid fever setting and implicates highly localized human waste as the major contributor to poor water quality in the Kathmandu Valley.     

Antimicrobial Resistance,Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata,India during 2009-2013

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (bla _TEM-1, catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.     

High-resolution genotyping of the endemic Salmonella Typhi population during a Vi (typhoid) vaccination trial in Kolkata

Background

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem especially in developing countries. Vaccines against typhoid are commonly used by travelers but less so by residents of endemic areas.

Methodology

We used single nucleotide polymorphism (SNP) typing to investigate the population structure of 372 S. Typhi isolated during a typhoid disease burden study and Vi vaccine trial in Kolkata, India. Approximately sixty thousand people were enrolled for fever surveillance for 19 months prior to, and 24 months following, Vi vaccination of one third of the study population (May 2003–December 2006, vaccinations given December 2004).

Principal Findings

A diverse S. Typhi population was detected, including 21 haplotypes. The most common were of the H58 haplogroup (69%), which included all multidrug resistant isolates (defined as resistance to chloramphenicol, ampicillin and co-trimoxazole). Quinolone resistance was particularly high among H58-G isolates (97% Nalidixic acid resistant, 30% with reduced susceptibility to ciprofloxacin). Multiple typhoid fever episodes were detected in 22 households, however household clustering was not associated with specific S. Typhi haplotypes.

Conclusions

Typhoid fever in Kolkata is caused by a diverse population of S. Typhi, however H58 haplotypes dominate and are associated with multidrug and quinolone resistance. Vi vaccination did not obviously impact on the haplotype population structure of the S. Typhi circulating during the study period.     

Functional selection of a type IV pili-binding peptide that specifically inhibits Salmonella Typhi adhesion to/invasion of human monocytic cells

Salmonella enterica serovar Typhi (S. Typhi) is an important pathogen which infects humans exclusively and causes typhoid or enteric fever. Recently it has been discovered that type IVB pili, encoded by the S. Typhi pil operon located in the major pathogenicity island, may be important in the pathogenesis of epidemic enteric fever. To further investigate the roles of type IVB pili of S. Typhi, a 12-mer peptide (RQERSSLSKPVV), binding to the structural protein PilS of the type IVB pili of S. Typhi, was isolated with a ribosome display system. This peptide was designated as peptide R. We found that peptide R inhibited adhesion to/invasion of human monocytic THP-1 cells by piliated S. Typhi bacteria, but had no effects on nonpiliated S. Typhi bacteria. A random 12-mer peptide, of size and solubility equal to peptide R, served as a control on the specificity of peptide R. The specific interaction and binding equilibrium between the 12-mer peptide R and PilS protein was determined by isothermal titration calorimetry (ITC) and a binding constant Ka determined to be between 0.4 x 10(5) and 2.2 x 10(5)L mol(-1). Our findings suggest that the type IV pili-binding peptide R holds potential as an antibacterial peptide effective against S. Typhi infections, both in terms of prevention and therapeutic treatment. The data further provide insights into the understanding of the pathogenic roles of the type IVB pili of S. Typhi.     

Linkage of avian and reproductive tract tropism with sequence divergence adjacent to the 5S ribosomal subunit rrfH of Salmonella enterica

The 183 bp between the end of the 23S rrlH rRNA gene and the start of the 5S rrfH rRNA gene (ISR-1) and the 197 bp between the end of the rrfH rRNA gene and the start of the transfer RNA aspU (ISR-2) of Salmonella enterica ssp. enterica serotypes Enteritidis, Typhimurium, Pullorum, Heidelberg, Gallinarum, Typhi and Choleraesuis were compared. ISR-1s of D1 serotypes (Pullorum, Gallinarum and Enteritidis), B serotypes (Typhimurium and Heidelberg) and the C2 serotype Newport and the enteric fever pathogens serotype A Paratyphi and serotype D1 Typhi formed three clades, respectively. ISR-2 further differentiated the avian-adapted serotype Gallinarum from avian-adapted Pullorum and Salmonella bongori from S. enterica. The results suggest that serotypes Heidelberg and Choleraesuis share some evolutionary trends with egg-contaminating serotypes. In addition, ISR-1 and ISR-2 sequences that confirm serotype appear to be linked to clinically relevant host associations of the Salmonellae.     

Construction of an attenuated Salmonella enterica serovar Paratyphi A vaccine strain harboring defined mutations in htrA and yncD

The global epidemic features of enteric fever have changed greatly in recent years. The incidence of enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased. In some areas of Asia, infections with S. Paratyphi A have exceeded those with S. Typhi, resulting in S. Paratyphi A becoming the main causative agent of enteric fever. However, two currently licensed typhoid vaccines do not confer adequate cross‐protection against S. Paratyphi A infection. Therefore, development of specific vaccines against enteric fever caused by S. Paratyphi A is urgently needed. In the present study, an attenuated strain was constructed by double deletion of the htrA and yncD genes in a wild‐type strain of S. Paratyphi A and its safety and immunogenicity assessed. In a mouse model, the 50% lethal dose of the double deletion mutant and the wild‐type strain were 3.0 × 10⁸ CFU and 1.9 × 10³ CFU, respectively, suggesting that the double deletion resulted in remarkably decreased bacterial virulence. Bacterial colonization of the double deletion mutant in the livers and spleens of infected mice was strikingly less than that of the wild‐type strain. A single nasal administration of the attenuated vaccine candidate elicited high concentrations of anti‐LPS and anti‐flagellin IgG in a mouse model and protected immunized mice against lethal challenge with the wild‐type strain. Thus, our findings suggest that the attenuated vaccine strain is a promising candidate worthy of further evaluation both as a human enteric fever vaccine and as a vaccine delivery vector for heterologous antigens.     

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.     

Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray

The genus Salmonella consists of over 2,200 serovars that differ in their host range and ability to cause disease despite their close genetic relatedness. The genetic factors that influence each serovar's level of host adaptation, how they evolved or were acquired, their influence on the evolution of each serovar, and the phylogenic relationships between the serovars are of great interest as they provide insight into the mechanisms behind these differences in host range and disease progression. We have used an Salmonella enterica serovar Typhimurium spotted DNA microarray to perform genomic hybridizations of various serovars and strains of both S. enterica (subspecies I and IIIa) and Salmonella bongori to gain insight into the genetic organization of the serovars. Our results are generally consistent with previously published DNA association and multilocus enzyme electrophoresis data. Our findings also reveal novel information. We observe a more distant relationship of serovar Arizona (subspecies IIIa) from the subspecies I serovars than previously measured. We also observe variability in the Arizona SPI-2 pathogenicity island, indicating that it has evolved in a manner distinct from the other serovars. In addition, we identify shared genetic features of S. enterica serovars Typhi, Paratyphi A, and Sendai that parallel their unique ability to cause enteric fever in humans. Therefore, whereas the taxonomic organization of Salmonella into serogroups provides a good first approximation of genetic relatedness, we show that it does not account for genomic changes that contribute to a serovar's degree of host adaptation.     

Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal

AIMS: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal. METHODS AND RESULTS: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent. CONCLUSIONS: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.