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.     

伤寒沙门菌和甲型副伤寒沙门菌分离株的外膜蛋白谱比较

目的:比较伤寒沙门菌和甲型副伤寒沙门菌流行菌株的外膜蛋白谱差异。方法:运用二维蛋白电泳方法,对我国伤寒沙门菌株XJ90和甲型副伤寒沙门菌株JX2005-92在实验室通用营养条件下培养提取的外膜蛋白进行分离,比对其差异,对差异蛋白点进行质谱鉴定,对鉴定蛋白点的基因序列也进行比较。结果:菌株XJ90中发现20个特异蛋白点,质谱鉴定出16个;菌株JX2005-92中发现29个特异蛋白点,鉴定出18个。在这些蛋白中,OmpA是数目最多的同种差异蛋白。这些差异蛋白点中的大部分编码基因在2种细菌中序列高度相似或相同。结论:伤寒沙门菌和甲型副伤寒沙门菌基因序列高度相似的外膜蛋白具有不同的修饰形式,提示其不同遗传背景在相同的环境条件下表现出精细的功能差异。     

Identification of Immunogenic Salmonella enterica Serotype Typhi Antigens Expressed in Chronic Biliary Carriers of S. Typhi in Kathmandu,Nepal

Background

Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.

Methodology/Principal Findings

To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.

Conclusions/Significance

Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.     

Unsuitability of Quantitative Bacteroidales 16S rRNA Gene Assays for Discerning Fecal Contamination of Drinking Water

Bacteroidales species were detected in (tap) water samples from treatment plants with three different PCR assays. 16S rRNA gene sequence analysis indicated that the sequences had an environmental rather than fecal origin. We conclude that assays for Bacteroidales 16S rRNA genes are not specific enough to discern fecal contamination of drinking water in the Netherlands.Drinking water in many countries is routinely monitored for recent fecal contamination by testing for fecal indicator organisms Escherichia coli, thermotolerant coliforms, and/or intestinal enterococci to demonstrate microbial safety (13, 21, 42). Although these indicator organisms have been used for many decades, they have some limitations: the number of E. coli/coliform/enterococcus bacteria in feces is relatively low (18, 38), and they sometimes might be able to grow in the environment (10, 11, 14, 27). Consequently, scientists have been searching for alternative indicator organisms to determine fecal contamination of water. In 1967, bacteria belonging to the genus Bacteroides were suggested as alternative indicator organisms (26). Bacteroides spp. might have some advantages over the traditional indicator organisms. The numbers of Bacteroides spp. in the intestinal tract of humans and animals are 10 to 100 times higher than the numbers of E. coli or intestinal enterococci (1, 2, 12, 26). However, the use of Bacteroides spp. as indicator organisms was hampered by the complex cultivation conditions required (1, 2). The introduction of molecular methods made it possible to detect bacterial species that belong to the order Bacteroidales, an order that includes the genus Bacteroides, without cultivation. As a result, real-time PCR methods were developed for the quantitative detection of Bacteroidales in surface and recreation water and the potential of Bacteroidales species as an indication of fecal contamination of recreational waters was demonstrated (6, 12, 16, 19, 20, 29). Bacteroidales species might be useful indicator organisms for fecal contamination of drinking water as well. However, methods to detect fecal contamination in drinking water should be more sensitive, because people ingest more drinking water and the quality assessments and standards for fecal contamination are stricter than for bathing water. Studies exploring real-time PCR for the detection of Bacteroidales genes in drinking water have not been published to our knowledge. The objective of our study was, therefore, to determine if assays for the detection of Bacteroidales 16S rRNA genes can be used to detect fecal contamination in drinking water.Unchlorinated tap water samples were obtained in November 2007 and February 2010 from one or more locations in the distribution systems of nine different drinking water treatment plants (plants A to I; Table ​Table1)1) that produced unchlorinated drinking water from confined (plants B, C, E, F, and G) and unconfined (plants A, D, H, and I) groundwater. The treatment plants are located in the central part of the Netherlands within 90 km of each other. In addition, untreated groundwater from extraction wells and/or untreated raw groundwater (mixture of groundwater from different extraction wells) was sampled in March 2008 (Table ​(Table1).1). Water samples (100 ml) were filtered over a 25-mm polycarbonate filter (0.22-μm pore size, type GTTP; Millipore, Netherlands) and a DNA fragment was added as internal control to determine the recovery efficiency of DNA isolation and PCR analysis (2a, 40). DNA was isolated using a FastDNA spin kit for soil (Qbiogene, United States) according to the supplier''s protocol. Primer sets AllBac 296f and AllBac 412r, resulting in a PCR product of 108 bp, were used in combination with TaqMan probe AllBac375Bhqr to quantitatively determine the number of Bacteroidales 16S rRNA gene copies in the water samples using a real-time PCR instrument (20). The PCR cycle after which the fluorescence signal of the amplified DNA was detected (threshold cycle [C_T]) was used to quantify the concentration of 16S rRNA gene copies. Quantification was based on comparison of the sample C_T value with the C_T values of a calibration curve graphed using known copy numbers of the Bacteroidales 16S rRNA gene, as previously described (12, 20). The correlation coefficient of the calibration curve was 0.99, and the efficiency of the PCR 95 to 105%. Finally, the Bacteroidales cell number was calculated by using the recovery rate of the internal standard and assuming five 16S rRNA gene copy numbers per cell (5). The detection limit of this gene assay was 50 Bacteroidales cells 100 ml⁻¹ (corresponding to 10 16S rRNA gene copies per reaction mixture). Furthermore, the 16S rRNA genes that were obtained from several water samples from treatment plant C with the AllBac and TotBac (12) primer sets were sequenced, and the nearest relatives were obtained from the GenBank database using BLAST searches.

TABLE 1.

Numbers of Bacteroidales cells in extraction wells, raw groundwater, and unchlorinated tap water of nine different groundwater plants in the Netherlands^a

Chemical Contamination of California Drinking Water

Drinking water contamination by toxic chemicals has become widely recognized as a public health concern since the discovery of 1,2-dibromo-3-chloropropane in California''s Central Valley in 1979. Increased monitoring since then has shown that other pesticides and industrial chemicals are present in drinking water. Contaminants of drinking water also include naturally occurring substances such as asbestos and even the by-products of water chlorination. Public water systems, commercially bottled and vended water and mineral water are regulated, and California is also taking measures to prevent water pollution by chemicals through various new laws and programs.     

CRISPR Is an Optimal Target for the Design of Specific PCR Assays for Salmonella enterica Serotypes Typhi and Paratyphi A

Background

Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms.

Methodology

Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats), as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers.

Principal findings

We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species.

Conclusions

The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.     

Basin-Wide Analysis of the Dynamics of Fecal Contamination and Fecal Source Identification in Tillamook Bay, Oregon

The objectives of this study were to elucidate spatial and temporal dynamics in source-specific Bacteroidales 16S rRNA genetic marker data across a watershed; to compare these dynamics to fecal indicator counts, general measurements of water quality, and climatic forces; and to identify geographic areas of intense exposure to specific sources of contamination. Samples were collected during a 2-year period in the Tillamook basin in Oregon at 30 sites along five river tributaries and in Tillamook Bay. We performed Bacteroidales PCR assays with general, ruminant-source-specific, and human-source-specific primers to identify fecal sources. We determined the Escherichia coli most probable number, temperature, turbidity, and 5-day precipitation. Climate and water quality data collectively supported a rainfall runoff pattern for microbial source input that mirrored the annual precipitation cycle. Fecal sources were statistically linked more closely to ruminants than to humans; there was a 40% greater probability of detecting a ruminant source marker than a human source marker across the basin. On a sample site basis, the addition of fecal source tracking data provided new information linking elevated fecal indicator bacterial loads to specific point and nonpoint sources of fecal pollution in the basin. Inconsistencies in E. coli and host-specific marker trends suggested that the factors that control the quantity of fecal indicators in the water column are different than the factors that influence the presence of Bacteroidales markers at specific times of the year. This may be important if fecal indicator counts are used as a criterion for source loading potential in receiving waters.     

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.     

The deadly bite of Salmonella Typhi

Pathogenic microorganisms overcome the host's innate and adaptive immune system and cause local or systemic infections, potentially leading to organ failure, sepsis, or even death. Some microorganisms can also directly or indirectly alter the differentiation and proliferation of host cells, promoting the development of tumors. A large number of oncogenic viruses have been identified and estimated to account for ~15% of human cancers. They do so by encoding oncogenes or through their intrinsic ability to manipulate the genomic stability of the host cell by integrating their own genetic elements. Also bacterial infections have been linked to carcinogenesis, although the underlying molecular mechanisms are less well understood. The best‐studied example is Helicobacter pylori, which has been classified as a class I carcinogen by the World Health Organization due to its ability to promote stomach cancer after chronic infection, which causes tissue inflammation and atrophy of the gastric mucosa. In a recent issue of Cell Host & Microbe, the Neefjes laboratory explores the association between Salmonella enterica subsp. enterica sv. Typhi (S. Typhi)—which is the causative agent of human typhoid fever—and gallbladder carcinoma 1 .     

A Systematic Review and Meta-Analysis of Fecal Contamination and Inadequate Treatment of Packaged Water

Background

Packaged water products provide an increasingly important source of water for consumption. However, recent studies raise concerns over their safety.

Objectives

To assess the microbial safety of packaged water, examine differences between regions, country incomes, packaged water types, and compare packaged water with other water sources.

Methods

We performed a systematic review and meta-analysis. Articles published in English, French, Portuguese, Spanish and Turkish, with no date restrictions were identified from online databases and two previous reviews. Studies published before April 2014 that assessed packaged water for the presence of Escherichia coli, thermotolerant or total coliforms were included provided they tested at least ten samples or brands.

Results

A total of 170 studies were included in the review. The majority of studies did not detect fecal indicator bacteria in packaged water (78/141). Compared to packaged water from upper-middle and high-income countries, packaged water from low and lower-middle-income countries was 4.6 (95% CI: 2.6–8.1) and 13.6 (95% CI: 6.9–26.7) times more likely to contain fecal indicator bacteria and total coliforms, respectively. Compared to all other packaged water types, water from small bottles was less likely to be contaminated with fecal indicator bacteria (OR = 0.32, 95%CI: 0.17–0.58) and total coliforms (OR = 0.10, 95%CI: 0.05, 0.22). Packaged water was less likely to contain fecal indicator bacteria (OR = 0.35, 95%CI: 0.20, 0.62) compared to other water sources used for consumption.

Conclusions

Policymakers and regulators should recognize the potential benefits of packaged water in providing safer water for consumption at and away from home, especially for those who are otherwise unlikely to gain access to a reliable, safe water supply in the near future. To improve the quality of packaged water products they should be integrated into regulatory and monitoring frameworks.     

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.     

Quantifying Salmonella Population Dynamics in Water and Biofilms

Members of the bacterial genus Salmonella are recognized worldwide as major zoonotic pathogens often found to persist in non-enteric environments including heterogeneous aquatic biofilms. In this study, Salmonella isolates that had been detected repeatedly over time in aquatic biofilms at different sites in Spring Lake, San Marcos, Texas, were identified as serovars Give, Thompson, Newport and -:z10:z39. Pathogenicity results from feeding studies with the nematode Caenorhabditis elegans as host confirmed that these strains were pathogenic, with Salmonella-fed C. elegans dying faster (mean survival time between 3 and 4 days) than controls, i.e., Escherichia coli-fed C. elegans (mean survival time of 9.5 days). Cells of these isolates inoculated into water at a density of up to 10⁶?ml^?1 water declined numerically by 3 orders of magnitude within 2 days, reaching the detection limit of our quantitative polymerase chain reaction (qPCR)-based quantification technique (i.e., 10³ cells ml^?1). Similar patterns were obtained for cells in heterogeneous aquatic biofilms developed on tiles and originally free of Salmonella that were kept in the inoculated water. Cell numbers increased during the first days to more than 10⁷ cells cm^?2, and then declined over time. Ten-fold higher cell numbers of Salmonella inoculated into water or into biofilm resulted in similar patterns of population dynamics, though cells in biofilms remained detectable with numbers around 10⁴ cells cm^?2 after 4 weeks. Independent of detectability by qPCR, samples of all treatments harbored viable salmonellae that resembled the inoculated isolates after 4 weeks of incubation. These results demonstrate that pathogenic salmonellae were isolated from heterogeneous aquatic biofilms and that they could persist and stay viable in such biofilms in high numbers for some time.     

Bacterial Contamination of Drinking Water Supplies in a Modern Rural Neighborhood

On six occasions during a 15-month period, the private well and spring water supplies in a modern rural neighborhood of 78 households were examined for total coliforms, fecal coliforms, Staphylococcus aureus, and standard plate count bacteria. More than one-third of the water supplies were unsatisfactory on at least one occasion as judged by standard plate counts over 10³/ml and the presence of coliforms, fecal coliforms, and/or S. aureus. Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli were the most frequently isolated total coliforms. At least 12 other genera of bacteria were identified from standard plate count agar. Coliform contamination was found to be higher after periods of rainfall, and high standard plate counts were more prevalent during warmer weather. These observations probably reflect leakage of surface water into improperly sealed wells or aquifer contamination during winter and the lack of chlorination to control microbial regrowth during the warm season. An inverse correlation was found between the presence of high standard plate counts and incidence of coliforms. Consumer education and at least a twice yearly monitoring of private water supplies (winter and summer) are suggested methods to signal that treatment may be necessary to reduce the risk of waterborne disease.     

A genomic snapshot of Salmonella enterica serovar Typhi in Colombia

Little is known about the genetic diversity of Salmonella enterica serovar Typhi (S. Typhi) circulating in Latin America. It has been observed that typhoid fever is still endemic in this part of the world; however, a lack of standardized blood culture surveillance across Latin American makes estimating the true disease burden problematic. The Colombian National Health Service established a surveillance system for tracking bacterial pathogens, including S. Typhi, in 2006. Here, we characterized 77 representative Colombian S. Typhi isolates collected between 1997 and 2018 using pulse field gel electrophoresis (PFGE; the accepted genotyping method in Latin America) and whole genome sequencing (WGS). We found that the main S. Typhi clades circulating in Colombia were clades 2.5 and 3.5. Notably, the sequenced S. Typhi isolates from Colombia were closely related in a global phylogeny. Consequently, these data suggest that these are endemic clades circulating in Colombia. We found that AMR in S. Typhi in Colombia was uncommon, with a small subset of organisms exhibiting mutations associated with reduced susceptibility to fluoroquinolones. This is the first time that S. Typhi isolated from Colombia have been characterized by WGS, and after comparing these data with those generated using PFGE, we conclude that PFGE is unsuitable for tracking S. Typhi clones and mapping transmission. The genetic diversity of pathogens such as S. Typhi is limited in Latin America and should be targeted for future surveillance studies incorporating WGS.     

A novel linear plasmid mediates flagellar variation in Salmonella Typhi

Unlike the majority of Salmonella enterica serovars, Salmonella Typhi (S. Typhi), the etiological agent of human typhoid, is monophasic. S. Typhi normally harbours only the phase 1 flagellin gene (fliC), which encodes the H:d antigen. However, some S. Typhi strains found in Indonesia express an additional flagellin antigen termed H:z66. Molecular analysis of H:z66+ S. Typhi revealed that the H:z66 flagellin structural gene (fljB(z66)) is encoded on a linear plasmid that we have named pBSSB1. The DNA sequence of pBSSB1 was determined to be just over 27 kbp, and was predicted to encode 33 coding sequences. To our knowledge, pBSSB1 is the first non-bacteriophage-related linear plasmid to be described in the Enterobacteriaceae.     

Characteristics and Dynamics of Salmonella Contamination along the Coast of Agadir,Morocco

The occurrence of Salmonella enterica in the environment of tropical and desert regions has remained largely uninvestigated in many areas of the world, including Africa. In the present study, we investigated the presence of Salmonella spp. along 122 km of the coastline of Agadir (southern Morocco) in relation to environmental parameters. A total of 801 samples of seawater (243), marine sediment (279), and mussels (279) were collected from six sites between July 2004 and May 2008. The overall prevalence of Salmonella spp. was 7.1%, with the highest occurrence in mussels (10%), followed by sediment (6.8%) and seawater (4.1%). Only three serotypes were identified among the 57 Salmonella sp. strains isolated. S. enterica serotype Blockley represented 43.8% of all Salmonella strains and was identified in mussel and sediment samples. S. enterica serotype Kentucky (29.8%) was found almost exclusively in mussels, whereas S. enterica serotype Senftenberg (26.3%) was detected in sediment and seawater. Statistical analysis using generalized additive models identified seawater temperature, environmental temperature, rainfall, and solar radiation as significant factors associated with the presence of Salmonella. Rainfall was the only variable showing a linear positive effect on the presence of Salmonella in the sea, whereas the remaining variables showed more complex nonlinear effects. Twenty-eight (49.1%) Salmonella isolates displayed resistance to ampicillin (22 isolates), nalidixic acid (9 isolates), sulfonamide compounds (2 isolates), and tetracycline (1 isolate), with six of these isolates displaying multiple resistance to two of these antimicrobial agents. Pulsed-field gel electrophoresis analysis revealed homogenous restriction patterns within each serotype that were uncorrelated with the resistance pattern profiles.Salmonella enterica bacteria are one of the most frequent causes of food-borne infections transmitted to humans, mainly from animal products (9). In addition to health concerns, the presence of Salmonella contamination in the food chain has serious economic consequences related to the costs of medical care and lost productivity (36). Thus, studies aimed at examining the capacity of Salmonella spp. to survive in different habitats are critical for controlling contamination and understanding the routes of colonization of new hosts (40).Salmonella bacteria display a high degree of resistance to a large variety of stress factors, which provides them with an enhanced capacity to persist in changing environments (40). However, the persistence of the organism outside of the host is not uniform among the different serogroups (3, 4, 13, 24, 32, 34). About 50 of the more than 2,500 different serotypes of Salmonella included in the current classification scheme (29) are dominantly identified in human and animal sources (34). Information about groups that predominate in a given environment and their relationship to potential human or animal origins remains scarce. In recent years, some studies carried out in aquatic environments have provided new insights into the ecological preferences of different Salmonella serogroups in these environments. These studies have revealed the presence of specific patterns of contamination in different geographical areas in association with environmental and oceanographic variables (13, 24, 32) and have identified major factors and conditions that favor the presence of the contaminating bacteria. Identification of the different Salmonella strains present in the environment at serotype level is an essential preliminary step in discriminating potentially clinically important strains among the Salmonella present in contaminated areas, providing invaluable information about the nature of the contamination and allowing the inference of potential routes of dissemination through microbial source tracking (31). All of this information is critical for an improved assessment of the potential risks to public health associated with Salmonella and for the evaluation of the ecological preferences of the diverse and heterogeneous group of organisms which comprise the genus Salmonella (26).The lack of information regarding the epidemiology, contamination, and potential routes of transmission of Salmonella is of particular concern in many regions of the world, such as Africa and Central America, where gastrointestinal diseases continue to be a major cause of illness, primarily due to poor sanitary conditions and nutritional deficiencies. In the present study, we investigated the dynamics of Salmonella contamination in the coastal areas of Agadir, a populous region of southern Morocco where shellfish production and maritime tourism are important to the local economy. Information concerning the biological characteristics of the isolates was correlated with environmental data in order to evaluate the climatic conditions that favor contamination of this region by this pathogen and to identify the potential sources of contamination.     

Sources and Distribution of Surface Water Fecal Contamination and Prevalence of Schistosomiasis in a Brazilian Village

Background

The relationship between poor sanitation and the parasitic infection schistosomiasis is well-known, but still rarely investigated directly and quantitatively. In a Brazilian village we correlated the spatial concentration of human fecal contamination of its main river and the prevalence of schistosomiasis.

Methods

We validated three bacterial markers of contamination in this population by high throughput sequencing of the 16S rRNA gene and qPCR of feces from local residents. The qPCR of genetic markers from the 16S rRNA gene of Bacteroides-Prevotella group, Bacteroides HF8 cluster, and Lachnospiraceae Lachno2 cluster as well as sequencing was performed on georeferenced samples of river water. Ninety-six percent of residents were examined for schistosomiasis.

Findings

Sequence of 16S rRNA DNA from stool samples validated the relative human specificity of the HF8 and Lachno 2 fecal indicators compared to animals. The concentration of fecal contamination increased markedly along the river as it passed an increasing proportion of the population on its way downstream as did the sequence reads from bacterial families associated with human feces. Lachnospiraceae provided the most robust signal of human fecal contamination. The prevalence of schistosomiasis likewise increased downstream. Using a linear regression model, a significant correlation was demonstrated between the prevalence of S. mansoni infection and local concentration of human fecal contamination based on the Lachnospiraceae Lachno2 cluster (r² 0.53) as compared to the correlation with the general fecal marker E. coli (r² 0.28).

Interpretation

Fecal contamination in rivers has a downstream cumulative effect. The transmission of schistosomiasis correlates with very local factors probably resulting from the distribution of human fecal contamination, the limited movement of snails, and the frequency of water contact near the home. In endemic regions, the combined use of human associated bacterial markers and GIS analysis can quantitatively identify areas with risk for schistosomiasis as well as assess the efficacy of sanitation and environmental interventions for prevention.     

Sequence Analysis of the Plasmid pGY1 Harbored in Salmonella enterica Serovar Paratyphi A

The cryptic plasmid pGY1, which is harbored by a clinical isolate of Salmonella enterica serovar Paratyphi A, was identified in a 9-year-old girl with paratyphoid fever in 2005, and its DNA sequence was determined. It is 3592 bp in length and had a G+C content of 43.3%. Three ORFs were predicted that share low similarity with hypothetical proteins in the GenBank database. pGY1 shared 36.6% sequence homology with the cryptic plasmid pIMVS1 from Salmonella typhimurium. Its unique sequence makes it attractive for further study to obtain insight into the evolutionary relationship of this plasmid with other Enterobacteriaceae plasmids.     

Pan-Genomic Analysis Provides Insights into the Genomic Variation and Evolution of Salmonella Paratyphi A

Salmonella Paratyphi A (S. Paratyphi A) is a highly adapted, human-specific pathogen that causes paratyphoid fever. Cases of paratyphoid fever have recently been increasing, and the disease is becoming a major public health concern, especially in Eastern and Southern Asia. To investigate the genomic variation and evolution of S. Paratyphi A, a pan-genomic analysis was performed on five newly sequenced S. Paratyphi A strains and two other reference strains. A whole genome comparison revealed that the seven genomes are collinear and that their organization is highly conserved. The high rate of substitutions in part of the core genome indicates that there are frequent homologous recombination events. Based on the changes in the pan-genome size and cluster number (both in the core functional genes and core pseudogenes), it can be inferred that the sharply increasing number of pseudogene clusters may have strong correlation with the inactivation of functional genes, and indicates that the S. Paratyphi A genome is being degraded.