Richelle C. Charles

The Origin of the Haitian Cholera Outbreak Strain

BACKGROUND Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti. METHODS We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of 23 diverse strains of V. cholerae to assess the likely origin of the cholera outbreak in Haiti. RESULTS Both single-nucleotide variations and the presence and structure of hypervariable chromosomal elements indicate that there is a close relationship between the Haitian isolates and variant V. cholerae El Tor O1 strains isolated in Bangladesh in 2002 and 2008. In contrast, analysis of genomic variation of the Haitian isolates reveals a more distant relationship with circulating South American isolates. CONCLUSIONS The Haitian epidemic is probably the result of the introduction, through human activity, of a V. cholerae strain from a distant geographic source. (Funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.).

Cholera in the 21st century.

Purpose of review This review will focus on recent advances in our understanding of biologic and environmental factors that shape current cholera outbreaks, advances in our understanding of host–pathogen interactions during cholera, and recent evolution of current treatment and cholera prevention strategies. Recent findings New research studies have improved our understanding of a number of dynamic factors that shape the ecology of Vibrio cholerae and influence its transmission, including the role of lytic bacteriophage, biofilm formation, a hyperinfectious state of human-passaged V. cholerae, and the impact of severe weather events. Provision of safe water and improved sanitation continue to be the mainstays of preventing cholera transmission; however, the role of cholera vaccination as a control measure in both endemic and epidemic settings is evolving. Recent advances in our understanding of long-lived protective immunity after natural infection may aid in the global efforts to control cholera. Summary Improved understanding of factors associated with protective immunity and dynamic factors associated with cholera outbreaks may lead to improved control and prevention strategies for cholera.

Social stress effects on territorial marking and ultrasonic vocalizations in mice

Acute social defeat (SD) leads to transient and persistent physiological and behavioral changes. We examined the effects of acute SD on territorial urine marking and ultrasonic courtship vocalizations in DBA/2 male mice. Both behaviors are considered androgen dependent and are influenced by social status, with dominant mice displaying more of both behaviors. In Experiment 1, male mice that received SD displayed prolonged inhibition of territorial urine marking, relative to nondefeated control mice (NOSD). In addition, territorial marking increased with repeated tests. In Experiment 2, male mice that received 3 successive days of SD displayed fewer ultrasonic courtship vocalizations at 30 min. post-SD1 and 30 min. post-SD2, relative to NOSD mice. In Experiment 2, we also observed decreased territorial marking 4 weeks post-SD. In sum, SD induced prolonged inhibition of territorial marking, but had only transient effects on ultrasonic courtship vocalizations, suggesting that different mechanisms may mediate the maintenance of these behaviors.

Salmonella chronic carriage: epidemiology, diagnosis, and gallbladder persistence.

Typhoid (enteric fever) remains a major cause of morbidity and mortality worldwide, causing over 21 million new infections annually, with the majority of deaths occurring in young children. Because typhoid fever-causing Salmonella have no known environmental reservoir, the chronic, asymptomatic carrier state is thought to be a key feature of continued maintenance of the bacterium within human populations. Despite the importance of this disease to public health, our understanding of the molecular mechanisms that catalyze carriage, as well as our ability to reliably identify and treat the Salmonella carrier state, have only recently begun to advance.

Cholera's western front

The cholera epidemics of the 19th century forged the way for the sanitation revolution and the provision of safe public water sources that are the hallmark of developed countries. Nevertheless, more than 1 billion people today, including much of the population in Haiti, lack access to improved water supply sources and hence remain vulnerable to epidemics of cholera 1. Through our work with non-governmental organizations and governmental agencies, we have been involved firsthand with the developing response to the cholera epidemic in Haiti. Here, we review the Haitian outbreak in the context of other recent cholera epidemics and discuss on-the-ground strategies for optimizing cholera case management. Prior to the current epidemic, cholera’s most recent appearance in the Western Hemisphere was in the early 1990s. After being effectively absent from the region for more than a century, cholera re-appeared in Peru in January 1991. The first cluster of patients was identified in a coastal village 80 kilometers north of Lima. By the end of 1991, over 390,000 cases of cholera and more than 4,000 deaths had occurred in 16 countries. Although the ferocity of the outbreaks first year was unmatched, cholera remained endemic in the hemisphere for ten years, with the last reported death in 2001. From a global perspective, the cholera epidemic of the 1990s in the Western Hemisphere was only one front in an ongoing pandemic of cholera that began in Indonesia in 1961. This is the seventh global pandemic of cholera that has occurred the last 200 years; the causative organism is an El Tor biotype of Vibrio cholerae serogroup O1. This El Tor biotype has been so successful that it has now completely replaced the previously circulating classical biotype. Pulse field gel electrophoresis performed at the CDC indicates that the epidemic strain of V. cholerae in Haiti is similar to the South Asian seventh pandemic El Tor strains 2. In the wake of the 1991 outbreak, the public health infrastructure was strengthened in many countries of Latin America and the Caribbean. Nevertheless, access to safe water in the hemisphere remains uneven, with Haiti being the most notable example. In 2008, only 12% of Haitians received piped, treated water, and only 17% had access to adequate sanitation 3. January’s earthquake effectively destroyed the already fragile infrastructure in Haiti and left more than a million homeless people living in makeshift encampments, primarily in Port-au-Prince. Concern for the possibility of diarrheal disease outbreaks was high in the aftermath of the earthquake, but no immediate event occurred.

Evolutionary consequences of intra-patient phage predation on microbial populations

The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, Vibrio cholerae, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient V. cholerae populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant V. cholerae populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of V. cholerae shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans. DOI: http://dx.doi.org/10.7554/eLife.03497.001

Comparative Proteomic Analysis of the PhoP Regulon in Salmonella enterica Serovar Typhi Versus Typhimurium

Background S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica. Methodology/Principal Findings Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP−/Q− mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions. Conclusions/Significance This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).

Circulating mucosal associated invariant T cells are activated in Vibrio cholerae O1 infection and associated with lipopolysaccharide antibody responses.

Background Mucosal Associated Invariant T (MAIT) cells are innate-like T cells found in abundance in the intestinal mucosa, and are thought to play a role in bridging the innate-adaptive interface. Methods We measured MAIT cell frequencies and antibody responses in blood from patients presenting with culture-confirmed severe cholera to a hospital in Dhaka, Bangladesh at days 2, 7, 30, and 90 of illness. Results We found that MAIT (CD3+CD4−CD161hiVα7.2+) cells were maximally activated at day 7 after onset of cholera. In adult patients, MAIT frequencies did not change over time, whereas in child patients, MAITs were significantly decreased at day 7, and this decrease persisted to day 90. Fold changes in MAIT frequency correlated with increases in LPS IgA and IgG, but not LPS IgM nor antibody responses to cholera toxin B subunit. Conclusions In the acute phase of cholera, MAIT cells are activated, depleted from the periphery, and as part of the innate response against V. cholerae infection, are possibly involved in mechanisms underlying class switching of antibody responses to T cell-independent antigens.

Characterization of anti-Salmonella enterica serotype Typhi antibody responses in bacteremic Bangladeshi patients by an immunoaffinity proteomics-based technology.

ABSTRACT Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.

Effects of social defeat and of diazepam on behavior in a resident-intruder test in male DBA/2 mice

Social stress induces robust behavioral and physiological changes, some of which may alter the responsiveness to pharmacological agents, including diazepam (DZP). We used a resident-intruder paradigm to (1) develop a comprehensive ethogram of behavioral changes following social defeat (SD) in the socially reactive strain, DBA/2 male mice, (2) determine whether acute exposure of DBA/2 mice to low-dose DZP would induce flight or aggressive behavior, both of which have been observed in other rodent models and (3) to test whether prior social stress affects responses to DZP. Behavioral responses to a nonaggressive intruder (NAI) mouse 24 h post-SD were measured in resident subject mice exposed to DZP (0, 0.5, 2.0 mg/kg, ip) either prior to the resident-intruder test (Experiment 1) or immediately post-SD (Experiment 2); control mice were not defeated (NOSD). In general, SD mice displayed increased passive and active avoidance, defense, immobility, and risk assessment relative to NOSD mice. In Experiment 1, mice treated acutely with 0.5 mg/kg DZP had more approach and flight behavior, while those treated with 2.0 mg/kg DZP had more avoidance than vehicle-treated mice, independent of SD. In Experiment 2, acute DZP (2 mg/kg) induced effects 24 h later, possibly secondary to withdrawal. In a nonsocial context (Experiment 3), DZP increased exploratory activity.