A variant in long palate, lung and nasal epithelium clone 1 is associated with cholera in a Bangladeshi population.

Vibrio cholerae causes a dehydrating diarrheal illness that can be rapidly fatal in the absence of specific treatment. The organism is an historic scourge and, like similar infectious diseases, may have influenced the evolution of the human genome. We report here the results of the first candidate gene association study of cholera. In a family-based study of 76 pedigrees from Dhaka, Bangladesh, we evaluated the association between cholera and five candidate genes-the cystic fibrosis transmembrane receptor; lactoferrin; long palate, lung and nasal epithelium clone 1 (LPLUNC1); estrogen-related receptor alpha and calcium-activated chloride channel 1. We found a significant association with a marker in the promoter region of LPLUNC1 (rs11906665), a member of a family of evolutionarily conserved innate immunity proteins. An earlier microarray-based study of duodenal biopsies showed significantly increased expression of LPLUNC1 in cholera patients compared with healthy control subjects. Our results suggest that variation in host innate immune responses may influence the outcome of exposure to V. cholerae in an endemic setting.

Crystallization of the B chain of Shiga-like toxin I from Escherichia coli.

Shiga-like toxin I (SLT-I) is produced by several pathogenic strains of Escherichia coli associated with diarrheal disease. The toxin consists of an A chain, which attacks eukaryotic ribosomes, inhibiting protein synthesis, and multiple copies of a 69 amino acid B chain. The B subunit mediates cell binding and uptake through its interactions with cell surface carbohydrate moieties. Here we report that the B chain has been crystallized in a form suitable for high-resolution X-ray analysis. The space group is P2(1)2(1)2(1), with a = 56.2 A, b = 59.9 A and c = 102.5 A. A rotation function using three-dimensional diffraction data suggests that the asymmetric unit is a tetramer.

Clostridium difficile--Associated diarrhea: A review.

Clostridium difficile causes 300 000 to 3 000 000 cases of diarrhea and colitis in the United States every year. Antibiotics most frequently associated with the infection are clindamycin, ampicillin, amoxicillin, and cephalosporins, but all antibiotics may predispose patients to C difficile infection. The clinical presentation varies from asymptomatic colonization to mild diarrhea to severe debilitating disease, with high fever, severe abdominal pain, paralytic ileus, colonic dilation (or megacolon), or even perforation. The most sensitive and specific test available for diagnosis of C difficile infection is a tissue culture assay for the cytotoxicity of toxin B. However, this test takes 1 to 3 days to complete and requires tissue culture facilities. Detection of C difficile toxin by means of enzyme-linked immunoassay is more rapid and inexpensive. A minority of patients may require more than 1 stool assay to detect toxin. Oral metronidazole or oral vancomycin hydrochloride for 10 to 14 days are equally effective at resolving clinical symptoms; oral metronidazole is preferred in most cases because of lowered cost and less selective pressure for vancomycin-resistant organisms. Approximately 15% of patients experience relapse after initial therapy and require retreatment, sometimes with an extended, tapering regimen. Immunity appears to be incomplete and predominantly mediated by serum IgG to toxin A. Measures for preventing the spread of the pathogen, appropriate diagnostic testing, and treatment may avert morbidity and mortality due to C difficile-associated diarrhea.

The interaction of the Vibrio cholerae transcription factors, Fur and IrgB, with the overlapping promoters of two virulence genes, irgA and irgB.

irgA, a virulence gene in Vibrio cholerae, encodes a 77kDa outer membrane protein. irgA expression is activated by irgB, which encodes a LysR-type transcription factor and is divergently transcribed from a promoter overlapping that of irgA. Expression of irgA and irgB is repressed by iron and Fur. A 200bp DNA fragment containing the irgA-irgB intergenic region was inserted between the Escherichia coli phoA and lacZ genes, respectively, to generate operon fusions to the two promoters, and this construct was crossed into the chromosomal lacZ gene of V. cholerae. This DNA fragment was sufficient to produce regulation of irgA-phoA and irgB-lacZ transcription by iron, Fur and IrgB. Purified V. cholerae Fur and IrgB overexpressed in E. coli bound simultaneously to this DNA fragment in gel shift experiments, and footprints of both proteins on the irgA-irgB intergenic region were observed using DNaseI footprinting. The Fur footprint overlapped a Fur box, previously identified by homology with the E. coli Fur box. The position of the IrgB footprint was consistent with activation of irgA transcription and repression of irgB transcription by IrgB. We present a model for the interaction of Fur and IrgB in transcriptional regulation of irgA.

Central nervous system infection with Listeria monocytogenes. 33 years' experience at a general hospital and review of 776 episodes from the literature.

We reviewed 776 previously reported and 44 new cases of CNS listeriosis outside of pregnancy and the neonatal period, and evaluated the epidemiologic, diagnostic, and therapeutic characteristics of this infection. Among patients with Listeria meningitis/meningoencephalitis, hematologic malignancy and kidney transplantation were the leading predisposing factors, but 36% of patients had no underlying diseases recognized. The infection occurred throughout life, with a higher incidence before the age of 3 and after the age of 45-50 years. Fever, altered sensorium, and headache were the most common symptoms, but 42% of patients had no meningeal signs on admission. Compared with patients with acute meningitis due to other bacterial pathogens, patients with Listeria infection had a significantly lower incidence of meningeal signs, and the CSF profile was significantly less likely to have a high WBC count or a high protein concentration. Gram stain of CSF was negative in two-thirds of cases of CNS listeriosis. One-third of patients had focal neurologic findings, and approximately one-fourth developed seizures over their course. Mortality was 26% overall, and was higher among patients with seizures and those older than 65 years of age. Relapse occurred in 7% of episodes. Ampicillin for a minimum of 15-21 days (with an aminoglycoside for at least the first 7-10 days) remains the treatment of choice. Cerebritis/abscess due to L. monocytogenes, without meningeal involvement, is less common but may be diagnosed by blood cultures and CNS imaging, or by stereotactic biopsy. Longer antibiotic therapy (at least 5-6 weeks) is needed in the presence of localized CNS involvement.

Native valve endocarditis due to coagulase-negative staphylococci. Clinical and microbiologic features.

Twenty-one patients with native valve endocarditis caused by coagulase-negative staphylococci were studied; 14 had pre-existing valvular or congenital heart disease. Although commonly subacute in presentation, complications of endocarditis were frequent: arterial emboli in five patients, new electrocardiographic conduction system abnormalities in nine, congestive heart failure in eight, annular or myocardial abscesses in five, and disruption of valve leaflets in three. Cures were achieved in 10 of 12 patients treated medically and seven of nine treated surgically. In microbiologic studies of 16 coagulase-negative staphylococci from patients with endocarditis, only eight were identified as Staphylococcus epidermidis. All isolates were susceptible to vancomycin. Antibiotic resistance (methicillin, four isolates; gentamicin, two isolates; rifampin, one isolate) was usually associated with nosocomial acquisition of endocarditis. Rather than representing contamination, coagulase-negative staphylococci in blood cultures may indicate life-threatening endocarditis. However, with careful attention to the selection of antibiotics for therapy and to the occurrence of heart failure due to intracardiac complications, treatment of this form of endocarditis is frequently successful. Organisms must always be tested for cryptic resistance to beta-lactam antibiotics. Valve replacement may be required frequently.

Cardiac conduction abnormalities complicating native valve active infective endocarditis.

Two hundred eleven episodes of native valve active infective endocarditis treated at the Massachusetts General Hospital between 1975 and 1983 were reviewed. The aortic (36%) and mitral (33%) valves were most frequently involved, but in 21% of the cases the site of infection could not be localized. Streptococcal (50%) and staphylococcal (35%) species were the most frequently isolated pathogens. New or changing ("unstable") conduction abnormalities developed in 9% of the patients, while an additional 7% had conduction abnormalities of "indeterminate" age. Unstable conduction block was more likely to develop in patients with aortic valve infective endocarditis than in those with mitral infection. Surgery was performed in 23% of the patients. Unstable conduction abnormalities were significantly associated with valve replacement, but in a multivariate analysis, this effect could be explained by the site of valvular infection. The mortality rate was 20%. Patients with unstable conduction abnormalities had a significantly higher mortality rate, even after other significant predictors of death (age, type of causative organism) were taken into account. Patients whose conduction changes persisted had a worse prognosis than those with transient conduction abnormalities. Although more hemodynamically compromised, patients with unstable conduction block who underwent valve replacement did at least as well as those given medical therapy alone. Patients with native valve active infective endocarditis in whom persistent, unstable conduction abnormalities develop without other identifiable cause, especially in the presence of aortic valve infection, should be considered for valve replacement.

Prosthetic valve endocarditis. Analysis of factors affecting outcome of therapy.

We analyzed the outcome of 116 patients with prosthetic valve endocarditis treated between 1975 and 1983 and used multivariate analysis to identify risk factors for in-hospital mortality and bad outcome during follow-up. Complicated prosthetic valve endocarditis was defined as the presence of a new or changing heart murmur, new or worsening heart failure, new or progressive cardiac conduction abnormalities, or prolonged fever during therapy. Complicated prosthetic valve endocarditis was present in 64% of patients; factors associated with complicated prosthetic valve endocarditis included aortic valve infection (odds ratio 4.3, p = 0.002) and onset of endocarditis within 12 months of the cardiac operation (odds ratio 5.5, p = 0.0001). The in-hospital mortality rate for prosthetic valve endocarditis was 23%; patients with complicated prosthetic valve endocarditis had a higher mortality than patients with uncomplicated infection (odds ratio 6.4, p = 0.0009). Combined medical-surgical therapy was used in 39% of patients; surgical therapy was more common in patients with complicated prosthetic valve endocarditis (odds ratio 16, p less than 0.0001) and in patients infected with coagulase-negative staphylococci (odds ratio 3.9, p = 0.003). Survival after initially successful therapy for prosthetic valve endocarditis was adversely affected by the presence of moderate or severe congestive heart failure at hospital discharge (p = 0.03). Bad outcome during follow-up (death, relapse of prosthetic valve endocarditis, or subsequent cardiac operation related to sequelae of the original infection) was more common in the medical than the medical-surgical therapy group (p = 0.02). The difference in long-term outcome between patients treated initially with medical or with medical-surgical therapy was particularly evident in those with complicated prosthetic valve endocarditis (p = 0.008). The presence of complicated prosthetic valve endocarditis is a central variable in assessing prognosis and planning therapy; the majority of patients with complicated prosthetic valve endocarditis are best treated with medical-surgical therapy. Those who are not treated surgically during their initial hospitalization are at high risk for progressive prosthesis dysfunction and require careful follow-up.

Distribution and incidence of viridans streptococcal species in routine clinical specimens.

Five hundred consecutive isolates of viridans streptococci were identified to the species level in an effort to determine their distribution and incidence in routine clinical specimens. Viridans streptococci accounted for significant percentages of streptococcal isolates from urine, wounds, body fluids, and blood. The most commonly isolated strains belonged to the Streptococcus milleri, Streptococcus mitis, Streptococcus sanguis I, and Streptococcus sanguis II species. Patient charts were reviewed in order to investigate the possible role as a urinary pathogen of strains belonging to a subgroup of S. milleri. Although these strains frequently are isolated from urine, they appear to play no pathogenic role in urinary tract infections.

Mortality, morbidity, and microbiology of endemic cholera among hospitalized patients in Dhaka, Bangladesh.

Mortality and morbidity associated with cholera acquired in a modern endemic setting have not been well defined. In Dhaka, Bangladesh from 1986 to 1996, we found that causative agents of cholera shifted over time, varying by serogroup, biotype, and serotype. At the International Centre for Diarrhoeal Disease Research (ICDDR,B: Centre for Health and Population Research) in 1996, 19,100 cholera patients were treated, 887 (4.6%) were admitted, and 33 died (mortality rate = 3.7% of cholera inpatients, 0.14% of all cholera patients). When cholera inpatients who were discharged improved were compared with those who died, bacteremia (odds ratio [OR] = 10.5, 95% confidence interval [CI] = 2.9-37.9), radiographic evidence of pneumonia (OR = 3.1, 95% CI = 1.2-7.7), and acidosis as estimated by the serum bicarbonate value (OR = 0.893, 95% CI = 0.825-0.963) were independently associated with death by multivariate analysis. Pneumonia was the leading cause of death and accounted for two-thirds of all deaths among individuals with cholera in this study. Death in hospitalized patients with cholera acquired in a modern endemic setting is, therefore, extremely rare, and most frequently due to concomitant infection, especially pneumonia.

Common adverse effects of antibacterial agents on major organ systems.

Antibiotics contribute significantly to the management of the surgical patient. However, their potential for adverse effects, both toxic and allergic, must always be kept in mind. We have reviewed the major adverse reactions to antibiotics, so that side effects may be promptly recognized and treated. Armed with this information, the surgeon can more effectively utilize this valuable class of drugs for the benefit of the patient.

Transcutaneous immunization with a Vibrio cholerae O1 Ogawa synthetic hexasaccharide conjugate following oral whole-cell cholera vaccination boosts vibriocidal responses and induces protective immunity in mice.

A shortcoming of currently available oral cholera vaccines is their induction of relatively short-term protection against cholera compared to that afforded by wild-type disease. We were interested in whether transcutaneous or subcutaneous boosting using a neoglycoconjugate vaccine made from a synthetic terminal hexasaccharide of the O-specific polysaccharide of Vibrio cholerae O1 (Ogawa) coupled to bovine serum albumin as a carrier (CHO-BSA) could boost lipopolysaccharide (LPS)-specific and vibriocidal antibody responses and result in protective immunity following oral priming immunization with whole-cell cholera vaccine. We found that boosting with CHO-BSA with immunoadjuvantative cholera toxin (CT) or Escherichia coli heat-labile toxin (LT) following oral priming with attenuated V. cholerae O1 vaccine strain O395-NT resulted in significant increases in serum anti-V. cholerae LPS IgG, IgM, and IgA (P < 0.01) responses as well as in anti-Ogawa (P < 0.01) and anti-Inaba (P < 0.05) vibriocidal titers in mice. The LPS-specific IgA responses in stool were induced by transcutaneous (P < 0.01) but not subcutaneous immunization. Immune responses following use of CT or LT as an adjuvant were comparable. In a neonatal mouse challenge assay, immune serum from boosted mice was associated with 79% protective efficacy against death. Our results suggest that transcutaneous and subcutaneous boosting with a neoglycoconjugate following oral cholera vaccination may be an effective strategy to prolong protective immune responses against V. cholerae.

Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus.

Shiga-like toxin is an iron-regulated cytotoxin quite similar to Shiga toxin from Shigella dysenteriae 1. The structural genes for Shiga-like toxin in Escherichia coli (sltA and sltB) appear to be transcribed as an operon from a promoter upstream of sltA. We used a gene fusion between the promoter and proximal portion of sltA with the gene for bacterial alkaline phosphatase to assess the regulation of toxin expression. Growth in low-iron conditions resulted in a 13- to 16-fold increase in alkaline phosphatase activity. In the presence of a null mutation in the fur locus, however, alkaline phosphatase activity was constitutively high regardless of the iron concentration. These data indicate negative regulation of the slt operon by the fur gene product. We used deletion analysis of the region upstream of the gene fusion to localize the promoter of the slt operon and to show that a region of DNA between the -35 and -10 boxes is necessary for iron regulation of slt expression. In this region, there is a 21-base-pair dyad repeat that is homologous to similar dyads in the promoter regions of three other fur-regulated genes. This region of dyad symmetry may represent an operator binding site for the Fur protein in the presence of iron.

Confirmation of the Fur operator site by insertion of a synthetic oligonucleotide into an operon fusion plasmid.

We constructed a synthetic oligonucleotide corresponding to the previously proposed consensus binding site for the Fur protein, a central iron-regulatory protein of Escherichia coli. When this oligonucleotide was introduced at the start of transcription of an operon fusion between the ompF promoter and the lacZ structural gene, beta-galactosidase activity became iron regulated. This consensus sequence is sufficient to function as an operator site for the binding of Fur protein in vivo.

Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange.

Vibrio vulnificus infections have been associated with iron overload and preexisting liver disease. Iron may play a major role in the pathogenesis of V. vulnificus infections. Many virulence genes, as well as genes involved in the transport of iron by bacteria, are regulated by iron, with increased expression under low-iron conditions. In Escherichia coli and Vibrio cholerae, transcriptional regulation by iron depends on the fur gene. We utilized Southern hybridization under low- and high-stringency conditions with both E. coli and V. cholerae fur gene probes to demonstrate that there are fur-homologous sequences in the DNAs of V. vulnificus, Vibrio fischeri, and Aeromonas sp. but not in the DNAs of the other bacterial species tested. We developed a restriction map and cloned the fur-homologous sequence from V. vulnificus. The hybridizing clone of V. vulnificus chromosomal DNA complemented a V. cholerae fur mutant. DNA sequence analysis confirmed the presence of a 149-amino-acid open reading frame that was 77% homologous to E. coli Fur and 93% homologous to V. cholerae Fur. Primer extension localized a single promoter for the V. vulnificus fur gene. Northern (RNA) blot analysis and beta-galactosidase assays of an operon fusion to lacZ suggested that there was not significant regulation of transcription of V. vulnificus fur by iron or the E. coli Fur protein. We used marker exchange to construct a V. vulnificus fur deletion mutant and confirmed its phenotype by observing overexpression of iron-regulated outer membrane proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fur deletion mutant of V. vulnificus will be helpful in future studies of the role of iron in V. vulnificus pathogenesis.

Identification, cloning, and sequencing of a gene required for ferric vibriobactin utilization by Vibrio cholerae.

Chromosomal DNA downstream of the Vibrio cholerae ferric vibriobactin receptor gene, viuA, was cloned and sequenced, revealing an 813-bp open reading frame encoding a deduced protein of 271 amino acids. In vitro transcription-translation of this DNA confirmed expression of a protein of the expected size. A deletion mutation of this gene, viuB, was created in the classical V. cholerae strain O395 by in vivo marker exchange. By cross-feeding studies, this mutant was unable to utilize exogenous ferric vibriobactin but synthesized the siderophore normally; synthesis of siderophore by the mutant was also confirmed by the Arnow assay. Complementation of the mutant with a plasmid encoding only viuB restored ferric vibriobactin utilization to normal. Unexpectedly, hydropathicity analysis of ViuB did not reveal a signal sequence or transmembrane domain, suggesting that ViuB is not a periplasmic or membrane protein but may be a cytoplasmic protein involved in ferric vibriobactin uptake and processing, perhaps analogous to the Escherichia coli protein Fes. ViuB was not, however, homologous to Fes or to other proteins in the database. Complementation studies revealed that the cloned V. cholerae viuB gene could complement an E. coli fes mutant but that the cloned E. coli fes gene could not complement a V. cholerae viuB mutant. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuB message that was negatively regulated by iron at the transcriptional level. The promoter of viuB was located by primer extension and contained a nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence, suggesting that expression of viuB is under the control of the V. cholerae fur gene.

Analysis of the complexity of gene regulation by fur in Vibrio cholerae.

Iron concentration influences the expression of a number of genes involved in iron uptake and virulence in bacteria. In Escherichia coli, coordinate regulation of these genes by iron depends on the product of the fur gene, which acts as an iron-responsive, DNA-binding repressor protein. Several genes in Vibrio cholerae are also repressed by iron; and a fur gene, homologous to E. coli fur, has been previously cloned from this organism. The present study was undertaken to define the roles of Fur and iron in regulating gene expression in V. cholerae. V. cholerae strains with a mutation in fur by virtue of suicide plasmid integration into this gene showed derepressed expression of two previously characterized, iron-regulated genes, irgA and viuA, in high concentrations of iron; even in the fur mutants, however, residual two- to threefold regulation by iron persisted. The fur mutant strains constructed by suicide plasmid integration required antibiotic selection to maintain the mutation. To analyze further the effect of Fur and iron on gene regulation in V. cholerae without the need for antibiotic selection, we used in vivo marker exchange to construct a nonrevertible V. cholerae fur mutant. This V. cholerae fur mutant grew significantly less well in Luria-Bertani medium than the wild-type parent but grew slightly better than the wild type under iron-restricted conditions. The V. cholerae fur mutant was unable to utilize a number of carbon sources including glycerol, acetate, succinate, lactate, and fumarate, that supported growth of the wild-type strain on minimal media. We utilized two-dimensional gel electrophoresis of whole-cell protein extracts from the fur mutant and wild-type strains following growth in conditions of either low or high concentrations of iron to identify proteins regulated by iron and/or Fur. Twenty-two proteins were negatively regulated by iron in the wild-type strain but constitutively expressed in the fur mutant, consistent with the model of Fur as an iron-dependent repressor. However, many other proteins were regulated in a different manner by iron and/or Fur. Seventeen proteins were negatively regulated by iron but independent of Fur, suggesting the presence of an additional iron-dependent repressor(s). Six proteins were strongly iron regulated in the fur mutant but hardly expressed at all in the wild-type strain regardless of the iron concentration, suggesting an interaction between Fur and another iron regulatory mechanism. There were 11 proteins that were induced rather than repressed by iron, in four different regulatory classes. Gene regulation in V. cholerae by Fur and iron is much more complex than previously thought and is reminiscent of the Lrp regulon in E.coli.

Cholera vaccines.

Cholera causes significant morbidity and mortality worldwide. For travelers, the risk of developing cholera per month of stay in a developing country is approximately 0.001%-0.01%, and cholera may present as traveler's diarrhea. In the United States, only a poorly tolerated, marginally effective, parenterally administered, phenol-inactivated vaccine is available. Outside the United States, 2 additional vaccines are commercially available: an oral killed whole cell-cholera toxin recombinant B subunit vaccine (WC-rBS) and an oral live attenuated Vibrio cholerae vaccine (CVD 103-HgR). These oral vaccines are well tolerated. In field trials, WC-rBS provides 80%-85% protection from cholera caused by V. cholerae serogroup O1 for at least 6 months. In volunteer studies, CVD 103-HgR provides 62%-100% protection against cholera caused by V. cholerae for at least 6 months. No commercially available cholera vaccine protects against disease caused by V. cholerae serogroup O139. New cholera vaccines are being developed.