Increased levels of inflammatory mediators in children and adults infected with Vibrio cholerae O1 and O139

Investigations were carried out to study the production of factors associated with the innate immune response in the systemic and mucosal compartments in adults and children infected with Vibrio cholerae O1 and V. cholerae O139. The levels of nonspecific mediators of the innate defense system, i.e., prostaglandin E(2) (PGE(2)), leukotriene B(4) (LTB(4)), and lactoferrin (Lf), as well as myeloperoxidase (MPO), were elevated at the acute stage of the disease in stools obtained from both O1- and O139-infected adults and children. In the systemic compartment, the levels of Lf were increased after onset of disease, which in children remained elevated up to convalescence compared to the healthy controls. Increased concentrations of C-reactive protein were seen in the sera of adult cholera patients at the acute stage of infection. Elevated levels of the nitric oxide (NO*) metabolites (nitrite and nitrate [NO(2)(-) and NO(3)(-)]) were detected in plasma but not in urine. The activity of the scavenger of reactive oxygen species, superoxide dismutase, was higher in the plasma of adults immediately after the onset of disease, suggesting that an active scavenging of reactive oxygen species was taking place. The concentration of 8-iso-prostaglandin F(2 alpha) remained unchanged in the systemic and mucosal compartments in the study subjects. After the recovery of patients from cholera, the concentration of the majority of the metabolites decreased to baseline levels by day 30 after the onset of infection. Immunohistochemical staining showed increased tissue expression of MPO, Lf, and inducible nitric oxide synthase at the acute stage in the duodenal biopsies of adults and rectal biopsies obtained from children with cholera. Very little difference was seen in the levels of the different inflammatory mediators in patients infected with V. cholerae O1 or the encapsulated V. cholerae O139. In summary, these results suggest that elevated concentrations of Lf, MPO, PGE(2), LTB(4), and NO*, as well as other metabolites, during the acute stage of the disease indicate that the innate defense system, as well as the inflammatory process, is activated in both adults and pediatric patients infected with V. cholerae O1 and O139.     

Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.

Vibrio cholerae serogroup O139, now considered to be the second organism capable of causing epidemic severe dehydrating cholera, contains a capsular polysaccharide which makes it difficult for it to be used in the conventional vibriocidal antibody assay optimized for V. cholerae O1. After modification of the procedure, which involved the use of specific bacterial strains, a lower bacterial inoculum, and increased amounts of complement, the vibriocidal antibody responses to V. cholerae O139 were measured in acute- and convalescent-phase sera from 33 V. cholerae O139-infected and 18 V. cholerae O1-infected patients and in single serum samples from 20 healthy control subjects. The responses in these individuals to V. cholerae O1 strains were also determined. Significant elevations in the homologous antibody response were found only in the convalescent-phase sera from both groups of patients with cholera. These findings may explain the basis for the lack of heterologous protection between the two serogroups of V. cholerae. Healthy controls had higher background levels of vibriocidal antibody to V. cholerae O1 than to V. cholerae O139.     

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.     

Induction of Fimbriated Vibrio cholerae O139

Several fimbriated phases of Vibrio cholerae O139 strains were selectively induced and compared immunologically and biochemically with those of V. cholerae O1. Fimbrial antigens were detected on the surfaces of vibrio cells colonizing the epithelial cells of a rabbit small intestine. Convalescent-phase sera from six individuals infected with V. cholerae O139 revealed the development of antibody against the fimbrillin. These findings suggest that the fimbriae of V. cholerae O1 and O139 are expressed in vivo during infection and that consideration must be given to the use of fimbrial antigens as components of vaccines against cholera.     

Phage specific for Vibrio cholerae O139 Bengal.

From the stool of a Vibrio cholerae O139 Bengal-infected patient, a phage that specifically lysed capsulated V. cholerae O139 strains only was isolated. The phage is useful for the confirmatory diagnosis of V. cholerae O139 infection and for the differentiation of variants that lack the capsule.     

The major subunit of the toxin-coregulated pilus TcpA induces mucosal and systemic immunoglobulin A immune responses in patients with cholera caused by Vibrio cholerae O1 and O139

Diarrhea caused by Vibrio cholerae is known to give long-lasting protection against subsequent life-threatening illness. The serum vibriocidal antibody response has been well studied and has been shown to correlate with protection. However, this systemic antibody response may be a surrogate marker for mucosal immune responses to key colonization factors of this organism, such as the toxin-coregulated pilus (TCP) and other factors. Information regarding immune responses to TCP, particularly mucosal immune responses, is lacking, particularly for patients infected with the El Tor biotype of V. cholerae O1 or V. cholerae O139 since highly purified TcpA from these strains has not been available previously for use in immune assays. We studied the immune responses to El Tor TcpA in cholera patients in Bangladesh. Patients had substantial and significant increases in TcpA-specific antibody-secreting cells in the circulation on day 7 after the onset of illness, as well as similar mucosal responses as determined by an alternate technique, the assay for antibody in lymphocyte supernatant. Significant increases in antibodies to TcpA were also seen in sera and feces of patients on days 7 and 21 after the onset of infection. Overall, 93% of the patients showed a TcpA-specific response in at least one of the specimens compared with the results obtained on day 2 and with healthy controls. These results demonstrate that TcpA is immunogenic following natural V. cholerae infection and suggest that immune responses to this antigen should be evaluated for potential protection against subsequent life-threatening illness.     

Phenotypic and genotypic changes in Vibrio cholerae O139 Bengal.

To find reasons for the recent decline of Vibrio cholerae O139 Bengal cholera in Bangladesh, phenotypic and genotypic changes in O139 isolates obtained from patients with cholera from 1993 to 1996 were studied. The isolates were tested for the presence of ctx and tcpA genes, hemagglutinin/protease (HA/P), capsule, D-mannose-sensitive hemagglutinin (MSHA), L-fucose-sensitive hemagglutinin (FSHA), tube test (tube) and CAMP test (CAMP) hemolytic activities, resistance to 2,4-diamino-6,7-diisopropyl pteridine (O/129) and trimethoprim-sulfamethoxazole (TMP-SMX), and genotype by pulsed-field gel electrophoresis (PFGE). All isolates possessed ctx and tcpA genes, HA/P, and a capsule. Most isolates were negative for FSHA, but although the majority of the isolates were positive for MSHA, no discernible trend in the activity was found during the study period. All early isolates were CAMP hemolysin positive and resistant to the vibriostatic compound O/129 and TMP-SMX, the two properties that could be used for the presumptive diagnosis of O139 cholera. However, subsequently, isolates that were CAMP hemolysin negative and susceptible to TMP-SMX and O/129 were increasingly encountered, with all the 1996 isolates being so, which suggested that these properties can no longer be used for the presumptive diagnosis of O139 cholera. V. cholerae O139 isolates that were CAMP hemolysin positive and resistant to O/129 and TMP-SMX produced a disease of greater severity than that caused by the CAMP hemolysin-negative and susceptible isolates on the basis of the lengths of stay of the hospitalized patients. The study period witnessed the evolution of four different genotypes by PFGE. All of these data suggested that the V. cholerae O139 isolates have undergone changes in some properties. However, how these changes influenced their prevalence relative to that of V. cholerae O1 in human infection is not clear. Studies of the environmental factors will provide the key for an understanding of the relative abundance of these vibrios.     

Rapid screening method for identification of cholera toxin-producing Vibrio cholerae O1 and O139.

A novel method of identifying cholera enterotoxin (CT)-producing Vibrio cholerae serogroups O1 and O139 was developed. The method uses degradation of NAD as a specific biochemical marker for the CT-producing strains. The substrate NAD at a concentration of 100 mumol/liter was markedly degraded when it was incubated at 37 degrees C for 2 h with the CT-producing stains at a final cell density equivalent to that of a twofold dilution of a McFarland no. 1 standard. NAD degradation was monitored by an enzyme-amplified color development assay. Subsequent tests conducted with a total of 119 strains of V. cholerae, including both clinical and environmental isolates, confirmed a significant correlation between NAD degradation and CT production for all V. cholerae strains belonging to serogroups O1 and O139. Since 2 of 11 non-O1, non-O139 V. cholerae strains not carrying the CT gene degraded NAD, serotyping of the strains prior to the test is recommended.     

Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae.

Vibrio cholerae causes epidemic diarrhea throughout the world. Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics. V. cholerae, like most bacteria, has developed resistance to some antibiotics. In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics. Bengal isolates showed unique trends in antimicrobial resistance. Many clinical laboratories use automated antibiotic susceptibility testing for V. cholerae. It is important to know if automated susceptibility test results for V. cholerae coincide with reported trends in antibiotic susceptibility. In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V. cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates. Vitek susceptibilities for V. cholerae showed a good correlation with preestablished epidemiological data. Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains. Regardless of serogroup, > or = 98% of the V. cholerae isolates tested were susceptible to most antibiotics tested by us. It is important to continue susceptibility testing of all new isolates of V. cholerae because of emerging resistant strains. However, V. cholerae remains susceptible to most of the available antibiotics.     

Increased Levels of Inflammatory Mediators in Children and Adults Infected with Vibrio cholerae O1 and O139

Investigations were carried out to study the production of factors associated with the innate immune response in the systemic and mucosal compartments in adults and children infected with Vibrio cholerae O1 and V. cholerae O139. The levels of nonspecific mediators of the innate defense system, i.e., prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄), and lactoferrin (Lf), as well as myeloperoxidase (MPO), were elevated at the acute stage of the disease in stools obtained from both O1- and O139-infected adults and children. In the systemic compartment, the levels of Lf were increased after onset of disease, which in children remained elevated up to convalescence compared to the healthy controls. Increased concentrations of C-reactive protein were seen in the sera of adult cholera patients at the acute stage of infection. Elevated levels of the nitric oxide (NO^·) metabolites (nitrite and nitrate [NO₂⁻ and NO₃⁻]) were detected in plasma but not in urine. The activity of the scavenger of reactive oxygen species, superoxide dismutase, was higher in the plasma of adults immediately after the onset of disease, suggesting that an active scavenging of reactive oxygen species was taking place. The concentration of 8-iso-prostaglandin F_2α remained unchanged in the systemic and mucosal compartments in the study subjects. After the recovery of patients from cholera, the concentration of the majority of the metabolites decreased to baseline levels by day 30 after the onset of infection. Immunohistochemical staining showed increased tissue expression of MPO, Lf, and inducible nitric oxide synthase at the acute stage in the duodenal biopsies of adults and rectal biopsies obtained from children with cholera. Very little difference was seen in the levels of the different inflammatory mediators in patients infected with V. cholerae O1 or the encapsulated V. cholerae O139. In summary, these results suggest that elevated concentrations of Lf, MPO, PGE₂, LTB₄, and NO^·, as well as other metabolites, during the acute stage of the disease indicate that the innate defense system, as well as the inflammatory process, is activated in both adults and pediatric patients infected with V. cholerae O1 and O139.     

霍乱弧菌O139菌毛提取及其疫苗的试制

目的　 寻求Ｏ１３９ 型霍乱弧菌菌毛提取纯化的最适方法和其疫苗研制。方法　ＳＤＳＰＡＧＥ,斑点酶免疫（ＤＢＩ） 检测和ＢＡＥＬＩＳＡ 法。采用ＰＥＬＡ 材料包封菌毛,成为缓释微球疫苗。结果电泳均存在相对分子质量为２０ ．５ ×１０３ 的条带;ＤＢＩ结果为阳性。小鼠血清ＩｇＧ 以皮下ＭＳＴＣＰ 组最高（１∶７ ６８０） ,唾液中的ｓＩｇＡ 以口服ＭＳＴＣＰ 组最高（１∶６０） 。保护力试验中口服组的保护力约为３０ ％ ～４０ ％ ,而皮下组为７０ ％ ～９０ ％ 。结论　 口服组主要诱导粘膜免疫,皮下组主要诱导全身性免疫。ＴＣＰ 是霍乱弧菌的共同抗原之一,可作为霍乱疫苗的候选抗原。     

Escalating association of Vibrio cholerae O139 with cholera outbreaks in India

Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other. In two of these outbreaks each, only V. cholerae O1 biotype ElTor or V. cholerae O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated. The interesting feature is the escalating association of V. cholerae O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139. The O139 strains from the six different outbreaks were genotypically closely related. These trends indicate a shift in the outbreak propensity of V. cholerae O139.     

Vibrio cholerae O139 conjugate vaccines: synthesis and immunogenicity of V. cholerae O139 capsular polysaccharide conjugates with recombinant diphtheria toxin mutant in mice

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to < or =50. Treatment with 2-ME reduced the titers of 17 of 20 patients to < or =50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.     

Characterization of Aeromonas trota strains that cross-react with Vibrio cholerae O139 Bengal.

It has previously been shown that Vibrio cholerae O139 Bengal shares antigens with V. cholerae serogroups O22 and O155. We detected six surface water isolates of Aeromonas trota that agglutinated in polyclonal antisera to V. cholerae O139 and V. cholerae O22 but not in antiserum to V. cholerae O155. On the basis of agglutinin-absorption studies, the antigenic relationship between the cross-reacting bacteria were found to be in an a,b-a,c fashion, where a is the common antigenic epitope and b and c are unique epitopes. The antigen sharing between A. trota strains and V. cholerae O139 was confirmed in immunoblot studies. However, A. trota strains did not react with two monoclonal antibodies specific for V. cholerae O139 and, consequently, tested negative in the Bengal SMART rapid diagnostic test for V. cholerae O139 which uses one of the monoclonal antibodies. A polyclonal antiserum to a cross-reacting A. trota strain cross-protected infant mice against cholera on challenge with virulent V. cholerae O139. All A. trota strains were cytotoxic for HeLa cells, positive for adherence to HEp-2 cells, and weakly invasive for HEp-2 cells; one strain was heat-stable toxin positive in the suckling mouse assay; however, all strains were negative for cholera toxin-like enterotoxin. Studies on bacteria that share somatic antigen with V. cholerae O139 may shed further light on the genesis of V. cholerae O139.     

Role of Vibrio cholerae O139 surface polysaccharides in intestinal colonization

Since the first occurrence of O139 Vibrio cholerae as a cause of cholera epidemics, this serogroup has been investigated intensively, and it has been found that its pathogenicity is comparable to that of O1 El Tor strains. O139 isolates express a thin capsule, composed of a polymer of repeating units structurally identical to the lipopolysaccharide (LPS) O side chain. In this study, we investigated the role of LPS O side chain and capsular polysaccharide (CPS) in intestinal colonization by with genetically engineered mutants. We constructed CPS-negative, CPS/LPS O side chain-negative, and CPS-positive/LPS O side chain-negative mutants. Furthermore, we constructed two mutants with defects in LPS core oligosaccharide (OS) assembly. Loss of LPS O side chain or CPS resulted in a approximately 30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process. The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize. To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro. These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile.     

Molecular Epidemiology of Reemergent Vibrio cholerae O139 Bengal in India

We report the prevalence of the O139 serogroup in Calcutta, India, after its reemergence in August 1996 and the spread of the reemerged clone to other parts of the country by using previously established molecular markers. Phenotypically, the reemerged Vibrio cholerae O139 displayed a difference compared to those that appeared in late 1992 and 1993 in that the current O139 strains are sensitive to co-trimoxazole. Ribotyping with the enzyme BglI produced two rRNA restriction patterns in the O139 strains isolated after August 1996, and these patterns were identical to those exhibited by strains of O139 isolated in 1992. Three clones of V. cholerae O139 are currently prevailing in the country, with strains exhibiting three bands after HindIII digestion and hybridization with a ctxA probe being dominant. The reemergence of V. cholerae O139 in Calcutta after a 32-month quiescent period reestablishes the O139 serogroup as an entity which is likely to play a crucial role in the temporal antigenic variations among the serogroups of V. cholerae causing cholera.