Changes in Cerebral Hemodynamics During Systemic Pulmonary Shunt and Pulmonary Artery Banding in Infants with Congenital Heart Disease.

Palliative surgery is often performed in the treatment of congenital heart disease. Two representative palliative procedures are the systemic pulmonary shunt and pulmonary artery banding. Dramatic changes in cerebral hemodynamics may occur in these operations due to changes in the pulmonary-to-systemic blood flow ratio and systemic oxygenation. However, there seem to be almost no studies evaluating them. Accordingly, we evaluated cerebral perfusion by transcranial Doppler ultrasonography and cerebral oxygenation by near infrared spectroscopy during these procedures. In the post hoc analysis of a previous prospective observational study, cerebral blood flow velocities of the middle cerebral artery measured by transcranial Doppler were compared between the start and end of surgery as were the pulsatility index and resistance index. The cerebral oxygenation values were also compared between the start and end of surgery. Twenty-two infants with systemic pulmonary shunt and 20 infants with pulmonary artery banding were evaluated. There were no significant differences of the flow velocities between the start and end of surgery in either procedure. The pulsatility index significantly increased after pulmonary artery banding, which may compete with the increase in cerebral perfusion due to the increase in systemic blood flow. The cerebral oxygenation decreased in both procedures, possibly due to an increase in body temperature. Arterial oxygen saturation was almost the same before and after both procedures. Contrary to our expectation, the changes in cerebral hemodynamics in the palliative operations were small if the management of physiological indices such as arterial oxygen saturation was properly performed during the procedures.

Cholera outbreaks in the El Tor biotype era and the impact of the new El Tor variants.

Vibrio cholerae O1, the causative agent of the disease cholera, has two biotypes namely the classical and El Tor. Biotype is a subspecific taxonomic classification of V. cholerae O1. Differentiation of V. cholerae strains into biotype does not alter the clinical management of cholera but is of immense public health and epidemiological importance in identifying the source and spread of infection, particularly when V. cholerae is first isolated in a country or geographic area. From recorded history, till date, the world has experienced seven pandemics of cholera. Among these, the first six pandemics are believed to have been caused by the classical biotype whereas the ongoing seventh pandemic is caused by the El Tor biotype. In recent years, new pathogenic variants of V. cholerae have emerged and spread throughout many Asian and African countries with corresponding cryptic changes in the epidemiology of cholera. In this chapter, we describe the outbreaks during the seventh pandemic El Tor biotype era spanning more than five decades along with the recent advances in our understanding of the development, evolution, spread, and impact of the new variants of El Tor strains.

Passive immunity with multi-serotype heat-killed Shigellae in neonatal mice.

The short- and long-term passive protective efficacy of a mixture of heat-killed cells of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6, S. boydii 4, and S. sonnei) were studied in neonatal mice. Neonatal mice from immunized dams exhibited significant short- and long-term passive protection against individual challenge by each of the six Shigella strains. High IgG and IgA titers against the lipopolysaccharide from each of the six Shigella strains were observed in sera from immunized dams.

The role of Vibrio cholerae genotyping in Africa.

Toxigenic Vibrio cholerae, the causative agent of the disease cholera, is prevalent in the African continent from the 1970s when the seventh pandemic spread from Asia to Africa. In the past decade, cholera has caused devastating outbreaks in much of Africa, illustrated by the recent cholera epidemics in Zimbabwe and regions of central Africa. Given the extent of cholera in Africa, a robust and efficient surveillance system should be in place to prevent and control the disease in this continent. Such a surveillance system would be greatly bolstered by use of molecular typing techniques to identify genetic subtypes. In this review, we highlight the role that modern molecular typing techniques can play in tracking and aborting the spread of cholera.

Vibrio cholerae non-O1, non-O139 serogroups and cholera-like diarrhea, Kolkata, India.

We identified 281 Vibrio cholerae non-O1, non-O139 strains from patients with diarrhea in Kolkata, India. Cholera-like diarrhea was the major symptom (66.0%); some patients (20.3%) had severe dehydration. These strains lacked the ctxA gene but many had hlyA, rtxA, and rtxC genes. Pulsed-field gel electrophoresis showed no genetic link among strains.

Protective immunity by oral immunization with heat-killed Shigella strains in a guinea pig colitis model.

The protective efficacy of and immune response to heat-killed cells of monovalent and hexavalent mixtures of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, Shigella boydii 4, and Shigella sonnei) were examined in a guinea pig colitis model. A monovalent or hexavalent mixture containing 1 × 10(7) of each serogroup/serotype of heat-killed Shigella cells was administered orally on Days 0, 7, 14 and 21. On Day 28, the immunized animals were challenged rectally with 1 × 10(9) live virulent cells of each of the six Shigella serogroups/serotypes. In all immunized groups, significant levels of protection were observed after these challenges. The serum titers of IgG and IgA against the lipopolysaccharide of each of the six Shigella serogroups/serotypes increased exponential during the course of immunization. High IgA titers against the lipopolysaccharide of each of the six Shigella serogroups/serotypes were also observed in intestinal lavage fluid from all immunized animals. These data indicate that a hexavalent mixture of heat-killed cells of the six Shigella serogroups/serotypes studied would be a possible broad-spectrum candidate vaccine against shigellosis.

Vibrio fluvialis in patients with diarrhea, Kolkata, India.

We identified 131 strains of Vibrio fluvialis among 400 nonagglutinating Vibrio spp. isolated from patients with diarrhea in Kolkata, India. For 43 patients, V. fluvialis was the sole pathogen identified. Most strains harbored genes encoding hemolysin and metalloprotease; this finding may contribute to understanding of the pathogenicity of V. fluvialis.

Development and evaluation of a PCR assay for tracking the emergence and dissemination of Haitian variant ctxB in Vibrio cholerae O1 strains isolated from Kolkata, India.

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata during April 2006, and 93.3% of strains isolated during 2011 carried the new allele. Dendrogram analysis showed a pulsed-field gel electrophoresis (PFGE) pattern of the new variant strains isolated recently that was distinct from the PFGE pattern of the strains carrying classical ctxB that closely matched the 2006 to 2007 variant strains.

Vibrio cholerae/mimicus in fecal microbiota of healthy children in a cholera endemic urban slum setting in Kolkata, India.

During a double-blind, randomized, placebo-controlled probiotic trial among 3758 children residing in an urban slum in Kolkata, India, Vibrio cholerae/mimicus was detected in fecal microbiota of healthy children. The importance of this finding in the local, regional and global transmission of cholera is discussed.

Production and properties of lipase of Aeromonas sobria.

Aeromonas have been isolated from a wide variety of aquatic environments. However the number of Aeromonas in sea water is extremely small compared to that in fresh water. In in vitro culture, Aeromonas can grow in mediums containing NaCl at a concentration of 3.0%, this concentration corresponding to that of sea water. It is unclear why the number of Aeromonas is low in sea water. Exoproteins of bacteria are thought to be important for bacterial growth and survival in the environment. Previously, the present authors have shown that mediums containing 3.0% NaCl suppress production of two proteases, serine protease and metalloprotease. In this experiment, other exoproteins whose production is influenced by the amount of NaCl in the medium were analyzed. A protein whose production is repressed in medium containing 3.0% NaCl was found and purified. Biological assay of the purified protein showed that it degrades tributyrin and hydrolyzes para-nitrophenyl-fatty acylesters. These results show that the protein is a lipase. Subsequently, the nucleotide sequence of the gene encoding the lipase was determined and the amount of mRNA of the lipase gene in the cells measured. It was found that transcription of the gene is not inhibited by NaCl in the medium. This result indicates that the lipase might be synthesized, but the folding process to become an active structure does not progress smoothly in a medium containing 3.0% NaCl.

Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells.

Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells.

Development of a sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli.

A sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was developed for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli by using specific primers. Counts of the enteric pathogens spiked in human stools were quantified at the lower detection limit of 10(3) cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 10(5) to 10(6) cells/g stool. The bacterial counts determined by RT-qPCR were almost equivalent to those determined by the culture method and fluorescence in situ hybridization (FISH) during the course of in vitro culture. Bacterial rRNA in the stools was stable for at least 4 weeks when the stools were kept as the suspensions in RNA-stabilizing agent, RNAlater®, even at 37(o) C. These data suggested that the rapid and high sensitive rRNA-targeted RT-qPCR was applicable for the accurate quantification of viable enteric pathogens, such as V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli.

Vibrio cholerae O139 genomes provide a clue to why it may have failed to usher in the eighth cholera pandemic.

Cholera is a life-threatening infectious disease that remains an important public health issue in several low and middle-income countries. In 1992, a newly identified O139 Vibrio cholerae temporarily displaced the O1 serogroup. No study has been able to answer why the potential eighth cholera pandemic (8CP) causing V. cholerae O139 emerged so successfully and then died out. We conducted a genomic study, including 330 O139 isolates, covering emergence of the serogroup in 1992 through to 2015. We noted two key genomic evolutionary changes that may have been responsible for the disappearance of genetically distinct but temporally overlapping waves (A-C) of O139. Firstly, as the waves progressed, a switch from a homogenous toxin genotype in wave-A to heterogeneous genotypes. Secondly, a gradual loss of antimicrobial resistance (AMR) with the progression of waves. We hypothesize that these two changes contributed to the eventual epidemiological decline of O139.

Diarrheagenic pathogens in polymicrobial infections.

During systematic active surveillance of the causes of diarrhea in patients admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata, India, we looked for 26 known gastrointestinal pathogens in fecal samples from 2,748 patients. Samples from about one-third (29%) of the patients contained multiple pathogens. Polymicrobial infections frequently contained Vibrio cholerae O1 and rotavirus. When these agents were present, some co-infecting agents were found significantly less often (p = 10 (-5) to 10 (-33), some were detected significantly more often (p = 10 (-5) to 10 (-26), and others were detected equally as often as when V. cholerae O1 or rotavirus was absent. When data were stratified by patient age and season, many nonrandom associations remained statistically significant. The causes and effects of these nonrandom associations remain unknown.

Live non-invasive Shigella dysenteriae 1 strain induces homologous protective immunity in a guinea pig colitis model.

A non-invasive live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed from a Shiga toxin gene deleted mutant of Shigella dysenteriae 1 by introducing a plasmid vector pPR1347 that carried a lipopolysaccharide biosynthesis gene (rfb and rfc) of Salmonella typhimurium. In guinea pigs, four successive oral administrations of LTSH Δstx showed complete protection against rectal challenge with wild type S. dysenteriae 1 strain. Exponential increase of the serum IgG and IgA titer against lipopolysaccharide of LTSH Δstx was observed during immunization, peaked on day 28 and remained at that level until day 35 after the initiation of the immunization. In intestinal lavage of the immunized animals, significant increase of IgA titer against lipopolysaccharide of LTSH Δstx was also observed. These data suggested that LTSH Δstx could be a useful candidate to induce protective immunity against S. dysenteriae 1 infection.

Vibrio parahaemolyticus, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli and Vibrio cholerae.

This review highlighted the following: (i) pathogenic mechanism of the thermostable direct hemolysin produced by Vibrio parahaemolyticus, especially on its cardiotoxicity, (ii) heat-labile and heat-stable enterotoxins produced by enterotoxigenic Escherichia coli, especially structure-activity relationship of heat-stable enterotoxin, (iii) RNA N-glycosidase activity of Vero toxins (VT1 and VT2) produced by enterohemorrhagic Escherichia coli O157:H7, (iv) discovery of Vibrio cholerae O139, (v) isolation of new variant of Vibrio cholerae O1 El Tor that carries classical ctxB, and production of high concentration of cholera toxin by these strains, and (vi) conversion of viable but nonculturable (VBNC) Vibrio cholerae to culturable state by co-culture with eukaryotic cells.

Bacterial exotoxins downregulate cathelicidin (hCAP-18/LL-37) and human beta-defensin 1 (HBD-1) expression in the intestinal epithelial cells.

Cathelicidin (hCAP-18/LL-37) and beta-defensin 1 (HBD-1) are human antimicrobial peptides (AMPs) with high basal expression levels, which form the first line of host defence against infections over the epithelial surfaces. The antimicrobial functions owe to their direct microbicidal effects as well as the immunomodulatory role. Pathogenic microorganisms have developed multiple modalities including transcriptional repression to combat this arm of the host immune response. The precise mechanisms and the pathogen-derived molecules responsible for transcriptional downregulation remain unknown. Here, we have shown that enteric pathogens suppress LL-37 and HBD-1 expression in the intestinal epithelial cells (IECs) with Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) exerting the most dramatic effects. Cholera toxin (CT) and labile toxin (LT), the major virulence proteins of V. cholerae and ETEC, respectively, are predominantly responsible for these effects, both in vitro and in vivo. CT transcriptionally downregulates the AMPs by activating several intracellular signalling pathways involving protein kinase A (PKA), ERK MAPKinase and Cox-2 downstream of cAMP accumulation and inducible cAMP early repressor (ICER) may mediate this role of CT, at least in part. This is the first report to show transcriptional repression of the AMPs through the activation of cellular signal transduction pathways by well-known virulence proteins of pathogenic microorganisms.

Vibrio cholerae O1 clinical strains isolated in 1992 in Kolkata with progenitor traits of the 2004 Mozambique variant.

Retrospective analysis led to the detection of two Vibrio cholerae variant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml(-1)) resistance, positivity in Voges-Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to Kolkata V. cholerae O1 strains isolated on or before 1992. NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.