Genetic diversity and antibiotic resistance of clinical and environmental Vibrio Cholerae suggests that many serogroups are reservoirs of resistance.

Vibrio cholerae is an important human pathogen and the cause of cholera. Since genetic variation and antibiotic resistance of strains have implications for effective treatment of the disease, we examined the genetic diversity and antibiotic resistance profile in 92 clinical strains (serogroup O1) and 56 environmental strains (O1 antigen, 42 strains; non-O1 antigen, 14 strains) isolated in Brazil between 1991 and 1999. Clinical and environmental O1 strains showed greater drug resistance compared to environmental non-O1 strains. Nearly all clinical O1 strains were resistant to one or more antibiotics while half of the environmental O1 and non-O1 strains were resistant to one or more antibiotics. No plasmids or class 1 integrons were detected in the strains by PCR analysis. Multilocus enzyme electrophoresis analysis (MLEE) suggests most of the O1 strains belong to a single (South American) clone that is related but different to seventh-pandemic strains isolated from other parts of the world. Our results show that there is a close genetic relationship between clinical and environmental O1 strains and that many serogroups and the environment can be a reservoir for antibiotic resistance.

Genetic diversity of Neisseria meningitidis strains isolated in Rio de Janeiro, Brazil, evaluated by multilocus enzyme electrophoresis.

AIMS: To analyse Neisseria meningitidis isolates from meningococcal meningitis cases in Rio de Janeiro (Brazil) from 1990 to 1993 and 1999-2002, to determine the genetic and relatedness with hypervirulent and epidemic strains. METHODS AND RESULTS: The isolates were analysed by multilocus enzyme electrophoresis (MEE) clustering into 83 electrophoretic types (ET). All isolates from 1999 to 2002, formed a cluster which included one strain of the ET-5 complex worldwide associated with epidemics. CONCLUSIONS: The overall results suggested a panmictic structure probably because of recombination events. The observation of a separated cluster including isolates from 1999 to 2002 and an ET-5 complex strain, also suggested the introduction of strains genetically related with this hypervirulent complex in the State of Rio de Janeiro (Brazil) over the last 5 years. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of strains related to the ET-5 complex in several states of Brazil was already described elsewhere, but this is the first time it was reported in the State of Rio de Janeiro. Our findings reinforce the necessity to genetically determine the clones which should be considered to produce a national vaccine against meningococcal meningitis.

Differentiation of environmental and clinical isolates of Vibrio mimicus from Vibrio cholerae by multilocus enzyme electrophoresis.

In this study, we demonstrated that analyzed strains of Vibrio mimicus and Vibrio cholerae could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci. We also showed that the combination of four enzymatic loci enables us to differentiate these two species. Our results showed that the ribosomal intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between V. mimicus and V. cholerae. On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical strains were analyzed.

Toxin-co-regulated pilus cluster in non-O1, non-toxigenic Vibrio cholerae: evidence of a third allele of pilin gene.

Polymerase chain reaction has been used to detect the presence of the virulence associated gene, tcpA and part of the promoter distal region of the toxin-co-regulated pilus cluster in non-O1, non-toxigenic, Vibrio cholerae. The amplified regions were characterised by restriction fragment length polymorphism and heteroduplex motility assay. We describe the nucleotide sequence of the tcpA gene fragment from non-toxigenic vibrios from clinical and environmental sources. The present study shows that there are at least three types of the tcpA gene among V. cholerae and the primers specific for the classical tcpA gene, amplify all biotypes. A sequence similarity in other regions of the toxin-co-regulated pilus cluster is suggested. The evidences for the presence of this cluster among non-toxigenic vibrios is, to our knowledge, reported for the first time. The use of restriction fragment length polymorphism for typing the tcpA and studying the alleles distribution is proposed.

Evolutionary control of infectious disease: prospects for vectorborne and waterborne pathogens.

Evolutionary theory may contribute to practical solutions for control of disease by identifying interventions that may cause pathogens to evolve to reduced virulence. Theory predicts, for example, that pathogens transmitted by water or arthropod vectors should evolve to relatively high levels of virulence because such pathogens can gain the evolutionary benefits of relatively high levels of host exploitation while paying little price from host illness. The entrance of Vibrio cholerae into South America in 1991 has generated a natural experiment that allows testing of this idea by determining whether geographic and temporal variations in toxigenicity correspond to variation in the potential for waterborne transmission. Preliminary studies show such correspondences: toxigenicity is negatively associated with access to uncontaminated water in Brazil; and in Chile, where the potential for waterborne transmission is particularly low, toxigenicity of strains declined between 1991 and 1998. In theory vector-proofing of houses should be similarly associated with benignity of vectorborne pathogens, such as the agents of dengue, malaria, and Chagas' disease. These preliminary studies draw attention to the need for definitive prospective experiments to determine whether interventions such as provisioning of uncontaminated water and vector-proofing of houses cause evolutionary reductions in virulence.

The Amazonia variant of Vibrio cholerae: molecular identification and study of virulence genes.

The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the E1 Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.

Aortic reconstruction with crimped bovine pericardial conduits.

BACKGROUND AND AIM OF THE STUDY: A bovine pericardial conduit processed in glutaraldehyde was designed, incorporating the principle of crimping used for synthetic vascular prostheses. The crimping process did not affect the integrity of collagen fibers and tissue structure. This conduit, designed for aortic reconstruction, is available in different sizes, with or without a biological valve. METHODS: Between October 1989 and May 1997, 40 patients with aortic dissection, aortic aneurysm, aortic coarctation or aortoiliac occlusive disease underwent aortic reconstruction using this vascular substitute. Procedures included total reconstruction of the ascending aorta and aortic valve with reimplantation of coronary arteries (nine patients), single ascending thoracic aorta (six), descending thoracic aorta (two), aortic arch (one) and thoracoabdominal aorta (one); the abdominal aorta was reconstructed in 21 cases, including those undergoing aortoiliac or aortofemoral bypass. RESULTS: The hospital mortality rate was 20% (eight patients); causes of deaths were low cardiac output, recurrence of aortic dissection, multiple organ failure and bleeding. Mean follow up was 3.6 years; total follow up was 114 patient-years. Late conduit-related complications occurred in four patients, including a limb obstruction in one patient subjected to aortofemoral bypass and infection of three resulting in pseudoaneurysm (incidence of 3.5 +/- 1.8% per patient-year). All underwent reoperation. There were four late deaths due to sudden death, coronary artery disease, pneumonia and metabolic complications of diabetes and renal failure (incidence of 3.5 +/- 1.8% per patient year). The eight-year actuarial survival rate was 63.7 +/- 11.6%, including hospital mortality, and the eight-year actuarial freedom from conduit failure due to primary tissue structural degeneration was 100%. CONCLUSIONS: The crimping design provides a circular tube which makes construction of the anastomosis easier, retains its shape with bending, and avoids kinking. The material is very soft, easy to handle and suture, coapts nicely to suture lines resulting in a hemostatic anastomosis. The eight-year follow up demonstrated a satisfactory performance without report of fibrosis, calcification or aneurysmal dilation.

Mitral valve replacement with glutaraldehyde preserved aortic allografts.

OBJECTIVE: To present long-term results after mitral valve replacement with stent mounted glutaraldehyde preserved aortic allografts in patients older than 15 years. The clinical support for this study was to combine the glutaraldehyde technique of biological tissue preservation with the advantages of allografts when compared to xenografts. This was demonstrated in previous studies using other methods of tissue processing. METHODS: Between September 1984 and November 1994, 70 patients aged 16-77 years (mean 35.4 years) underwent mitral valve replacement with this preserved and mounted allograft. Of these, 40 patients (57.2%) were aged 16-35 years and 15 (21.4%) were 20 years old or younger; 46 (65.7%) were females and 24 (34.3%) males. Single mitral valve replacement was performed in 60 patients and 10 were also subjected to other combined cardiac procedures. Human aortic valves were obtained during routine autopsy, processed in glutaraldehyde and mounted into flexible stents, using the same technique as that used for porcine bioprostheses. RESULTS: Hospital mortality was 1.4%. Total follow-up was 543.1 patient-years, corresponding to a mean follow-up of 7.9 years per patient. Echocardiography demonstrated a hemodynamic performance similar to porcine bioprostheses. Late mortality was 0.7 +/- 0.6% per patient-year and the causes were congestive heart failure in 2, prosthetic endocarditis in 1 and acute myocardial infarction in 1. The 12-year actuarial survival was 92.4 +/- 3.2%. The incidence of late complications was 5.2 +/- 1.2% per patient-year, including congestive heart failure, prosthetic endocarditis, periprosthetic leak, thromboembolic episodes, recurrence of rheumatic disease, coronary artery disease and allograft failure. Complications related to heart disease represented 2.8 +/- 0.6% and allobioprosthesis-related 2.4 +/- 0.5% per patient-year. The 12-year actuarial freedom from primary valve failure was 81.0 +/- 15.0%. The incidence of reoperations was 1.5 +/- 0.8% per patient-year and the main indication was prosthetic endocarditis. Other causes were periprosthetic leak, aortic insufficiency in the native aortic valve and allobioprosthesis dysfunction. Functional results demonstrated a significant improvement in patients clinical condition. CONCLUSION: This 12-year follow-up shows a very low incidence of primary allograft failure for patients older than 15 years undergoing mitral valve replacement, and much superior than our results with porcine bioprosthesis in the same age group. This supports our assumption that this investigational valve represents a new advance in cardiac valve surgery.

Detection of Vibrio cholerae and V. mimicus heat-stable toxin gene sequence by PCR.

Previously the heat-stable enterotoxin in Vibrio cholerae and V. mimicus has been detected by suckling mouse assay, a non-specific approach, and by DNA probes, a time-consuming method. This report describes a polymerase chain reaction (PCR) procedure for the detection of the stn (NAG-ST) and sto (O1-ST) gene sequences that is rapid and specific, allowing toxin gene molecular characterisation. A total of 34 V. cholerae and V. mimicus isolates was examined for ST and CT genes. The NAG-ST gene sequence was amplified in 13 of 22 non-O1/non-O139 V. cholerae and three of five V. mimicus strains. A new enterotoxin gene sequence pattern was found with MseI and TaqI restriction endonuclease PCR fragment digestion of two V. cholerae isolates, in addition to the pattern anticipated from the Genbank sequence, and found with the other ST+. These results show that ST-PCR detection is useful for the characterisation of V. cholerae and V. mimicus.

The distinction of pathogenic Vibrio cholerae groups using arbitrarily primed PCR fingerprints.

Pathogenic Vibrio cholerae strains were compared by fingerprinting with arbitrarily primed polymerase chain reaction (AP-PCR). They were O1 classical and El Tor strains and recent non-O1 Bengal strains. Ten oligonucleotides from a total of fifty-two tested gave distinctive patterns, and these strains were separated into four groups. A second technique, amplification of 16S/23S rRNA spacers with a pair of oligonucleotides, was also used. Various bands were obtained, and the result can be treated as an additional fingerprint with a different pattern for each of the groups. The method of AP-PCR fingerprinting is fast and sensitive. A test of the stability of the El Tor patterns was done with a set of strains isolated during the present Brazilian epidemics. Examples of AP-PCRs with non-O1 strains are given. A typing scheme is proposed in which oligo 1 is first used, and depending on the fingerprint obtained, additional oligonucleotides are used to confirm the classification of the strain. It is proposed that the AP-PCR technique be used for epidemiological studies, analysing strains reaching new locations or environmental isolates suspected of being pathogenic. It will be particularly helpful in cases in which traditional methods cannot clearly classify the strain.

New variant of Vibrio cholerae O1 from clinical isolates in Amazonia.

A survey of pathogenic Vibrio cholerae O1 strains from the north of Brazil by using arbitrarily primed PCR fingerprints revealed a group of strains with similar fingerprint patterns that are distinct from those of the current El Tor epidemic strain. These strains have been analyzed by in vivo and in vitro techniques and the group has been denominated the Amazonian variant of V. cholerae O1.

Vibrio cholerae O139 synonym bengal is closely related to Vibrio cholerae El Tor but has important differences.

Although Vibrio cholerae O139 synonym Bengal strains, from the current epidemics in India and Bangladesh, are closely related to seventh-pandemic strains, as shown by multilocus enzyme electrophoresis, Bengal strains are encapsulated and portions of the O1 antigen biosynthetic complex genes found in O1 strains are altered or lacking. Encapsulated Bengal strains showed resistance to killing by normal human serum. The presence of the capsule suggests the potential for bloodstream invasion in susceptible hosts and has profound implications for vaccine development.

An analysis of the V1 and V2 regions of Vibrio cholerae and Vibrio mimicus 16S rRNA.

The V1 and V2 variable regions of the 16S rRNA gene of three strains of V. cholerae and one strain of V. mimicus were amplified by PCR. Fragments containing both regions were cloned into M13mp18 using Smal and sequenced by the dideoxy method. The 263-bp sequence from a strain isolated during the 1991 cholera outbreak in Brazil was deposited in Genbank under the accession number L05178. Except for an extra G in one of the strains, the three V. cholerae sequences were identical. The V. mimicus sequence was very similar, with only two substitutions. We compared these sequences with the Vibrio 16S rRNA sequences described by Dorsch et al. in 1992. It was noted that the V1 region, including helix 6 and its associated loop, comprised two different sizes and sequences in the various Vibrio species. While V. cholerae, V. mimicus, V. vulnificus, V. anguillarum and V. diazotrophicus had a 46-nucleotide V1, other species such as V. parahaemolyticus, V. proteolyticus, V. alginolyticus, V. campbellii and V. hollisae had longer 54- or 55-nucleotide regions, with a different consensus sequence. The phylogeny of Vibrio was analysed using the sequenced region and its equivalent in other species, by means of the "Phylip" software package. Species with a short helix 6 were grouped together, as were species with a long helix. Dorsh et al.'s analysis is discussed in relation to this "helix 6 split".

Identification of Vibrio cholerae by enzyme electrophoresis.

Zymovar analysis of 260 strains of Vibrio cholerae plus 3 reference strains of V. mimicus, using 13 structural loci, led to the grouping of strains in 73 zymovars (strain or group of strains sharing the same alleles). Effective separation of strains, distinction of V. cholerae strains from closely related V. mimicus and the detection of 2 vibrio strains, including one with two O1 serovars, in supposedly pure collection cultures, illustrate the potential of zymovar analysis in the identification of V. cholerae isolates. Two El Tor strains from USA, one CT+ and the other CT-, shared the same zymovar 71, while 127 typical El Tor strains belonged to zymovar 14.

Early experience with crimped bovine pericardial conduit for arterial reconstruction.

A bovine pericardial conduit was developed in the laboratory incorporating the principle of crimping used for synthetic vascular prostheses. The pericardium was processed in glutaraldehyde and the tube was crimped by a technique which preserves the integrity of collagen fibres. This vascular substitute presents a non-thrombogenic and non-porous inner surface which does not require preclotting and does not leak. The material is very soft, easy to handle and suture, coapts nicely to suture lines resulting in a hemostatic anastomosis. The crimping design provides longitudinal elasticity and resistance to collapsing, retains its shape with bending and avoids kinking. Crimping provides a circular tube which makes the construction of the anastomosis easier. Experimental studies in dogs demonstrated absence of thromboembolism with the conduit implanted in the abdominal aorta. Fibrin accumulation was not noted in the convexities of the crimps. This conduit was designed for aortic and pulmonary reconstruction and available in different sizes with or without a biological valve. Initial clinical experience included its use in 10 patients with aortic dissections or aortic aneurysms from August 1989 to March 1990. A reconstruction of the abdominal aorta was performed in 2 patients, the descending thoracic aorta in 2, the ascending aorta in 2 and the ascending aorta including the aortic valve and reimplantation of coronary arteries in 4. For the latter 4, composite crimped pericardial tubes containing a porcine bioprosthesis were used. An additional patient with a single ventricle underwent a Fontan type operation also employing a valved crimped pericardial conduit.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac valve replacement with glutaraldehyde preserved aortic allografts.

From September 1984 to December 1988, 144 patients underwent cardiac valve replacement using glutaraldehyde preserved stent mounted aortic allografts. The mean age was 21.4 years (54.9% were 15 years old or younger). The mitral valve was replaced in 125 patients, the aortic valve in 7, the pulmonary valve in 3, the tricuspid in 1, the mitral and tricuspid in 1, and the aortic and mitral in 7. Hospital mortality was 2.8% (4 patients). Total follow-up was 396.3 patient-years and the mean follow-up was 2.8 years per patient. The 5-year actuarial survival was 73.8% +/- 11.9%. The 4-year actuarial survival for patients aged 15 or younger was 81.4% +/- 7.1%. The overall mortality was 2.8% +/- 0.8%/per patient-year. The incidence of valve-related complications was 4.8% +/- 1.1%/per patient-year, and the calcification rate was 3.0% +/- 0.9%/per patient-year and was the main complication reported in 12 patients, all under the age of 15 years. It occurred 14-47 months after implantation (mean 32.7 months). Five-year actuarial freedom from valve dysfunction due to calcification was 82.6% +/- 5.0% and for patients aged 15 or younger was 69.9% +/- 8.8%. The incidence of reoperation was 3.3% +/- 0.9%/per patient-year. These initial results demonstrate a 5-year actuarial freedom from primary valve failure due to fibrocalcification superior to the results obtained with xenobioprostheses in the paediatric age group.

A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis.

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains, B. cereus (8), including two of var. mycoides, and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus. Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.

A zymovar analysis of Vibrio cholerae isolated in Australia.

Zymovar analysis was used to study 50 strains of Vibrio cholerae O1 and 40 strains of V. cholerae non-O1 isolated in Australia. The strains were assigned to 42 zymovars; the O1 strains to 9 types and the non-O1 strains to 33 types, with no overlapping between serovars. All the human O1 isolates, regardless of their ability to produce cholera toxin (CT), and all the CT-producing O1 environmental isolates, were type Z14. The remaining O1 strains and the non-O1 strains belonged to a variety of zymovars, and more than one zymovar was present in some rivers.