Are natural reservoirs important for cholera surveillance? The case of an outbreak in a Brazilian estuary.

UNLABELLED: Paranaguá Bay is one of the largest estuarine systems on the Southern Brazilian coast. The only recorded cholera outbreak in this region since the early 20th century occurred in 1999 and resulted in 467 cases and at least three reported deaths in a population of approx. 150 000 people. This short communication reports historical, unpublished data related to that outbreak. Water, zooplankton and bivalve samples were collected and evaluated using direct fluorescence assay to determine whether Vibrio cholerae serogroups O1 and O139 were present in the estuarine system at that time. Most of the water (83%) and zooplankton samples (75%) were positive for V. cholerae O1, while V. cholerae O139 was not detected. Shellfish (Mytella sp.) were also positive for V. cholerae O1. These results indicate that the estuary, including biological vectors such as copepods and bivalves, comprise an important reservoir of V. cholerae O1 and a probable waterborne pathway for the disease, in addition to contamination with untreated sewage. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite most of the cholera cases that occurred in Brazil during the 7th pandemic were located in the northern areas of the country, a significant outbreak in Paranaguá, an estuary in the south coast, resulted in at least three deaths in 1999. We report here the detection of Vibrio cholerae O1 in water, zooplankton and bivalve samples during the outbreak, using direct fluorescence assay as an alternative method for the traditional plate culture employed at the time by the Brazilian Sanitary Agency. Results demonstrate that aquatic natural reservoirs comprise a potential route of transmission of cholera, in addition to untreated sewage and routine monitoring is recommended.

Diversity of somatic coliphages in coastal regions with different levels of anthropogenic activity in São Paulo State, Brazil.

Bacteriophages are the most abundant and genetically diverse viruses on Earth, with complex ecology in both quantitative and qualitative terms. Somatic coliphages (SC) have been reported to be good indicators of fecal pollution in seawater. This study focused on determining the concentration of SC and their diversity by electron microscopy of seawater, plankton, and bivalve samples collected at three coastal regions in São Paulo, Brazil. The SC counts varied from <1 to 3.4 × 10(3) PFU/100 ml in seawater (73 samples tested), from <1 to 4.7 × 10(2) PFU/g in plankton (46 samples tested), and from <1 to 2.2 × 10(1) PFU/g in bivalves (11 samples tested). In seawater samples, a relationship between the thermotolerant coliforms and Escherichia coli and SC was observed at the three regions (P = 0.0001) according to the anthropogenic activities present at each region. However, SC were found in plankton samples from three regions: Baixada Santista (17/20), Canal de São Sebastião (6/14), and Ubatuba (3/12). In seawater samples collected from Baixada Santista, four morphotypes were observed: A1 (4.5%), B1 (50%), C1 (36.4%), and D1 (9.1%). One coliphage, Siphoviridae type T1, had the longest tail: between 939 and 995 nm. In plankton samples, Siphoviridae (65.8%), Podoviridae (15.8%), Microviridae (15.8%), and Myoviridae (2.6%) were found. In bivalves, only the morphotype B1 was observed. These SC were associated with enteric hosts: enterobacteria, E. coli, Proteus, Salmonella, and Yersinia. Baixada Santista is an area containing a high level of fecal pollution compared to those in the Canal de São Sebastião and Ubatuba. This is the first report of coliphage diversity in seawater, plankton, and bivalve samples collected from São Paulo coastal regions. A better characterization of SC diversity in coastal environments will help with the management and evaluation of the microbiological risks for recreation, seafood cultivation, and consumption.

Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil.

The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were alphaA (65%), followed by alphaB (17.5%), alphaD (9%), alphaAaD hybrids (5%), and alphaC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.

Genotypes associated with virulence in environmental isolates of Vibrio cholerae.

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.

Characterisation of diarrhoeagenic Escherichia coli clones by ribotyping and ERIC-PCR.

The ability of ribotyping and enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) to discriminate diarrhoeagenic Escherichia coli clones of 122 strains belonging to 26 distinct serotypes was evaluated. The 26 serotypes corresponded to 24 ribotypes and 25 ERIC-types. Correlation between multilocus enzyme electrophoresis, ERIC-PCR and ribotyping was c. 90% for the dominant ribotypes. Related clones such as O55:H7 and O157:H7 presented similar ribotypes and clustered together in a dendrogram, and the two divergent clonal groups of enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) were included in distinct branches. The results suggest the possibility of applying these two simpler techniques as tools to identify clones of diarrhoeagenic E. coli.