DETECTION AND TEST SUSCEPTIBILITY SANITIZERS OF

DIARRHEAGENIC Escherichia coli STRAINS ISOLATED BEACHES OF SÃO LUÍS – MARANHÃO

ALMEIDA, N.C.1*, FIGUEIREDO, P.M.S.2*

1*_{Graduação em Engenharia Ambiental, Centro Universitário do Maranhão – UniCeuma. Rua Josué} Montello, 01, Renascença II. São Luís-MA. 65075-120. 1* [email protected] 2*_{Doutora em Microbiologia, Centro Universitário do Maranhão.} 2*_{[email protected]}

ABSTRACT

The marine environment is composed of major importance for society because the uses that this ecosystem provides. Thus, the water quality of this environment is key to its uses are not harmful to health. The city of São Luís located in the state of Maranhão - Brazil, has no effective treatment of wastewater, which are released directly into rivers or on beaches. This is strong evidence that the coastal environment can be indicators of fecal contamination (FIB). Samples contaminated with fecal coliform from the main beaches of São Luís, such as Ponta d'Areia, São Marcos, Calhau and Olho d’Água, were analyzed with the objective of determining which pathogens and which diarrheagenic strains of

Escherichia coli are present on beaches and in addition, be tested for susceptibility to

sanitizers strain of E. coli. The isolation and identification of pathogenic bacterial strains of the samples were performed by means of selective and EnteroKit B, whereas the identification of diarrheagenic strains and toxin production of Escherichia coli were performed by PCR using specific primers. To test the susceptibility of strains of E. coli sanitizers was performed by the method of successive dilutions, which were made in five different dilutions. In all sites were identified species of Enterobacteria such as E. coli,

Serratia liquefaciens, Hafnia alvei, Salmonella spp., Serratia sp., and the predominance of

the first kind. Strains of E. coli subjected to PCR were identified mostly as ETEC, equivalent to 82%, and only on the beaches of Ponta d'Areia and Olho d’Água subtypes were identified EHEC and EAEC. The sanitizers were tested two brands of detergents, alcohol and two brands of household bleach. The latter presented a higher efficiency in presenting five dilutions tested bactericidal for all strains of E. coli. The detergent brand II showed greater efficiency in comparison with detergent brand I, since the first one presented a more bactericidal and bacteriostatic against the second. But the domestic alcohol for all strains of Escherichia coli tested, inhibited the growth of microorganisms, and their bactericidal action at much lower compared to other sanitizers.

Keywords: Enterobacteria; Diarrheagenic Escherichia coli; Sanitizers; beaches of São Luís – MA.

DETECTION OF 1,3-PROPANEDIOL BIOSYNTHESIS GENES IN Klebsiella

pneumoniae GLC29

FLORA, A.B.1*,CONTIERO, J.2, SILVA, L.F.1; GOMEZ, J.G.C.1

1

Depto de Microbiologia, Universidade de São Paulo Av. Professor Lineu Prestes, nº 1374, Lab 172, ICB II São Paulo - SP. *[email protected]

2_{Depto. de Bioquímica e Microbiologia, Universidade Estadual Paulista “Julio de Mesquita Filho” Av. 24 A,} nº 1515 Bela Vista. Rio Claro – SP

Keywords: 1,3-propanediol, glycerol, biodiesel, metabolic engineering Introduction:

Nowadays, researchers are focusing on new ways to produce energy, due to the limited sources of petroleum available on the planet. Biodiesel, derived from vegetable oils, is a new kind of green fuel that is being introduced in the energy market and its production process generates large amounts of glycerol as by-product. Glycerol can be used as carbon source in microbial bioprocesses, such as the production of 1,3-propanediol. The 1,3- propanediol is used in the manufacture of plastic materials, fibres and it is easily recycled.

Klebsiella pneumoniae GLC29 was showed a good performance as 1,3-propanediol

producer since it does not depend on vitamin B12 addition in the culture medium and its growth is not restricted under anaerobic conditions. The biosynthesis of 1,3-propanediol from glycerol is performed in two enzymatic steps. First glycerol is dehydrated to 3- hydroxypropionaldehyde by glycerol dehydratase encoded by dhaB, dhaC and dhaE. 3- Hydroxypropionaldehyde is reduced to 1,3-propanediol by 1,3-propanediol dehydrogenase (encoded by dhaT). The gene dhaF encodes a glycerol dehydratase reactivation factor and DhaR is a transcriptional regulator of the operon dhaTBCEF. In this study genomic data from Klebsiella were used to design primers for the detection of dhaB and dhaT. These primers were successfully used to detect such genes in K. pneumoniae GLC29.

Material and Methods:

K. pneumoniae cells were cultivated in LB medium and its DNA was extracted using the

QIAprep Spin Miniprep Kit (QIAgen USA). The sequences of dhaB (glycerol dehydratase, large subunit) and dhaT (1,3-propenodiol oxireductase) were retrieved from sequenced genomes at GenBank. The primers were designed considering conserved regions using the Fast PCR software. The genes were amplified by PCR and the amplicons were separated by agarose gel electrophoresis and detected after staining with ethidium bromide.

Results and Discussion:

The primers to detect dhaB (dhaBF and dhaBR) were designed from genome sequences from nine Klebsiella spp. and two Citrobacter spp. Only one amplicon of 619 bp was obtained and the sequence of this fragment showed about 98% similarity to part of dhaB genes from other Klebsiella. For the amplification of dhaT, primers (dhaTF and dhaTR) were designed based on genome sequences of six Klebsiella spp. and one Citrobacter sp. Only one amplicon of 420 bp was obtained and its sequence presented about 98% similarity to part of dhaT orthologs from other Klebsiella. Thus, the primers designed were successfully used to detect dhaB and dhaT. These primers are now being used to detect plasmids harbouring the complete dha operon in a genomic library.

DETECTION OF ROTAVIRUS AND ADENOVIRUS IN FECAL SAMPLES FROM