Maria Helena Matté

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

ABSTRACT A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI andtoxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive fortcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA,zot, ace, tcpA, andtcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specificst gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that theV. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.

Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov.

Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy. Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%). The temperature for growth was 4-30 degrees C. Both strains were rod-shaped, with a single flagellum. However, strain KMM 231T revealed a single long fimbrium. Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. 70%). Also present were saturated and monounsaturated straight-chain fatty acids. Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization. The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles. Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and Idiomarina zobellii sp. nov. (type strain is KMM 231T).

Genetic Diversity of Clinical and Environmental Isolates of Vibrio cholerae Determined by Amplified Fragment Length Polymorphism Fingerprinting

ABSTRACT Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- andTaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. choleraestrains that appear to have been derived locally from environmental O1 or non-O1 strains.

Silent dissemination of colistin-resistant Escherichia coli in South America could contribute to the global spread of the mcr-1 gene.

During a Brazilian multicentric antimicrobial resistance surveillance study, colistin resistance was investigated in 4,620 Enterobacteriaceae isolated from human, animal, food and environmental samples collected from 2000 to 2016. We present evidence that mcr-1-positive Escherichia coli has been emerging in South America since at least 2012, supporting a previous report on the possible acquisition of mcr-1-harbouring E. coli by European travellers visiting Latin American countries.

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

ABSTRACT Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

First Report of the Globally Disseminated IncX4 Plasmid Carrying the mcr-1 Gene in a Colistin-Resistant Escherichia coli Sequence Type 101 Isolate from a Human Infection in Brazil

ABSTRACT A colistin-resistant Escherichia coli strain was recovered from a patient with a diabetic foot infection in Brazil. Whole-genome analysis revealed that the E. coli isolate belonged to the widespread sequence type (ST) 101 and harbored the mcr-1 gene on an IncX4 plasmid that was highly similar to mcr-1-bearing IncX4 plasmids that were recently identified in Enterobacteriaceae from food, animal, and human samples recovered on different continents. These results suggest that self-transmissible IncX4-type plasmids may represent promiscuous plasmids contributing to the intercontinental spread of the mcr-1 gene.

Distribution of Potentially Pathogenic Vibrios in Oysters from a Tropical Region

Oysters ( Crassostrea gigas ) originating from the southern coast of the State of São Paulo-Brazil were analyzed for Vibrio species. Most Probable Numbers (MPN/100 g) were obtained for Vibrio alginolyticus (<3-1,500), Vibrio parahaemolyticus (<3-1,200), Vibrio fluvialis (<3-150), Vibrio cholerae non 01 (<3-40), Vibrio furnissii (<3-40), Vibrio mimicus (<3-40) and Vibrio vulnificus (<3-30). The highest incidence was observed for V. alginolyticus (81%), followed by V. parahaemolyticus (77%), V. cholerae non 01 (31 %), V. fluvialis (27%), V. furnissii (19%), V. mimicus (12%), and V. vulnificus (12%). Forty-eight percent of the isolates tested were positive for enterotoxins in the rabbit ileal loop (RIL) test and 11.1% in the suckling mice test. Vibrio parahaemolyticus (1.1 %) was positive in the Kanagawa test. Vibrio vulnificus (25%) showed lethality in young adult mice. During the field inspection it was observed that generally the conditions of storage were not adequate as 65% of the samples were maintained in temperatures ranging from 25 to 40°C, 19% were frozen, 12% refrigerated and 4% was immersed in contaminated seawater. These results emphasize the great potential for food poisoning by inadequately preserved seafood, and the necessity to upgrade the standards for food quality assessment.

Cellulomonas persica sp. nov. and Cellulomonas iranensis sp. nov., mesophilic cellulose-degrading bacteria isolated from forest soils.

Two newly described species of mesophilic, cellulose-degrading, aerobic bacteria were isolated from forest humus soils along the southern border of the Caspian Sea. Cellulomonas persica and Cellulomonas iranensis are proposed as new specific epithets based on comparative sequence analyses of 16S rDNA, DNA-DNA hybridization and phenotypic characteristics. Formal species descriptions are provided.

Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers

Department of Microbiology, Institute of Biomedical Sciences, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Department of Clinical Analysis, School of Pharmacy, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Institute of Biological Sciences, Universidade Federal do Para, Belem, PA, Brazil; School of Public Health, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Environmental Company of Sao Paulo State (CETESB), Sao Paulo, SP, Brazil

An overview of antimicrobial resistance and its public health significance

Multiple papers have been published regarding the bacterial resistance theme over the last years. A variety of information has reached general and scientific public, daily bringing up data on new resistant microorganisms, new drugs, outbreaks, epidemiological news, resistance gene dissemination, and the lack of information in a particular field has caught our attention: the public health department. Most of researchers, physicians and government employees interpret the public health field as a separate department, not linked to this antibiotic resistance era that we are living nowadays. In this paper we carefully tried to fill in the blanks between public health and the bacteria resistance issue, also considering historical, social, economical and biological problematic that come with this possible pre-antibiotic era.