Romilio T. Espejo

Vibrio parahaemolyticus diarrhea, Chile, 1998 and 2004.

Analysis of clinical isolates of Vibrio parahaemolyticus from outbreaks in Chile in the cities of Puerto Montt in 2004 and in Antofagasta in 1998 indicated that 23 of 24 isolates from Puerto Montt and 19 of 20 from Antofagasta belonged to the pandemic clonal complex that emerged in Southeast Asia in 1996.

Determination of Molecular Phylogenetics of Vibrio parahaemolyticus Strains by Multilocus Sequence Typing

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.

Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio.

Analysis of the 16S rDNAs obtained from cultures of single colonies of either type collection strains or environmental strains of the genus Vibrio revealed the presence of polymorphism in every one of the strains examined. Polymorphism was detected by visualization of heteroduplexes produced after 16S rDNA PCR amplification, a procedure that allows for the screening of a large number of isolates. Amplified 16S rDNAs obtained from both Vibrio parahaemolyticus and an environmental strain were cloned. Their nucleotide sequences revealed differences of up to 2% among 16S rDNAs from the same strain. Polymorphic sites were concentrated in a recognized variable stem-loop of bacterial 16S rRNA that contained in some cases up to 83% of the total mismatches observed. Most of the substitutions present in the stem-loop region showed compensating base covariation. The accumulation of so many compensating changes in the stem-loop region implies that the divergence of the different versions of this stem-loop is relatively ancient. This divergence could be the result of either a selection process or a lateral transfer of independently evolved genes.

Bacterial 16S rRNA Gene Analysis Revealed That Bacteria Related to Arcobacter spp. Constitute an Abundant and Common Component of the Oyster Microbiota (Tiostrea chilensis)

To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.

PCR-TTGE Analysis of 16S rRNA from Rainbow Trout (Oncorhynchus mykiss) Gut Microbiota Reveals Host-Specific Communities of Active Bacteria

This study assessed the relative contributions of host genetics and diet in shaping the gut microbiota of rainbow trout. Full sibling fish from four unrelated families, each consisting of individuals derived from the mating of one male and one female belonging to a breeding program, were fed diets containing either vegetable proteins or vegetable oils for two months in comparison to a control diet consisting of only fish protein and fish oil. Two parallel approaches were applied on the same samples: transcriptionally active bacterial populations were examined based on RNA analysis and were compared with bacterial populations obtained from DNA analysis. Comparison of temporal temperature gradient gel electrophoresis (TTGE) profiles from DNA and RNA showed important differences, indicating that active bacterial populations were better described by RNA analysis. Results showed that some bacterial groups were significantly (P<0.05) associated with specific families, indicating that microbiota composition may be influenced by the host. In addition, the effect of diet on microbiota composition was dependent on the trout family.

Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile

One-sentence summary for table of contents: Outbreaks are decreasing and the O3:K6 pandemic strain is being replaced by a new serotype and new strains.

Primary structure of the cleavage site associated with trypsin enhancement of rotavirus SA11 infectivity

The primary structure of the trypsin cleavage site in the outer layer protein VP3 of rotavirus SA11 was determined. This cleavage enhances the infectivity of rotavirus SA11. Both VP8, one of the polypeptides generated by the cleavage, and VP3 had their alpha-NH2 blocked. Only VP5, the other polypeptide produced by the cleavage, was susceptible to sequential Edman degradation, indicating that it contained the new alpha-NH2 terminus generated by trypsin hydrolysis. The results indicated that purified VP5 is composed of two polypeptides with the following amino acid sequence at their N terminus: (a) ??VYTRAQPNQDAVVSKTS...; (b) AQPNQDAVVSKTS.... Sequencing of the DNA complementary to ds RNA segment 4 revealed a nucleotide sequence encoding the amino acid sequences indicated above, with only one different amino acid. From these results, the amino acid sequence of the site cleaved by trypsin was extended to cover the C termini (present in VP8). The following sequence, which contains two sites (indicated with asterisks) and can be cleaved by trypsin was deduced: ... VPVSIVSR*NIVYTR*AQPNQDIVVSKTS....

Dynamics of Clinical and Environmental Vibrio parahaemolyticus Strains during Seafood-Related Summer Diarrhea Outbreaks in Southern Chile

ABSTRACT Seafood consumption-related diarrhea became prevalent in Chile when the pandemic strain of Vibrio parahaemolyticus serotype O3:K6 reached a region in the south of Chile (Region de los Lagos) where approximately 80% of the countrys seafood is produced. In spite of the large outbreaks of clinical infection, the load of V. parahaemolyticus in shellfish of this region is relatively low. The pandemic strain constitutes a small but relatively stable group of a diverse V. parahaemolyticus population, composed of at least 28 genetic groups. Outbreaks in Region de los Lagos began in 2004 and reached a peak in 2005 with 3,725 clinical cases, all associated with the pandemic strain. After 2005, reported cases steadily decreased to a total of 477 cases in 2007. At that time, 40% of the clinical cases were associated with a pandemic strain of a different serotype (O3:K59), and 27% were related to V. parahaemolyticus isolates unrelated to the pandemic strain. In the results published here, we report that in the summer of 2008, when reported cases unexpectedly increased from 477 to 1,143, 98% of the clinical cases were associated with the pandemic strain serotype O3:K6, a change from 2007. Nevertheless, in 2009, when clinical cases decreased to 441, only 64% were related to the pandemic strain; the remaining cases were related to a nonpandemic tdh- and trh-negative strain first identified in shellfish in 2006. Overall, our observations indicate that the pandemic strain has become a relatively stable subpopulation and that when the number of diarrhea cases related to the pandemic strain is low, previously undetected V. parahaemolyticus pathogenic strains become evident.

Effect of Associated Bacteria on the Growth and Toxicity of Alexandrium catenella

ABSTRACT Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na+ channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.

Oxytetracycline Treatment Reduces Bacterial Diversity of Intestinal Microbiota of Atlantic Salmon

The effect of oxytetracycline (OTC) treatment on intestinal bacterial populations in juvenile Atlantic salmon Salmo salar was evaluated. Oxytetracycline was administered by way of medicated feed to fish held in experimental tanks. Restriction fragment length polymorphism and sequencing of 16S rDNA from isolates were used to analyze the intestinal microbiota before, during, and after OTC administration. The microbiota from untreated fish was more diverse, consisting mainly of Pseudomonas, Acinetobacter, Bacillus, Flavobacterium, Psycrobacter, and Brevundimonas spp. In contrast, the microbiota of the OTC-treated group was characterized by lower diversity and consisted only of Aeromonas, clustering with A. sobria and A. salmonicida. Antibiotic-resistant isolates were identified as Aeromonas spp.; sequencing the resistance determinant showed it to be the tetE gene. Overall, OTC treatment changed the composition of the intestinal microbiota of Atlantic salmon, as evidenced by a reduction in bacterial diversity. These results support the current concern that antibiotic treatment can facilitate the proliferation of opportunistic bacteria by eradicating competing microorganisms.