Isolation Of Colonies Research Articles

Mycoremediation of Hydrocarbon and its products using Fusarium oxysporum

Oil spillage is one of the major causes of environmental pollution which diversely affects the ecosystem. Mycoremediation plays a significant role in degrading or removing substances like hydrocarbons, polychlorinated compounds, heavy metals and others toxicants from the affected environment using fungi. The aim was to isolate the fungi from the contaminated soil and evaluate its degradative potential. The contaminated soil sample was collected from an automobile shop in Padi, Chennai. The fungi were grown in Bushnell Haas Agar medium and isolation of pure colonies was done. Two fungi species were grown. Hydrocarbon degradation assay was performed with the isolated fungi and the hydrocarbon chosen for degradation was naphthalene. The growth profiles were monitored and the absorbance was measured up to 9 days. One of the isolated fungi STRAIN-2 (C1-P) efficiently degraded 1% naphthalene and was confirmed by methylene blue assay. The molecular characterization of STRAIN-2 (C1-P) was done to generate DNA barcode for identifying the organism. The organism was identified as Fusarium oxysporum using morphological observations and genomic DNA sequencing. The identified fungi achieved 98% similarity with accession number KR0470601 from NCBI.

Widespread detection of antibiotic-resistant bacteria from natural aquatic environments in southern Ontario.

To elucidate how widespread antibiotic resistance is in the surface water environment, we studied the prevalence of antibiotic resistance bacteria at four locations in southern Ontario. We found that the percentage of bacteria resistant to the antibiotic tetracycline was higher at the river site, which flows through agricultural land, and lower at the lake sites. A total of 225 colonies were selected for further testing of antibiotic disc susceptibility to eight different antibiotics to calculate the multiple antibiotic resistance (MAR) score and the antibiotic resistance index for each site. Although the isolates from the lake site outside the city displayed resistance to fewer antibiotics, their MAR scores were not significantly different from that of the lake sites adjacent to urban beaches, showing that MAR was widespread in the natural water environments tested. Isolation of colonies under selection pressure to tetracycline was found to have a significant effect on the likelihood that the isolates would contain multiple resistance traits for other antibiotics. Identification of isolates selected on tetracycline was compared with that of isolates that were sensitive to tetracycline, and the community composition was found to be distinctly different, although isolates from the genera Chryseobacterium, Pseudomonas, and Stenotrophomonas were found in both communities.

Evaluating the preservation and isolation of stool pathogens using the COPAN FecalSwab™ Transport System and Walk-Away Specimen Processor

The isolation of stool pathogens is difficult due to their fastidious nature and the rapid overgrowth of fecal flora. In this study, we evaluate the preservation and isolation of enteric pathogens from stool using the automated COPAN Walk-Away Specimen Processor (WASP®) in conjunction with FecalSwab™ and selenite media. Pathogen viability and fecal commensal abundance were stable in FecalSwab™ media under both room-temperature and refrigerated incubation conditions, resulting in a significantly increased number of well-isolated pathogen colonies observed when compared to samples incubated in modified Cary–Blair media. Isolation of individual pathogen colonies was improved via WASP® planting compared to those planted using the Isoplater system. Furthermore, preincubation using the newly formulated COPAN selenite media significantly enhanced the yield of Salmonella enterica serovar Typhimurium. Together, the automated WASP® system combined with FecalSwab™ and selenite media represents a rapid and efficient approach for the processing of stool specimens compared to standard methods.

Gene Knockouts in Goats Using CRISPR/Cas9 System and Somatic Cell Nuclear Transfer.

The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos into recipient females. In this procedure, the transfection conditions for goat fibroblasts were optimized due to their high sensitivity to electrotransfection, which enabled the isolation of single-cell colonies carrying simultaneous disruption of multiple genes for SCNT with a single co-transfection of pooled CRISPR/Cas9 targeting vectors.

Short communication: Occurrence of methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci in dairy goat herds in Ohio, United States

In light of the scarcity of information about the occurrence and epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MRCNS) in small ruminants in general, and particularly dairy goats, we launched this limited-scope study. The findings reported here show the detection of MRSA and MRCNS in goat milk and teat skin samples from dairy goat herds in the state of Ohio. A total of 120 milk samples and 120 teat-swab samples were collected from 5 farms. After conventional isolation and phenotypic characterization of the staphylococci colonies, bacterial isolates were tested by PCR assay targeting the genes nuc to identify Staphylococcus aureus and mecA to detect MRSA and MRCNS. The clonal complexes of MRSA isolates was also determined by multiloccus sequence typing. Fifteen (6.2%) positive S. aureus samples were found in this study: 9 from milk and 6 from teat skin samples. Four (2%) MRSA isolates were detected and, using multiloccus sequence typing genotyping, these were designated to clonal complexes CC133 (n = 2; milk samples) and CC5 (n = 2; teat skin). Three (1.25%) coagulase-negative staphylococci isolates from the teat skin also harbored the mecA gene. Although, the MRSA isolated from milk samples is not a typical human-associated lineage, the CC5 clone isolated from teat skin is a common and widespread clonal complex associated with humans, suggesting that this extramammary niche could be a relevant reservoir of methicillin-resistant staphylococci. Furthermore, the fact that 75% of MRSA were recovered from 1 farm showing poor hygiene practices strengthens the hypothesis that good hygiene practices could be useful to prevent persistence and spread of MRSA at a farm level.

Electrode plate-culture methods for colony isolation of exoelectrogens from anode microbiomes

Exoelectrogens play central roles in microbial fuel cells and other bioelectrochemical systems (BESs), yet their physiological diversity remains largely elusive due to the lack of efficient methods for the isolation from naturally occurring microbiomes. The present study developed an electrode plate-culture (EPC) method that facilitates selective colony formation by exoelectrogens and used it for isolating them from an exoelectrogenic microbiome enriched from paddy-field soil. In an EPC device, the surface of solidified agarose medium was spread with a suspension of a microbiome and covered with a transparent fluorine doped tin oxide (FTO) electrode (poised at 0 V vs. the standard hydrogen electrode) that served as the sole electron acceptor. The medium contained acetate as the major growth substrate and Coomassie Brilliant Blue as a dye for visualizing colonies under FTO. It was shown that colonies successfully appeared under FTO in association with current generation. Analyses of 16S rRNA gene sequences of colonies indicated that they were affiliated with genera Citrobacter, Geobacter and others. Among them, Citrobacter and Geobacter isolates were found to be exoelectrogenic in pure-culture BESs. These results demonstrate the utility of the EPC method for colony isolation of exoelectrogens.

A new CHO (Chinese hamster ovary)-derived cell line expressing anti-TNFα monoclonal antibody with biosimilar potential.

Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn's disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. Our goal was to develop the first stage of an adalimumab biosimilar candidate with potential for national production, through the generation of a productive and stable cell line and assess its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRγ did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding and functional activity analyses performed with selected adabut clones in comparison to reference adalimumab represent an important status of "non-inferiority," part of the process required for a biosimilar development. We generated and selected high-quality adabut clones which mAbs may be further developed as the first in-house made Brazilian biosimilar, demonstrating a success case for our incipient biotechnology industry, or also modified as biobetters, thus representing an innovative strategy for the patients' welfare.

Genetic diversity and sex ratio of naked mole rat, Heterocephalus glaber, zoo populations.

The naked mole rat, Heterocephalus glaber, is a highly unusual mammal that displays a complex social system similar to that found in eusocial insects. Colonies of H. glaber are commonly maintained in zoo collections because they represent fascinating educational exhibits for the public. However, little is known about the genetic structure or sex ratio of captive populations of H. glaber. In this study, we developed a set of microsatellite markers to examine genetic variation in three captive zoo populations of H. glaber. We also studied sex ratio of these captive populations. Our goal was to determine levels of genetic variation within, and genetic differences between, captive populations of H. glaber. Overall, we found modest levels of genetic variation in zoo populations. We also uncovered little evidence for inbreeding within the captive populations. However, zoo populations did differ genetically, which may reflect the isolation of captive naked mole rat colonies. Finally, we found no evidence of biased sex ratios within colonies. Overall, our study documents levels of genetic variation and sex ratios in a captive eusocial mammalian population. Our results may provide insight into how to manage captive populations of H. glaber.

Identification and variability of Monilinia spp. isolates from peach

The brown rot caused by the fungus Monilinia spp. is the main disease of peach (Prunus persica) that occurs from flowering to post-harvest fruit. The objective of this study was to identify the species that causes brown rot in Rio Grande do Sul state using molecular method based on specific primers and study the morphological variability of Monilinia isolates using classic characterization methods. The isolates were collected in the main producing regions of peaches and determination of species was performed by using specific primers to the genus Monilinia. The culture media BDA?, peach juice and Pagnocca were used for the morphological characterization of isolates. Molecular study showed that all isolates from Rio Grande do Sul are the species M. fructicola. In the morphological characterization, such as mycelial diameter, color, margin, shape and elevation of the colonies and dry mass of mycelium, there are variability in the analyzed characteristics of the isolates in the three-culture media used. In the comparing the culture media, the higher dry matter values and diameter of colony isolates were obtained in Pagnocca and peach juice means.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based ‘library vs library’ Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome–lysosome fusion. This concept can also be applied to other systems to screen protein–RNA and protein–DNA interactions and delineate signaling landscape in cells.

Coadministration of the Campylobacter jejuni N-Glycan-Based Vaccine with Probiotics Improves Vaccine Performance in Broiler Chickens.

Source attribution studies report that the consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimization of the vaccine for commercial broiler chickens has great potential to prevent the entry of the pathogen into the food chain. Here, we tested the same vaccination approach in broiler chickens and observed similar efficacies in pathogen load reduction, stimulation of the host IgY response, the lack of C. jejuni resistance development, uniformity in microbial gut composition, and the bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of Clostridiales cluster XIVa, Anaerosporobacter mobilis, that was significantly more abundant in responder birds. In broiler chickens, coadministration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody responses, and weight gain. To investigate whether the responder-nonresponder effect was due to the selection of a C. jejuni "supercolonizer mutant" with altered phase-variable genes, we analyzed all poly(G)-containing loci of the input strain compared to nonresponder colony isolates and found no evidence of phase state selection. However, untargeted nuclear magnetic resonance (NMR)-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels that is possibly linked to increased microbial diversity in this subgroup. The comprehensive methods used to examine the bimodality of the vaccine response provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy.IMPORTANCECampylobacter jejuni is a common cause of human diarrheal disease worldwide and is listed by the World Health Organization as a high-priority pathogen. C. jejuni infection typically occurs through the ingestion of contaminated chicken meat, so many efforts are targeted at reducing C. jejuni levels at the source. We previously developed a vaccine that reduces C. jejuni levels in egg-laying chickens. In this study, we improved vaccine performance in meat birds by supplementing the vaccine with probiotics. In addition, we demonstrated that C. jejuni colonization levels in chickens are negatively correlated with the abundance of clostridia, another group of common gut microbes. We describe new methods for vaccine optimization that will assist in improving the C. jejuni vaccine and other vaccines under development.

Phage therapy of corrosion-producing bacterium Stenotrophomonas maltophilia using isolated lytic bacteriophages

PurposeCorrosion-producing microorganisms have different physiology and include sulfate-reducing bacteria, iron oxidizers and magnesium oxidizers. Biocorrosion has been seen in various industries, especially the petrochemicals and oil industries. One proposal to solve this problem is the use of bacteriophages to treat the bacteria-caused corrosion. The aims of this study were isolation and identification of corrosion-producing bacteria from petroleum pipeline corrosion as well as finding their specific bacteriophages for phage therapy purposes.Design/methodology/approachThe sample pipes with the corrosion were obtained from the Gandomkar petroleum pipeline station, Chaharmahal and Bakhtiari, Iran. For screening the corrosion-producing bacteria, the rusted pipe samples were cultured in a selective culture medium, manganese agar. The purified individual colonies were subjected to molecular examinations. For isolating bacteriophages from silversmithing workshops wastewater in Isfahan, whole plate titration methods and transmission electron microscopy were used to isolate and detect phages.FindingsThe cultivation of corrosion-based material on manganese agar after 18 hours incubation at 30°C resulted in the isolation of cream-colored colonies. The microscopic examinations showed Gram-negative coccobacilli. Based on molecular examinations, the isolated bacteria were identified as Stenotrophomonas maltophilia strain PBM-IAUF-2 with Genebank accession number of KU145278.1. The found bacteriophage was related to the Siphoviridae family of phages.Originality/valueThis paper is the first report of isolation and identification of corrosion-producing bacteria and its specific lytic phages from Gandomkar petroleum pipeline station, Chaharmahal and Bakhtiari, Iran. The biological procedures for preventing the microbial corrosion could be an asset and considered as a potential in the petroleum and industrial microbiology. Phage therapy is considered as one of the economical methods for reducing the biocorrosion.

Sequential screening by ClonePix FL and intracellular staining facilitate isolation of high producer cell lines for monoclonal antibody manufacturing

Screening and characterization of cell lines for stable production are critical tasks in identifying suitable recombinant cell lines for the manufacture of protein therapeutics. To aid this essential function we have developed a methodology for the selection of antibody expressing cells using fluorescence based ClonePix FL colony isolation and flow cytometry analysis following intracellular staining for immunoglobulin G (IgG). Our data show that characterization of cells by flow cytometry early in the clone selection process enables the identification of cell lines with the potential for high productivity and helps to eliminate unstable cell lines. We further demonstrate a correlation between specific productivity (qP) and intracellular heavy chain (HC) content with final productivity. The unique combination of screening using ClonePix FL and the flow cytometry approaches facilitated more efficient isolation of clonal cell lines with high productivity within a 15week timeline and which can be applied across NS0 and CHO host platforms. Furthermore, in this study we describe the critical parameters for the ClonePix FL colony based selection and the associated calculations to provide an assessment of the probability of monoclonality of the resulting cell lines.

Properties of Dental Pulp–derived Mesenchymal Stem Cells and the Effects of Culture Conditions

Dental pulp mesenchymal stem cells (DPMSCs) highly express mesenchymal stem cell markers and possess the potential to differentiate into neural cells, osteoblasts, adipocytes, and chondrocytes. Thus, DPMSCs are considered suitable for tissue regeneration.The colony isolation method has commonly been used to collect relatively large amounts of heterogeneous DPMSCs. Homogenous DPMSCs can be isolated by fluorescence-activated cell sorting using antibodies against mesenchymal stem cell markers, although this method yields a limited number of cells. Both quality and quantity of DPMSCs are critical to regenerative therapy, and cell culture methods need to be improved. We thus investigated the properties of DPMSCs cultured with different methods.DPMSCs in a three-dimensional spheroid culture system, which is similar to the hanging drop culture for differentiation of embryonic stem cells, showed upregulation of odonto-/osteoblastic markers and mineralized nodule formation. This suggests that this three-dimensional spheroid culturing system for DPMSCs may be suitable for inducing hard tissues. We further examined the effect of cell culture density on the properties of DPMSCs because the properties of stem cells can be altered depending on the cell density. DPMSCs cultured under the confluent cell density condition showed slight downregulation of some mesenchymal stem cell markers compared with those under the sparse condition.The ability of DPMSCs to differentiate into hard tissue–forming cells was found to be enhanced in the confluent condition, suggesting that the confluent culture condition may not be suitable for maintaining the stemness of DPMSCs. When DPMSCs are to be used for hard tissue regeneration, dense followed by sparse cell culture conditions may be a better alternative strategy.

The predation paradox: Synergistic and antagonistic interactions between grazing by crustacean predator and infection by cyanophages promotes bloom formation in filamentous cyanobacteria

AbstractIn this study, we assessed the impact of synergistic/antagonistic interactions between grazing by crustacean predator (Daphnia magna) and infection by cyanophage (Vb‐AphaS‐CL131) on the population dynamics of the harmful bloom‐forming filamentous cyanobacterium Aphanizomenon flos‐aquae. We observed synergistic effect of cyanophage infection on D. magna survival and grazing trough the lysis‐induced shift in cyanobacteria population structure toward the shorter filaments. However, lysis‐mediated‐ and grazing‐enhanced removal of short A. flos‐aquae filaments resulted in the dominance of grazing‐ and infection‐resistant A. flos‐aquae population. In addition, the presence of D. magna generated a trait‐mediated response in the A. flos‐aquae population by promoting the aggregation of filaments into colony‐like structures. Experiments using temperate A. flos‐aquae colony isolates from natural environment demonstrated that colony‐embedded A. flos‐aquae filaments are insensitive to the addition of both D. magna and cyanophages. Therefore, we propose that interactions between crustacean grazers and cyanophages may promote the emergence of defensive A. flos‐aquae genotypes and population traits that eventually lead to bloom formation in aquatic environments.

Strategies and Methodologies for Developing Microbial Detoxification Systems to Mitigate Mycotoxins.

Mycotoxins, the secondary metabolites of mycotoxigenic fungi, have been found in almost all agricultural commodities worldwide, causing enormous economic losses in livestock production and severe human health problems. Compared to traditional physical adsorption and chemical reactions, interest in biological detoxification methods that are environmentally sound, safe and highly efficient has seen a significant increase in recent years. However, researchers in this field have been facing tremendous unexpected challenges and are eager to find solutions. This review summarizes and assesses the research strategies and methodologies in each phase of the development of microbiological solutions for mycotoxin mitigation. These include screening of functional microbial consortia from natural samples, isolation and identification of single colonies with biotransformation activity, investigation of the physiological characteristics of isolated strains, identification and assessment of the toxicities of biotransformation products, purification of functional enzymes and the application of mycotoxin decontamination to feed/food production. A full understanding and appropriate application of this tool box should be helpful towards the development of novel microbiological solutions on mycotoxin detoxification.

Presence and distribution of yeasts in the reproductive tract in healthy female horses.

Yeasts are commensal organisms found in the reproductive and gastrointestinal tracts, and on the skin and other mucosa in mammals. The purpose of this study was to isolate and identify yeast flora in the caudal reproductive tract in healthy female horses. Longitudinal study. A total of 453 samples were collected using double-guarded swabs from the vestibule, clitoral fossa and vagina in 151 horses. All samples were cultured on Sabouraud 4% dextrose agar and incubated at 35°C for 7-10 days. Isolates were identified according to their morphological characteristics and biochemical profiles. Yeast colonies were isolated from 60 (39.7%) of the 151 horses. The isolated yeasts belonged to nine genera, and included Candida spp. (53.2%), Cryptococcus spp. (12.2%), Saccharomyces spp. (10.5%), Geotrichum spp. (8.0%), Rhodotorula spp. (7.1%), Malassezia spp. (3.7%), Trichosporon spp. (2.6%), Kluyveromyces spp. (2.6%) and Sporothrix spp. (0.2%). Candida krusei (43.1%) was the most frequent Candida species isolated. There was a significant difference in prevalence between C. krusei and other Candida species (P<0.05). The vestibule contained more yeast isolates (48.0%) than the vagina (18.3%). The isolation of yeast colonies from multiparous females (76.8%) was significantly higher than from maiden mares (P<0.05). The study was limited by the difficulty of distinguishing between normal flora and potential pathogens. Candida spp., in particular C. krusei, represent important flora resident in the caudal reproductive tract in healthy female horses. This is particularly important in contexts that require the initiation of empirical treatment prior to the completion of culture results.

Vibrio harveyi, V. parahaemolyticus, and V. vulnificus detection in Thai shellfishes by the triplex PCR method

Context: Shellfish sold in Southeast Asian markets are highly contaminated. Uncooked seafood samples were collected from markets in Bangkok, Thailand, which were contaminated with Vibrio species (27%) and in which antibiotic resistance was relatively high. Aims: To simultaneously detect V. harveyi, V. parahaemolyticus, and V. vulnificus in shellfish samples including life mussel (Perna viridis), blood clam (Tegillarca granosa), and baby clam (Paphia undulata) in the local markets in Bangkok. The sensitivity, accuracy, and specificity of triplex PCR method were also evaluated. Materials and Methods: The 150 frozen shellfishes were purchased from five local markets in Bangkok. Each sample was homogenized, enriched, and prepared for colony counting and isolation. Three Vibrio species were identified according to biochemical tests and then later confirmed by triplex PCR. The accuracy and specificity of triplex PCR were evaluated and compared with conventional tests. The sensitivity of triplex PCR was explained as total count (CFU/mL) of Vibrio species. Results: Using the biochemical test and triplex PCR method in the three Vibrio species of marine shellfishes, 14 and 15 isolates of V. harveyi, 8 isolates of V. parahaemolyticus, and undetectable and 1 isolate for V. vulnificus were identified, respectively. The accuracy of triplex PCR was higher than the conventional method. Triplex PCR was shown to be sensitive as total bacterial count for three Vibrio detection in marine shellfishes was 1.2 × 107-2 × 106 CFU/mL. Conclusions: Triplex PCR assay was higher in sensitivity, accuracy, and specificity, which proved to be convenient, simple, and effective method for Vibrio detection and identification in shellfishes.

High Prevalence of Diarrheagenic Escherichia coli among Children with Diarrhea in Kenya.

Diarrheagenic Escherichia coli (DEC) is an important agent of endemic and epidemic diarrhea worldwide, particularly in developing countries. DEC cannot be differentiated from commensal E. coli on selective media, although there are a few exceptions. Most studies use the colony isolation method, which cannot detect low numbers of DEC, and therefore, these studies might underestimate the incidence of DEC. In the present study, we employed a colony sweep method with real-time PCR targeting virulence genes of 5 categories of DEC; this technique can detect very low numbers of DEC among hundreds of commensal E. coli. DEC was detected in 171 (55.9%) of 306 children with diarrhea in Kenya. The prevalence of DEC in Kenya was notably higher than that (30 in 143, 21.0%) in Indonesia. Occurrences of multiple DEC infection in Kenya were frequent (69 in 306, 23.2%), suggesting that the source of DEC infection may be related to grossly contaminated food and water. In contrast, only 9 (6.0%) of 150 healthy adults in Kenya carried DEC. Considering that healthy adults naturally harbor non-DEC, it is interesting how children exclude DEC but not non-DEC as they grow up. Several mechanisms, such as mucosal immunity and intestinal microbiota, might be involved in the exclusion of DEC.

Quantification of Vibrio species in oysters from the Gulf of Mexico with two procedures based on MPN and PCR

Oysters can accumulate potentially pathogenic water bacteria. The objective of this study was to compare two procedures to quantify Vibrio species present in oysters to determine the most sensitive method. We analyzed oyster samples from the Gulf of Mexico, commercialized in Mexico City. The samples were inoculated in tubes with alkaline peptone water (APW), based on three tubes and four dilutions (10-1 to 10-4). From these tubes, the first quantification of Vibrio species was performed (most probable number (MPN) from tubes) and bacteria were inoculated by streaking on thiosulfate-citrate-bile salts-sucrose (TCBS) petri dishes. Colonies were isolated for a second quantification (MPN from dishes). Polymerase chain reaction (PCR) was used to determine species with specific primers: ompW for Vibrio cholerae, tlh for Vibrio parahaemolyticus, and VvhA for Vibrio vulnificus. Simultaneously, the sanitary quality of oysters was determined. The quantification of V. parahaemolyticus was significantly higher in APW tubes than in TCBS dishes. Regarding V. vulnificus counts, the differences among both approaches were not significant. In contrast, the MPNs of V. cholerae obtained from dishes were higher than from tubes. The quantification of MPNs through PCR of V. parahaemolyticus and V. vulnificus obtained from APW was sensitive and recommendable for the detection of both species. In contrast, to quantify V. cholerae, it was necessary to isolate colonies on TCBS prior PCR. Culturing in APW at 42°C could be an alternative to avoid colony isolation. The MPNs of V. cholerae from dishes was associated with the bad sanitary quality of the samples.