First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds

Background Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner. Methods Total genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5–V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the ‘number of AluI bands’ and ‘type of eletropherogram’. Results Two types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity. Conclusion The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

Differentiation of polyvalent bacteriophages specific to uropathogenic Proteus mirabilis strains based on the host range pattern and RFLP.

Urinary tract infections (UTIs) caused by P. mirabilis are difficult to cure because of the increasing antimicrobial resistance of these bacteria. Phage therapy is proposed as an alternative infection treatment. The aim of this study was to isolate and differentiate uropathogenic P. mirabilis strain specific polyvalent bacteriophages producing polysaccharide depolymerases (PDs). 51 specific phages were obtained. The plaques of 29 bacteriophages were surrounded by halos, which indicated that they produced PDs. The host range analysis showed that, except phages 58B and 58C, the phage host range profiles differed from each other. Phages 35 and 45 infected all P. mirabilis strains tested. Another 10 phages lysed more than 90% of isolates. Among these phages, 65A, 70, 66 and 66A caused a complete lysis of the bacterial lawn formed by 62% to 78% of strains. Additionally, phages 39A and 70 probably produced PDs. The phages' DNA restriction fragment length polymorphism (RFLP) analysis demonstrated that genomes of 51 isolated phages represented 34 different restriction profiles. DNA of phage 58A seemed to be resistant to selected EcoRV endonuclease. The 33 RFLP-EcoRV profiles showed a Dice similarity index of 38.8%. 22 RFLP patterns were obtained from single phage isolates. The remaining 12 restriction profiles consisted of 2 to 4 viruses. The results obtained from phage characterization based on the pattern of phage host range in combination with the RFLP method enabled effective differentiation of the studied phages and selection of PD producing polyvalent phages for further study.

Listeria monocytogenes in Five Sardinian Swine Slaughterhouses: Prevalence, Serotype, and Genotype Characterization

In a 3-year study (2008 to 2011) to estimate the prevalence and the contamination sources of Listeria monocytogenes in pork meat in Sardinia, Italy, 211 samples were collected from five Sardinian swine slaughterhouses: 171 samples from slaughtered pigs and 40 from the slaughterhouse environment. Fifty L. monocytogenes isolates were characterized by PCR-based serotyping, presence of virulence-associated genes, and pulsed-field gel electrophoresis restriction analysis. The overall prevalence of L. monocytogenes was 33% in swine carcasses, 7% in cecal material, 23% on meat contact surfaces, and 25% on noncontact surfaces. Only two serotypes were detected: 1/2c (78%) and 1/2a (22%). In all, based on the presence of virulence-associated genes, eight pathogenic profiles were detected. Only 42% of all isolates carried the full complement of virulence-associated genes and were allotted to profile 1. Six pulsed-field gel electrophoresis profiles persisted in the slaughterhouses; restriction profiles appeared to be specific to each plant.

Characterization ofblaCMY-2Plasmids in Salmonella and Escherichia coli Isolates from Food Animals in Canada

ABSTRACTOne hundred thirty-fourblaCMY-2plasmids fromSalmonellaandEscherichia colistrains from animals and food in Canada were characterized. Five plasmid groups were identified based on replicon type and restriction profiles. Three groups containedE. coliplasmids only. IncA/C plasmids included most multiresistant plasmids and all those of bovine origin.

Plasmids from freshwater environments capable of IncQ retrotransfer are diverse and include pQKH54, a new IncP-1 subgroup archetype

Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2.1×10−4 to 1.75×10−5 were obtained for five of the nine plasmids – the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1β subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1γ. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.

Use of Restriction Fragment Length Polymorphism, Immunoblotting, and Polymerase Chain Reaction in the Differentiation of Avian Poxviruses

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.