Diversity of Salmonella Isolates from Central Florida Surface Waters

ABSTRACTIdentification ofSalmonellaserotypes is important for understanding the environmental diversity of the genusSalmonella. This study evaluates the diversity ofSalmonellaisolates recovered from 165 of 202 Central Florida surface water samples and investigates whether the serotype of the environmentalSalmonellaisolates can be predicted by a previously published multiplex PCR assay (S. Kim, J. G. Frye, J. Hu, P. J. Fedorka-Cray, R. Gautom, and D. S. Boyle, J. Clin. Microbiol. 44:3608–3615, 2006,http://dx.doi.org/10.1128/JCM.00701-06). Multiplex PCR was performed on 562Salmonellaisolates (as many as 36 isolates per water sample) to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings before isolates were serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of theSalmonella-positive water samples, allSalmonellaisolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex patterns of 495 isolates (88.1%) did not match any previously reported pattern. The remaining 68 isolates matched reported patterns but did not match the serotypes for those patterns. The use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-threeSalmonella entericaserotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:−. A majority (141/223) ofSalmonellaisolates clustered into one genotypic group.Salmonellaisolates in Central Florida surface waters are serotypically, genotypically, and phenotypically (in terms of antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict theSalmonellaserotype.

Levels of Candidatus Liberibacter asiaticus and Xanthomonas citri in Diverse Citrus Genotypes and Relevance to Potential Transmission from Pollinations

The diseases huanglongbing [HLB, associated with Candidatus Liberibacter asiaticus (CLas)] and Asian citrus canker [ACC, caused by Xanthomonas citri (Xcc)] are widespread in Florida and many other citrus-growing areas, presenting unprecedented challenges for citrus breeding. Because HLB and ACC weaken trees and compromise cropping, breeding is much less efficient using seed parents that have been exposed to these diseases. Therefore, it would be highly desirable to use unique disease-exposed selections only as pollen parents with pollen applied to disease-free trees. However, there may be a risk of introducing these diseases using such pollen sources. To assess this potential, abundance of the pathogens associated with these diseases was assessed in anthers and flowers using quantitative polymerase chain reaction. Because CLas is systemic, levels on mature leaves from the flower source trees were assessed to see if the presence of CLas in flowers was associated with leaf levels. Disease-exposed trees were tested in 10 genotypes from each of three broad genotypic categories, which reflect different levels of susceptibility to the diseases associated with the pathogens studied: Poncirus trifoliata hybrids (most resistant to HLB), Citrus maxima and hybrids (susceptible to both diseases), and C. reticulata and hybrids (considerable resistance to ACC). Of the 30 samples of each tissue type analyzed for CLas, 88% of mature leaves, 69% of flowers, and 88% of anthers had one or more CLas bacterium per sample. The trifoliate genotypic group had significantly lower levels of CLas than the pummelo and mandarin groups in mature leaf samples, but CLas levels were more similar between groups in anther and flower samples, and the pathogen was present in most of the trifoliate hybrids tested. Mean numbers of CLas detected per nanogram nucleic acid were 100 to 800 times higher in mature leaf samples, most characteristic of HLB symptoms, compared with anther samples. Xcc DNA was detected in 30% of flower samples and 23% of anther samples. No significant differences in Xcc levels were found between tissue type or genotypic group. However, regressions between Xcc levels in flowers and percent of plant pedigree derived from mandarin had a negative correlation and an r2 of 0.159 (P = 0.029). The biology of CLas is consistent with the pathogen being present in anthers from unopened flowers, whereas the ACC pathogen detected inside flowers was likely the result of contamination despite great care in sample collection and handling. Where exceptional diligence to exclude HLB and ACC is appropriate, results suggest that there may be a risk of spreading these pathogens through use of pollen from trees on infected farms.

Insulin Receptor (IR) Pathway Hyperactivity in IGF-IR Null Cells and Suppression of Downstream Growth Signaling Using the Dual IGF-IR/IR Inhibitor, BMS-754807

The biology of IGF-IR/IR signaling was studied in normal mouse embryonic fibroblasts (MEFs) that were either wild type (wt), heterozygous (het), or null for the IGF-IR. The ability of IGF-I, IGF-II, or insulin to stimulate serum-starved MEFs was characterized by gene expression profiling and biochemical analyses for activation of downstream signals. Each genotypic group of MEFs exhibited distinct patterns of expression both while resting and in response to stimulation. The insulin receptor (IR) pathway in IGF-IR null MEFs was hypersensitive to insulin ligand stimulation resulting in greater AKT phosphorylation than in wt or het MEFs stimulated with the same ligand. Interestingly, the IR pathway hypersensitivity in IGF-IR null MEFs occurred with no observed changes in the levels of IR isoforms A or B. A new small molecule IGF-IR inhibitor (BMS-754807), having equipotent activity against both IGF-IR and IR, proved effective in suppressing both AKT and ERK phosphorylation from both the IGF-IR and IR pathways by all three ligands tested in wt, het, and null MEFs. The use of a dual IGF-IR/IR inhibitor addresses concerns about the use of growth inhibiting therapies directed against the IGF-IR receptor in certain cancers. Lastly, comparison of the antiproliferative effects (IC50s) of various compounds in wt vs. null MEFs demonstrates that genetically characterized MEFs provide a simple and inexpensive tool with which to define compounds as having mostly on-target or off-target IGF-IR activities because off-target compounds affect both wt and null MEFs equally.

Blood-based CHRNA3 single nucleotide polymorphisms (SNPs) and outcome in advanced non-small cell lung cancer (NSCLC) patients (p)

8033 Background: Nicotonic acetylcholine receptors (nAChRs) are associated with resistance to gemcitabine (gem), cisplatin (cis) and paclitaxel in NSCLC cell lines. Three SNPs of CHRNA3, CHRNA5 and LOC123688 increase lung cancer risk. These SNPs may have influenced outcome in p treated in our phase III trial (Cobo et al. J Clin Oncol 2007;25:2747–54). Methods: Stage IV NSCLC p were treated with customized chemotherapy based on ERCC1 mRNA expression. p in the control arm received docetaxel (doc)/cis; p in the genotypic arm with low ERCC1 levels (low genotypic group [LG]) received doc/cis; p in the genotypic arm with high ERCC1 levels (high genotypic group [HG]) received doc/gem. DNA was extracted from lymphocytes, and CHRNA3 (rs1051730), CHRNA5 (rs16969968) and LOC123688 (rs8034191) SNPs were genotyped with the Taqman allele discrimination assay. Results: A significant interaction was found for CHRNA3 and PS (P = 0.02). In p with PS 0, CT p had a better response than both CC (P = 0.01) and TT (P = 0.02) p, and LG p also had a better response (P = 0.01). When the CHRNA3 genotype was added in the multivariate analysis for progression-free survival (PFS), an improvement was observed in the LG in PS 0 p (P = 0.02). PS 0 p in the LG with the CT genotype attained an 84% response, 12.1-month PFS, and 19-month median survival (MS) ( Table ). Conclusions: CHRNA3 genotyping can improve customized chemotherapy based on tumor ERCC1 mRNA in stage IV NSCLC p with PS 0. [Table: see text] No significant financial relationships to disclose.

Treatment of arterial hypertension in prognosis of acute myocardial ishemia. Clinical and genetic analysis

The aim of the study was to evaluated the role of ID ACE gene polymorphism in the development of acute myocardial ishemia in patients with arterial hypertension (HT). Patients with DD genotype were demonstrated a higher risk of myocardial infarction then patients with ID and II genotype. Conversely, the course of HT in II genotypic group is more favorable, but the efficacy of their treatment by traditional agents is minimal.

Molecular Characterization of Isoniazid-Resistant Mycobacterium tuberculosis Isolates Collected in Australia

ABSTRACT Elucidation of the molecular basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has led to the development of different genotypic approaches for the rapid detection of INH resistance in clinical isolates. Mutations in katG, in particular the S315T substitution, are responsible for INH resistance in a large proportion of tuberculosis cases. However, the frequency of the katG S315T substitution varies with population samples. In this study, 52 epidemiologically unrelated clinical INH-resistant M. tuberculosis isolates collected in Australia were screened for mutations at katG codon 315 and the fabG1-inhA regulatory region. Importantly, 52 INH-sensitive isolates, selected to reflect the geographic and genotypic diversity of the isolates, were also included for comparison. The katG S315T substitution and fabG1-inhA −15 C-to-T mutation were identified in 34 and 13 of the 52 INH-resistant isolates, respectively, and none of the INH-sensitive isolates. Three novel katG mutations, D117A, M257I, and G491C, were identified in three INH-resistant strains with a wild-type katG codon 315, fabG1-inhA regulatory region, and inhA structural gene. When analyzed for possible associations between resistance mechanisms, resistance phenotype, and genotypic groups, it was found that neither the katG S315T nor fabG1-inhA −15 C-to-T mutation clustered with any one genotypic group, but that the −15 C-to-T substitution was associated with isolates with intermediate INH resistance and isolates coresistant to ethionamide. In total, 90.4% of unrelated INH-resistant isolates could be identified by analysis of just two loci: katG315 and the fabG1-inhA regulatory region.

comparison of protein polymorphisms in milk produced by two dairy farms in West Pomerania

The aim of the study was to compare the frequencies of protein polymorphisms in milk produced by cows with various proportion of HF genes within their genotypes, managed in two farms. Frequency analyses of beta-lactoglobulin (BLG) and three casein fractions (CSN1S1, CSN2, and CSN3) in individual genetic groups of animals were carried out in milk of 342 cows. Also, milk content of casein and whey proteins was assayed. The highest level of casein was found in farm B, in all the genotypic groups of cows, while the proportion of whey proteins in milk was highest in farm A, in the genotypic group of cows with 25% to 50% of HF genes. As far as the remaining groups of cows are concerned, the level of whey proteins was equal in both studied farms. A genetic differentiation of polymorphisms in milk protein was found in the analysed farms. Frequency of kappa-casein B (CSN3 B) genetic variants, which are desired in processing, was higher in farm A.

Koch's Bacillus – a look at the first isolate of Mycobacterium tuberculosis from a modern perspective

Using molecular methods the authors have studied mycobacterial DNA taken from a 19th century victim of tuberculosis. This was the case from which Robert Koch first isolated and cultured the organism responsible for tuberculosis. The mycobacteria were preserved within five glass culture tubes as abundant bacterial colonies on slopes of a gelatinous culture medium of unknown composition. Originally presented by Koch to surgical laryngologist Walter Jobson Horne in London in 1901, the relic has, since 1983, been in the care of the Royal College of Surgeons of England. Light and electron microscopy established the presence of acid-fast mycobacteria but showed that morphological preservation was generally poor. Eleven different genomic loci were successfully amplified by PCR. This series of experiments confirmed that the organisms were indeed Mycobacterium tuberculosis and further showed that the original strain was in evolutionary terms similar to ‘modern’ isolates, having undergone the TB D1 deletion. Attempts to determine the genotypic group of the isolate were only partially successful, due in part to the degraded nature of the DNA and possibly also to a truncation in the katG gene, which formed part of the classification scheme. Spoligotyping resulted in amplification of DR spacers consistent with M. tuberculosis but with discrepancies between independent extracts, stressing the limitations of this typing method when applied to poorly preserved material.

Molecular genetic characterization of the Fennoscandian cervid strain, a new genotypic group (G10) of Echinococcus granulosus

The northern biotype of Echinococcus granulosus occurs in North America and northern Eurasia in life-cycles involving cervids. Previously, cervid isolates of E. granulosus from North America have been characterized using molecular genetic techniques as the G8 genotype. In this study, 5 isolates of E. granulosus were collected from 4 reindeer and 1 moose in north-eastern Finland. DNA sequences within regions of mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NDI) genes and the internal transcribed spacer 1 (ITS-1) fragment of the ribosomal DNA were analysed. The mitochondrial nucleotide sequences were identical in all isolates, but high sequence variation was found in the ITS-1 region. Mitochondrial and nuclear sequences of the Finnish cervid E. granulosus and the camel strain (G6) of E. granulosus resembled closely each other. According to phylogenetic analyses, the Finnish isolates have close relationships also with the pig (G7) and cattle (G5) strains. Although some similarities were found with the previously published North American cervid strain (G8), particularly in the NDI sequence and some of the ITS-1 clones, the Finnish E. granulosus form represents a distinct, previously undescribed genotype of E. granulosus. The novel genotype is hereby named as the Fennoscandian cervid strain (G10).

Taxonomic relationships in the genus <i>Ammonia</i> (Foraminifera) based on ribosomal DNA sequences

The genus Ammonia is a common benthic foraminifer which is widely distributed in nearshore marine environments. Its large morphological variability causes considerable difficulties in species identification. In the present study, we investigated taxonomic relationships in Ammonia by using a molecular approach based on ribosomal DNA sequences. We obtained 149 partial large subunit ribosomal DNA (LSU rDNA) sequences and 23 small subunit ribosomal DNA (SSU rDNA) sequences from 88 living Ammonia specimens which were collected from free-living populations in 14 localities. Sequence analysis revealed the presence of eight distinct genotypic groups (T1–T7, T9) and one distinct genotype that is represented by one specimen (T8). Examination of morphological characters shows that only one genotypic group can be clearly distinguished by its morphology. Biogeographical and ecological features are used for an additional characterization and it seems that the different groups live in relatively well defined environmental conditions and that only one genotypic group is cosmopolitan, while the others have a rather restricted geographical distribution. According to our study, three of the genotypic groups can be regarded as distinct species.