Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Genetic diversity in fluoroquinolone and macrolide-resistant Campylobacter coli from pigs

The genetic diversity of 115 Campylobacter coli strains, isolated from pigs of 59 geographical distant farms in Switzerland, were characterized on the basis of their DNA fingerprints and resistance to macrolides and fluoroquinolones. Sequence analysis showed that the macrolide-resistant isolates had a point mutation in the 23S ribosomal RNA (rRNA) genes (A2075G) and that the fluoroquinolone-resistant isolates had a point mutation in the gyrase gene gyrA (C257T). One fluoroquinolone-resistant strain had an additional transition mutation in the gyrB gene (A1471C). The flaA restriction fragment length polymorphism (RFLP) genotyping revealed that 57% of the isolates were genetically different. Point mutations in the 23S rRNA and gyrA genes could be found in both genetically distant and genetically related isolates. Additionally, isolates with and without point mutations were found within individual farms and on different farms. This study showed that the ciprofloxacin and erythromycin-resistant C. coli population present on the pig farms is not issued from a common ancestral clone, but individual Campylobacter strains have most likely mutated independently to acquire resistances under the selective pressure of an antibiotic.

CYP2E1 genotype and isoniazid-induced hepatotoxicity in patients treated for latent tuberculosis

OBJECTIVE: To determine whether pharmacogenetic tests such as N-acetyltransferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1) genotyping are useful in identifying patients prone to antituberculosis drug-induced hepatotoxicity in a cosmopolite population. METHODS: In a prospective study we genotyped 89 patients treated with isoniazid (INH) for latent tuberculosis. INH-induced hepatitis (INH-H) or elevated liver enzymes including hepatitis (INH-ELE) was diagnosed based on the clinical diagnostic scale (CDS) designed for routine clinical practice. NAT2 genotypes were assessed by fluorescence resonance energy transfer probe after PCR analysis, and CYP2E1 genotypes were determined by PCR with restriction fragment length polymorphism analysis. RESULTS: Twenty-six patients (29%) had INH-ELE, while eight (9%) presented with INH-H leading to INH treatment interruption. We report no significant influence of NAT2 polymorphism, but we did find a significant association between the CYP2E1 *1A/*1A genotype and INH-ELE (OR: 3.4; 95% CI:1.1-12; p = 0.02) and a non significant trend for INH-H (OR: 5.9; 95% CI: 0.69-270; p = 0.13) compared with other CYP2E1 genotypes. This test for predicting INH-ELE had a positive predictive value (PPV) of 39% (95% CI: 26-54%) and a negative predictive value (NPV) of 84% (95% CI: 69-94%). CONCLUSION: The genotyping of CYP2E1 polymorphisms may be a useful predictive tool in the common setting of a highly heterogeneous population for predicting isoniazid-induced hepatic toxicity. Larger prospective randomized trials are needed to confirm these results.

Distribution and genetic variability among Campylobacter spp. isolates from different animal species and humans in Switzerland

In Switzerland, a national database with 1028 Campylobacter isolates from poultry, pigs, cats, dogs, cattle, humans, zoo animals and water has been created. The database contains the genetic fingerprint and background information of each Campylobacter isolate. Dominant species could be identified in the different sources with a majority of Campylobacter jejuni in poultry (73%), humans (79%), cattle (95%), zoo animals (40%) and water (100%), of Campylobacter coli in pigs (72%), and of Campylobacter upsaliensis/helveticus in cats and dogs (55%). The comparison of three genotyping methods, amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis and restriction fragment length polymorphism, revealed that AFLP allows discrimination between the different Campylobacter species and is the most appropriate method to distinguish specific strains within the same species. Genotyping analysis demonstrated that the Campylobacter population is heterogeneous among the different sources and that no dominant clone is spread in the country. Genotyping and the resulting database are useful tools to trace back future Campylobacter infections.

Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae

Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.

Japanese-US common-arm analysis of paclitaxel plus carboplatin in advanced non-small-cell lung cancer: a model for assessing population-related pharmacogenomics

PURPOSE To explore whether population-related pharmacogenomics contribute to differences in patient outcomes between clinical trials performed in Japan and the United States, given similar study designs, eligibility criteria, staging, and treatment regimens. METHODS We prospectively designed and conducted three phase III trials (Four-Arm Cooperative Study, LC00-03, and S0003) in advanced-stage, non-small-cell lung cancer, each with a common arm of paclitaxel plus carboplatin. Genomic DNA was collected from patients in LC00-03 and S0003 who received paclitaxel (225 mg/m(2)) and carboplatin (area under the concentration-time curve, 6). Genotypic variants of CYP3A4, CYP3A5, CYP2C8, NR1I2-206, ABCB1, ERCC1, and ERCC2 were analyzed by pyrosequencing or by PCR restriction fragment length polymorphism. Results were assessed by Cox model for survival and by logistic regression for response and toxicity. Results Clinical results were similar in the two Japanese trials, and were significantly different from the US trial, for survival, neutropenia, febrile neutropenia, and anemia. There was a significant difference between Japanese and US patients in genotypic distribution for CYP3A4*1B (P = .01), CYP3A5*3C (P = .03), ERCC1 118 (P < .0001), ERCC2 K751Q (P < .001), and CYP2C8 R139K (P = .01). Genotypic associations were observed between CYP3A4*1B for progression-free survival (hazard ratio [HR], 0.36; 95% CI, 0.14 to 0.94; P = .04) and ERCC2 K751Q for response (HR, 0.33; 95% CI, 0.13 to 0.83; P = .02). For grade 4 neutropenia, the HR for ABCB1 3425C-->T was 1.84 (95% CI, 0.77 to 4.48; P = .19). CONCLUSION Differences in allelic distribution for genes involved in paclitaxel disposition or DNA repair were observed between Japanese and US patients. In an exploratory analysis, genotype-related associations with patient outcomes were observed for CYP3A4*1B and ERCC2 K751Q. This common-arm approach facilitates the prospective study of population-related pharmacogenomics in which ethnic differences in antineoplastic drug disposition are anticipated.

Detection of Streptococcus pneumoniae strain cocolonization in the nasopharynx

Colonization with more than one distinct strain of the same species, also termed cocolonization, is a prerequisite for horizontal gene transfer between pneumococcal strains that may lead to change of the capsular serotype. Capsule switch has become an important issue since the introduction of conjugated pneumococcal polysaccharide vaccines. There is, however, a lack of techniques to detect multiple colonization by S. pneumoniae strains directly in nasopharyngeal samples. Two hundred eighty-seven nasopharyngeal swabs collected during the prevaccine era within a nationwide surveillance program were analyzed by a novel technique for the detection of cocolonization, based on PCR amplification of a noncoding region adjacent to the pneumolysin gene (plyNCR) and restriction fragment length polymorphism (RFLP) analysis. The numbers of strains and their relative abundance in cocolonized samples were determined by terminal RFLP. The pneumococcal carriage rate found by PCR was 51.6%, compared to 40.0% found by culture. Cocolonization was present in 9.5% (10/105) of samples, most (9/10) of which contained two strains in a ratio of between 1:1 and 17:1. Five of the 10 cocolonized samples showed combinations of vaccine types only (n = 2) or combinations of nonvaccine types only (n = 3). Carriers of multiple pneumococcal strains had received recent antibiotic treatment more often than those colonized with a single strain (33% versus 9%, P = 0.025). This new technique allows for the rapid and economical study of pneumococcal cocolonization in nasopharyngeal swabs. It will be valuable for the surveillance of S. pneumoniae epidemiology under vaccine selection pressure.

Clustering subspecies of Aeromonas salmonicida using IS630 typing.

BACKGROUND The insertion element IS630 found in Aeromonas salmonicida belongs to the IS630-Tc1-mariner superfamily of transposons. It is present in multiple copies and represents approximately half of the IS present in the genome of A. salmonicida subsp. salmonicida A449. RESULTS By using High Copy Number IS630 Restriction Fragment Length Polymorphism (HCN-IS630-RFLP), strains of various subspecies of Aeromonas salmonicida showed conserved or clustering patterns, thus allowing their differentiation from each other. Fingerprints of A. salmonicida subsp. salmonicida showed the highest homogeneity while 'atypical' A. salmonicida strains were more heterogeneous. IS630 typing also differentiated A. salmonicida from other Aeromonas species. The copy number of IS630 in Aeromonas salmonicida ranges from 8 to 35 and is much lower in other Aeromonas species. CONCLUSIONS HCN-IS630-RFLP is a powerful tool for subtyping of A. salmonicida. The high stability of IS630 insertions in A. salmonicida subsp. salmonicida indicates that it might have played a role in pathoadaptation of A. salmonicida which has reached an optimal configuration in the highly virulent and specific fish pathogen A. salmonicida subsp. salmonicida.

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA:rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C:N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision.

Phylogenetic position of Riemerella anatipestifer based on 16S rRNA gene sequences

Riemerella anatipestifer, the causative agent of septicemia anserum exsudativa (also called new duckling disease), belongs to the family Flavobacteriaceae of gram-negative bacteria. We determined the DNA sequences of the rrs genes encoding the 16S rRNAs of four R. anatipestifer strains by directly sequencing PCR-amplified rrs genes. A sequence similarity analysis confirmed the phylogenetic position of R. anatipestifer in the family Flavobacteriaceae in rRNA superfamily V and allowed fine mapping of R. anatipestifer on a separate rRNA branch comprising the most closely related species, Bergeyella zoohelcum, as well as Chryseobacterium balustinum, Chryseobacterium indologenes, and Chryseobacterium gleum. The sequences of the rrs genes of the four R. anatipestifer strains varied between 0.5 and 1.0%, but all of the strains occupied the same position on the phylogenetic tree. In general, differences in rrs genes were observed among R. anatipestifer strains, even within a given serotype, as shown by restriction fragment length polymorphism of PCR-amplified rrs genes.

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype

BACKGROUND/AIM To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.

The -308 tumour necrosis factor-alpha gene polymorphism predicts therapeutic response to TNFalpha-blockers in rheumatoid arthritis and spondyloarthritis patients

OBJECTIVE: To examine whether the G-to-A polymorphism at position -308 in the promoter of the tumour necrosis factor-alpha (TNFalpha) gene influences the therapeutic response to TNFalpha-blockers in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS). METHODS: A total of 54 patients with RA, 10 with PsA and 22 with AS were genotyped by polymerase chain reaction for the -308 TNFalpha promoter polymorphism. They were treated with infliximab (n = 63), adalimumab (n = 10) or etanercept (n = 13). Clinical response was assessed after 24 weeks by the Disease Activity Score in 28 joints (DAS28) for RA and PsA, and the Bath Ankylosing Spondylitis Activity Index (BASDAI) for AS patients. RESULTS: All patients with the A/A genotype (n = 3, all RA) and two patients with the A/G genotype (AS) failed to respond to anti-TNF treatment. Irrespective of the underlying disease, moderate response (n = 44) was predominantly associated with the A/G genotype (A/G 18/22, G/G 4/22), whereas good response (n = 59) was exclusively seen in patients with the G/G genotype. The average improvement in the DAS28 score was 0.83 in the A/A, 1.50 in the A/G and 2.64 in the G/G group of RA and PsA patients (P < 0.0001). The BASDAI score in AS improved on average by 1.21 in the A/G and by 3.30 in the G/G group (P < 0.005). CONCLUSIONS: The data suggest that humans with a TNFalpha -308 G/G genotype are better responders to anti-TNFalpha treatment than those with A/A or A/G genotypes independent of the treated rheumatic disease (RA, PsA or AS).

Alcohol and colorectal cancer: the role of alcohol dehydrogenase 1C polymorphism

BACKGROUND: Chronic alcohol consumption is a risk factor for colorectal cancer. Animal experiments as well as genetic linkage studies in Japanese individuals with inactive acetaldehyde dehydrogenase leading to elevated acetaldehyde concentrations following ethanol ingestion support the hypothesis that acetaldehyde may be responsible for this carcinogenic effect of alcohol. In Caucasians, a polymorphism of alcohol dehydrogenase 1C (ADH1C) exists resulting in different acetaldehyde concentrations following ethanol oxidation. METHODS: To evaluate whether the association between alcohol consumption and colorectal tumor development is modified by ADH1C polymorphism, we recruited 173 individuals with colorectal tumors diagnosed by colonoscopy and 788 control individuals without colorectal tumors. Genotyping was performed using genomic DNA extracted from whole blood followed by polymerase chain reaction. RESULTS: Genotype ADH1C*1/1 was more frequent in patients with alcohol-associated colorectal neoplasia compared to patients without cancers in the multivariate model controlling for age, gender, and alcohol intake (odds ratio = 1.674, 95% confidence interval = 1.110-2.524, 2-sided p from Wald test = 0.0139). In addition, the joint test of the genetic effect and interaction between ADH1C genotype and alcohol intake (2-sided p = 0.0007) indicated that the difference in ADH1C*1 polymorphisms between controls and colorectal neoplasia is strongly influenced by the alcohol consumption and that only individuals drinking more than 30 g ethanol per day with the genotype ADH1C*1/1 had an increased risk for colorectal tumors. CONCLUSIONS: These data identify ADH1C homozygosity as a genetic risk marker for colorectal tumors in individuals consuming more than 30 g alcohol per day and emphasize the role of acetaldehyde as a carcinogenic agent in alcohol-related colorectal carcinogenesis.