Generating amplicon reads for microbial community assessment with next-generation sequencing.

Marker gene amplicon sequencing is often preferred over whole genome sequencing for microbial community characterization, due to its lower cost while still enabling assessment of uncultivable organisms. This technique involves many experimental steps, each of which can be a source of errors and bias. We present an up-to-date overview of the whole experimental pipeline, from sampling to sequencing reads, and give information allowing for informed choices at each step of both planning and execution of a microbial community assessment study. When applicable, we also suggest ways of avoiding inherent pitfalls in amplicon sequencing.

Effect of the 5-HTTLPR polymorphism on affective temperament, depression and body mass index in obesity.

BACKGROUND AND AIM: Many studies show high prevalence of affective disorders in obese patients. Affective temperament is a subclinical manifestation of such conditions. The 5-HTT gene encoding the serotonin transporter may be involved in both mood and eating dysregulation. The aim of this study was to investigate the influence of a polymorphism in the 5-HTT gene on affective temperament types, depressive symptoms and Body Mass Index (BMI) in obese patients. METHODS: This study involved 390 patients (237 females, and 153 males) with obesity. The TEMPS-A questionnaire, Beck Depression Inventory (BDI) and Hamilton Depression Rating Scale (HDRS) were used to evaluate affective temperaments and prevalence of depression. DNA was obtained for serotonin transporter gene-linked polymorphism (5-HTTLPR) genotyping. RESULTS: In obese patients S/S genotype was associated with depressive and L/L with cyclothymic temperament. Subjects with L/L genotype presented significantly higher BMI and greater intensity of depressive symptoms in BDI and HDRS. Females scored higher in anxious and depressive, while males in hyperthymic, cyclothymic and irritable temperaments. Females scored higher in BDI (subjective depression) while males in HDRS (objective depression). LIMITATIONS: TEMPS-A, BDI and HDRS are frequently used in studies on affective disorders. However, these methods do not examine all dimensions of mood and personality. CONCLUSIONS: In obese patients S allele of 5-HTTLPR was associated with development of depressive temperament while L allele corresponded with greater obesity and prevalence of depression. Different mechanisms may be involved in manifestation of depression in males and females with obesity.

Caspase recruitment domain 15 gene haplotypes in sarcoidosis.

Sarcoidosis is a systematic granulomatous disorder. Genetic susceptibility could play a central role in the disease development and progression. In this study, we investigated whether caspase recruitment domain 15 (CARD15) gene haplotypes are associated with the onset or the clinical course of sarcoidosis. Three hundred Caucasian sarcoidosis patients and 381 matched controls were included. Eight haplotype-tagging single nucleotide polymorphisms (SNPs) in the CARD15 gene were examined by mass spectrometry-based SNP genotyping. By haplotype analysis, mutations located in between tested SNPs can also be identified. Therefore, we can conclude that there is no association between the CARD15 gene and the development or a special phenotype of sarcoidosis in our cohort.

Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3.

Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum beta-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla(CTX-M-3), it also bears the bla(TEM-1), aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of beta-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla(CTX-M-3), accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

Mosaic structure of p1658/97, a 125-kilobase plasmid harboring an active amplicon with the extended-spectrum beta-lactamase gene blaSHV-5.

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

[The value of structural neuroimaging in the selection of patients for epileptic surgery]. / Znaczenie strukturalnych badan neuroobrazujacych, w kwalifikacji chorych do leczenia operacyjnego padaczki.

UNLABELLED: The aim of this study was to investigate the value of structural neuroimaging with MRI in the selection of patients for epilepsy surgery. We sought to determine whether MRI influenced decision concerning resective surgery and whether MRI provided much more useful information than enhanced CT. MATERIALS AND METHODS: Neuroimaging studies, MRI and CT, of 300 patients; 265 with partial and 35 with primary generalized seizures, evaluated for surgical treatment of epilepsy were analysed. The MRIs and CTs were interpreted using visual diagnostic criteria and findings were correlated with the EEG changes and clinical semiology. RESULTS: MRIs identified structural lesions in 142, CTs in 96 of all patients. The clinical semiology (partial seizures), MRI, CT and EEG focal findings were concordant in 72 cases. The group of 34 patients had resective surgery. The 7 patients were also operated with MRI and CT focal abnormalities discordant with EEG changes. Also one patient with primary generalized epilepsy and temporal lobe lesion (glioma) had resective surgery. MRI studies revealed structural lesions in 48 patients with normal CT studies. The 43 patients with partial epilepsy had normal CTs and lesions in MRIs; the 34 cases revealed correlation with the EEG findings in 29 temporal and 5 extratemporal regions. Surgery were performed in 23 cases. Also one with partial seizures and MRI detected hippocampal atrophy was operated, despite of generalized EEG patterns. In contrast CT revealed two patients with normal MRI and focal changes. The patients with partial seizures and only CT abnormalities (focal calcifications) were not operated due to discordant EEG findings. In group of 132 patients with normal neuroimaging studies and EEG identified seizure focus only 27 had anterior temporal lobectomy. CONCLUSION: MRI studies gave additional information in case of 16% patients with intractable epilepsy in comparison with CT findings. Resective epilepsy surgery was almost twice as often performed when MRIs revealed structural abnormality. In operated patients, diagnostic sensitivity of structural MRI, CT and EEG to neurophatology were 70.6%, 46.7 and 92.4% respectively.