Evaluating gut microbiota profiles from archived fecal samples.

BACKGROUND: Associations between colorectal cancer and microbiota have been identified. Archived fecal samples might be valuable sample sources for investigating causality in carcinogenesis and biomarkers discovery due to the potential of performing longitudinal studies. However, the quality, quantity and stability of the gut microbiota in these fecal samples must be assessed prior to such studies. We evaluated i) cross-contamination during analysis for fecal blood and ii) evaporation in stored perforated fecal immunochemical tests (iFOBT) samples, iii) temperature stability as well as iv) comparison of the gut microbiota diversity and composition in archived, iFOBT and fresh fecal samples in order to assess feasibility of large scale microbiota studies. METHODS: The microbiota profiles were obtained by sequencing the V3-V4 region of 16S rDNA gene. RESULTS: The iFOBT does not introduce any cross-sample contamination detectable by qPCR. Neither could we detect evaporation during freeze-thaw cycle of perforated iFOBT samples. Our results confirm room temperature stability of the gut microbiome. Diverse microbial profiles were achieved in 100% of fresh, 81% of long-term archived and 96% of iFOBT samples. Microbial diversity and composition were comparable between fresh and iFOBT samples, however, diversity differed significantly between long-term archived, fresh and iFOBT samples. CONCLUSION: Our data showed that it is feasible to exploit archived fecal sample sets originally collected for testing of fecal blood. The advantages of using these sample sets for microbial biomarker discovery and longitudinal observational studies are the availability of high-quality diagnostic and follow-up data. However, care must be taken when microbiota are profiled in long-term archived fecal samples.

Genotypic diversity of anogenital human papillomavirus in women attending cervical cancer screening in Harare, Zimbabwe.

Although anogenital cancers have been on a gradual rise in developing countries in the past few decades, they have been understudied. The objective was to investigate genotypic diversity of anogenital HPV amongst women reporting for routine cervical cancer screening in Harare in Zimbabwe. A cross-sectional study that enrolled 144 women ≥18 years from a cervical cancer-screening clinic was performed. Each woman provided a self-collected cervico-vaginal swab (VS) and a clinician-collected anal swab (CCAS). HIV testing was offered and cervical cytology was performed. Both VS and CCAS samples were HPV genotyped, using amplicon sequencing of the L1 gene region with Illumina technology. Mean age of the women was 39.9 (range 18-83 years, SD ± 11.0). HPV prevalence was 72% (104/144) in VS and 48% (69/144) in CCAS. The most common genotypes detected in both VS and CCAS were HPV18, HPV52, and HPV16. Sixty two percent of the subjects had multiple genotypic HPV infections. The odds of being HPV-positive among HIV-infected women were higher than in HIV-negative women in both the vagina and the anus (CCAS OR = 4.8; CI 2.4-9.8, P < 0.001) and (VS OR = 2.9; CI 1.3-6.4, P = 0.005). High HPV prevalence and diverse genotypes were detected in both the vagina and anus. Anal oncogenic HPV infection was common. HPV 52 was one of the most common oncogenic genotypes in both the vagina and anus. HIV co-infection played a significant role in the prevalence of HPV. These data have implications for design of primary and secondary programs for prevention of anogenital cancer in Zimbabwe.

erm gene distribution among Norwegian Bacteroides isolates and evaluation of phenotypic tests to detect inducible clindamycin resistance in Bacteroides species.

The aims of this study were to describe the distribution of the most common erm genes in a collection of Norwegian Bacteroides isolates and to investigate whether the phenotypic tests for determining inducible clindamycin resistance among Bacteroides species recommended by EUCAST, NordicAST and the manufacturer of E-test®, are effective. We investigated 175 unique Bacteroides isolates for the presence of erm(B), erm(F) and erm(G) genes, determined their minimum inhibitory concentrations (MICs) to clindamycin and categorised their susceptibility according to EUCAST breakpoints. 27 isolates were resistant to clindamycin. Furthermore, we investigated whether these recommended methods could detect inducible resistance in the Bacteroides isolates: 1) EUCAST recommendation: Dissociated resistance to erythromycin (clindamycin susceptible with erythromycin MIC > 32 mg/L), 2) NordicAST recommendation: Double disk diffusion test (DDD) or 3) Manufacturer of E-test®'s recommendation: prolonged incubation of clindamycin E-test® for 48 h. erm genes were detected in 30 (17%, 95% CI 12%-23%) of 175 Bacteroides isolates with erm(F) as the dominating gene. There were six (4%, 95% CI 1%-7%) of 148 clindamycin susceptible isolates harbouring erm genes, they were considered inducibly resistant to clindamycin. None of the methods for phenotypic detection of inducible clindamycin resistance performed satisfactory with sensitivities of 33%, 17% and 0% and specificities of 90%, 99% and 97% for dissociated resistance, DDD and prolonged incubation of clindamycin E-test®, respectively. In our view, the scientific basis for investigating every Bacteroides isolate for inducible resistance to clindamycin is weak. Molecular detection of erm genes may prove a better option than the phenotypic methods we evaluated.

Massive parallel sequencing provides new perspectives on bacterial brain abscesses.

Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture- and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications. Aggregatibacter aphrophilus, Fusobacterium nucleatum, and Streptococcus intermedius or combinations of them were found in all spontaneous polymicrobial abscesses. F. nucleatum was systematically detected in samples with anaerobic flora. The increased detection rate for Actinomyces spp. and facultative Gram-negative rods further revealed several species associations. We suggest that A. aphrophilus, F. nucleatum, and S. intermedius are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition, F. nucleatum seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop.

Streptococcus pyogenes isolates causing severe infections in Norway in 2006 to 2007: emm types, multilocus sequence types, and superantigen profiles.

To investigate the epidemiological patterns and genetic characteristics of disease caused by group A Streptococcus (GAS), all available isolates from invasive cases in Norway during 2006 to 2007 (262 isolates) were subjected to antimicrobial susceptibility testing, T serotyping, emm typing, and multilocus sequence typing and screened for known streptococcal pyrogenic exotoxin (Spe) genes, smeZ, and ssa. The average incidence rate was 3.1 cases per 100,000 individuals. The most prevalent sequence types (STs) were STs 52, 28, and 334. In association with emm types 28, 77, and 87, the serotype T-28 comprised 24.8% of the strains. emm types 28, 1, and 82 were dominating. In 2007, a sharp increase in the number of emm-6 strains was noted. All strains were sensitive to penicillin and quinupristin-dalfopristin, while 3.4% and 6.1% of the strains were resistant to macrolides and tetracycline, respectively. Furthermore, the emm-6 strains had intermediate susceptibility to ofloxacin. Isolates displayed a wide variety of gene profiles, as shown by the presence or absence of the Spe genes, smeZ, and ssa, but 48% of the isolates fell into one of three profiles. In most cases, an emm type was restricted to one gene profile. Although the incidence decreased during this study, invasive GAS disease still has a high endemic rate, with involvement of both established and emerging emm types displaying variability in virulence gene profiles as well as differences in gender and age group preferences.

A core microbiota dominates a rich microbial diversity in the bovine udder and may indicate presence of dysbiosis.

The importance of the microbiome for bovine udder health is not well explored and most of the knowledge originates from research on mastitis. Better understanding of the microbial diversity inside the healthy udder of lactating cows might help to reduce mastitis, use of antibiotics and improve animal welfare. In this study, we investigated the microbial diversity of over 400 quarter milk samples from 60 cows sampled from two farms and on two different occasions during the same lactation period. Microbiota analysis was performed using amplicon sequencing of the 16S rRNA gene and over 1000 isolates were identified using MALDI-TOF MS. We detected a high abundance of two bacterial families, Corynebacteriaceae and Staphylococcaceae, which accounted for almost 50% of the udder microbiota of healthy cows and were detected in all the cow udders and in more than 98% of quarter milk samples. A strong negative correlation between these bacterial families was detected indicating a possible competition. The overall composition of the udder microbiota was highly diverse and significantly different between cows and between quarter milk samples from the same cow. Furthermore, we introduced a novel definition of a dysbiotic quarter at individual cow level, by analyzing the milk microbiota, and a high frequency of dysbiotic quarter samples were detected distributed among the farms and the samples. These results emphasize the importance of deepening the studies of the bovine udder microbiome to elucidate its role in udder health.

A preliminary study on the potential of Nanopore MinION and Illumina MiSeq 16S rRNA gene sequencing to characterize building-dust microbiomes.

There is a growing awareness of the importance of indoor microbiomes for human health. Given their complexity, these microbiomes can only be adequately surveyed using high throughput sequencing techniques. Oxford Nanopore's MinION is the newest third generation sequencing technology on the market. With its many advantages such as portability, user friendliness, simplicity, speed of sequencing and long read length, the technology is now an actual contender to established sequencing platforms. MinION's main disadvantage is a relatively low read accuracy compared to several other platforms, although this is constantly improving. The present study, which appears to be the first of its kind, provides the results of a preliminary analysis of the microbial communities in indoor environments based on 16S rRNA gene amplicon sequencing, using both the Oxford Nanopore Technologies (ONT) MinIOn and the Illumina MiSeq DNA sequencers. At the level of family and above, there was no significant difference between the microbial compositions as revealed by the two platforms. However, at the genus, and particularly at the species level, the ONT MinION reported greater taxonomic resolution than Illumina MiSeq.

Whole Genome Sequencing and Characterization of Multidrug-Resistant (MDR) Bacterial Strains Isolated From a Norwegian University Campus Pond.

The presence of extended-spectrum ß-lactamase (ESBL)-producing bacteria in environmental sources has been reported worldwide and constitutes a serious risk of community-acquired infections with limited treatment options. The current study aimed to explore the presence of these worrisome bacteria in a pond located at the Norwegian University of Life Sciences in Ås, Norway. A total of 98 bacterial isolates survived growth on selective chromogenic media and were identified by 16S rRNA Sanger sequencing. All strains were evaluated for the presence of the most commonly found ß-lactamases and ESBLs in clinical settings (bla CTX-M groups 1, 2, and 9, bla CMY, bla SHV, and bla TEM) and carbapenemases (bla IMP, bla KPC, bla NDM, bla OXA, bla SFC1, bla VIM) through multiplex PCR. A total of eight strains were determined to contain one or more genes of interest. Phenotypic resistance to 18 antimicrobial agents was assessed and isolates were subjected to whole genome sequencing through a combination of Oxford Nanopore's MinION and Illumina's MiSeq. Results revealed the presence of ß-lactamase and ESBL-producing Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and a Paraburkholderia spp. Identified ß-lactamases and ESBLs include bla CTX-M, bla TEM, bla CMY, bla SHV and a possible bla KPC-like gene, with both documented and novel sequences established. In addition, two inducible ß-lactamases were found, a class A ß-lactamase (L1) and a cephalosporinase (L2). All strains were determined to be multidrug resistant and numerous resistance genes to non-ß-lactams were observed. In conclusion, this study demonstrates that environmental sources are a potential reservoir of clinically relevant ESBL-producing bacteria that may pose a health risk to humans upon exposure.

TaME-seq: An efficient sequencing approach for characterisation of HPV genomic variability and chromosomal integration.

HPV genomic variability and chromosomal integration are important in the HPV-induced carcinogenic process. To uncover these genomic events in an HPV infection, we have developed an innovative and cost-effective sequencing approach named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). TaME-seq combines tagmentation and multiplex PCR enrichment for simultaneous analysis of HPV variation and chromosomal integration, and it can also be adapted to other viruses. For method validation, cell lines (n = 4), plasmids (n = 3), and HPV16, 18, 31, 33 and 45 positive clinical samples (n = 21) were analysed. Our results showed deep HPV genome-wide sequencing coverage. Chromosomal integration breakpoints and large deletions were identified in HPV positive cell lines and in one clinical sample. HPV genomic variability was observed in all samples allowing identification of low frequency variants. In contrast to other approaches, TaME-seq proved to be highly efficient in HPV target enrichment, leading to reduced sequencing costs. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. Efficient identification of both HPV variability and integration sites will be important for the study of HPV evolution and adaptability and may be an important tool for use in cervical cancer diagnostics.

Sequence type and emm type diversity in Streptococcus pyogenes isolates causing invasive disease in Norway between 1988 and 2003.

The incidence of invasive group A streptococcal disease has increased in Norway since the 1980s. Analysis of 100 isolates recovered from 1988 to 2003 showed an increased genotypic diversity over time, while the prevalence of the strain that dominated in 1988, sequence type (ST)-28/emm-1, decreased. Necrotizing fasciitis was often associated with ST-15/emm-3.

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

Human papillomavirus prevalence and type distribution in urine samples from Norwegian women aged 17 and 21 years: A nationwide cross-sectional study of three non-vaccinated birth cohorts.

BACKGROUND: The aim of the current study was to assess the HPV prevalence in unscreened and unvaccinated young women living in Norway, to provide important baseline data for early estimation of the impact of the HPV vaccination program. METHODS: A total of 13,129 self-sampled urine samples from two complete birth-cohorts of 17-year old women born in 1994 and 1996 and one third of a birth-cohort of 21-year old women born in 1990, were analysed for the presence of 37 HPV types using PCR and a DNA hybridization technique. RESULTS: In the two birth cohorts of 17-year old women, HPV was detected in 19.9% (95% CI 18.8-20.9) and 15.4% (95% CI 14.5-16.3), respectively. High-risk HPV types were detected in 11.2% (95% CI 10.3-12.0) and 7.6% (95% CI 6.9-8.2), respectively, while vaccine types were detected in 7.4% (95% CI 6.7-8.1) and 6.0% (95% CI 5.4-6.6), respectively. Among the 21-year old women HPV was detected in 45.4% (95% CI 42.9-47.8), whereas high-risk types were detected in 29.8% (95% CI 27.5-32.0). Vaccine types (HPV 6, 11, 16, 18) were detected in 16.2% (95% CI 14.4-18.1). CONCLUSION: This large population based study confirms that HPV testing in urine samples is easy and highly feasible for epidemiological studies and vaccine surveillance in young women. HPV was very common and a broad spectrum of HPV types was identified. Differences in HPV prevalence was seen both between age groups and between the two birth cohorts of 17-year old women.

Multilocus sequence typing scheme for bacteria of the Bacillus cereus group.

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.