c High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common "benchtop" sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.

Nosocomial infections pose a significant threat to patient health; however, the gold standard laboratory method for determining bacterial relatedness (pulsed-field gel electrophoresis [PFGE]) remains essentially unchanged 20 years after its introduction. Here, we explored bacterial whole-genome sequencing (WGS) as an alternative approach for molecular strain typing. We compared WGS to PFGE for investigating presumptive outbreaks involving three important pathogens: vancomycin-resistant Enterococcus faecium (n ‫؍‬ 19), methicillin-resistant Staphylococcus aureus (n ‫؍‬ 17), and Acinetobacter baumannii (n ‫؍‬ 15). WGS was highly reproducible (average < 0.39 differences between technical replicates), which enabled a functional, quantitative definition for determining clonality. Strain relatedness data determined by PFGE and WGS roughly correlated, but the resolution of WGS was superior (P ‫؍‬ 5.6 ؋ 10 ؊8 to 0.016). Several discordant results were noted between the methods. A total of 28.9% of isolates which were indistinguishable by PFGE were nonclonal by WGS. For A. baumannii, a species known to undergo rapid horizontal gene transfer, 16.2% of isolate pairs considered nonidentical by PFGE were clonal by WGS. Sequencing whole bacterial genomes with single-nucleotide resolution demonstrates that PFGE is prone to false-positive and false-negative results and suggests the need for a new gold standard approach for molecular epidemiological strain typing.

Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.

Biases in standard, nonselective microbiological culture lead to a distorted characterization of polymicrobial mixtures. NGS16S demonstrates enhanced reproducibility, quantification, and classification accuracy compared with standard culture, providing a more comprehensive, accurate, and culture-free analysis of clinical specimens.

Some bacterial infections involve potentially complex mixtures of species that can now be distinguished using next-generation DNA sequencing. We present a case of mastoiditis where Gram stain, culture, and molecular diagnosis were nondiagnostic or discrepant. Next-generation sequencing implicated coinfection of Fusobacterium nucleatum and Actinomyces israelii, resolving these diagnostic discrepancies. CASE REPORTA 46-year-old developmentally delayed man presented to an outside hospital in September 2013 with swelling of the left mastoid, bleeding gingiva, and increased oral secretions. A computed tomography (CT) scan demonstrated extensive involvement of the left temporal bone and evidence of abscess formation in the temporalis muscle. The abscess was large, ranging medially through the temporal bone to the clivus, with suggestion of osteomyelitis over a portion of the skull base. A chest X-ray indicated concurrent left lower-lobe pneumonia. Incision and drainage of the abscess were performed, and the patient was administered vancomycin (1 g intravenously [i.v.] every 24 h [q24h]) and piperacillin-tazobactam (3.375 g i.v. q6h). He was then transferred to Harborview Medical Center for further care.After transfer, the patient was febrile (38.9°C) and hypotensive (blood pressure of 91/58) and experienced transient hypoxic episodes (oxygen saturations of 73% on room air). He was consequently intubated for hypoxic respiratory failure.The patient underwent extensive surgical debridement of the temporalis muscle and mastoid space, revealing multiple pockets of necrotic tissue. Gram stain of one intraoperative tissue specimen showed rare (1ϩ) branching Gram-positive rods, suggesting Nocardia or Actinomyces species, while a Gram stain of other specimens (three swabs and one bone fragment) revealed no organisms. Postoperatively, the patient's treatment regimen was transitioned to vancomycin (0.75 g i.v. q8h), ceftriaxone (2 g i.v. q12h), and metronidazole (500 mg i.v. q8h). The patient had difficulty weaning from ventilator support, and 4 days after the procedure a CT scan revealed a left-sided hemithoracic opacity. The patient was switched to piperacillin-tazobactam (3.375 g i.v. q8h) to offer empirical coverage of both tissue infection and suspected ventilator-associated pneumonia. Bronchoalveolar lavage was later culture positive for Pseudomonas aeruginosa (determined to be sensitive to amikacin, cefepime, ceftazidime, ciprofloxacin, gentamicin, imipenem, levofloxacin, meropenem, piperacillin-tazobactam, and tobramycin).Patient tissue and bone specimens were separately homogenized in 3 ml of sterile saline using a Seward Stomacher 80 (Davie, FL). For bacterial cultures, material was inoculated onto sheep blood (Trypticase soy agar with 5% sheep blood), chocolate, MacConkey, and brucella agar plates and into brain heart infusion broth (all media from Remel, Lenexa, KS). Brucella agar plates were incubated anaerobically (35°C, 90% N 2 , 5% CO 2 , 5% H 2 ), and all other media were incubated aerobically (35°C, room air su...

The differential diagnosis of diarrhea in immunocompromised patients encompasses many intestinal parasites including the coccidian Cystoisospora belli. Gastrointestinal infection with C. belli leads to cystoisosporiasis with diarrhea and, depending on host immune status, can cause extraintestinal disease. C. belli is usually diagnosed by examination of stool or intestinal biopsy specimens; however, the organism may be undetected using these test methods. Thus, more sensitive molecular tools for detection of pathogenic parasites are desirable. Herein is described a patient with AIDS who had persistent diarrhea of unknown cause. Microscopic examinations of stool and ileal biopsy specimens were initially unremarkable for any specific pathogen. Screening of DNA extracted from biopsy material using extendedrange PCR primers recognizing conserved DNA sequences found in many fungi and parasites revealed infection with C. belli, which was confirmed at repeat histologic analysis. Extended-range PCR screening was used because the differential diagnosis was broad and other tools were not applied, yet this molecular approach led to the appropriate diagnosis and treatment of the condition. Thus, this approach offers a promising test for diagnosis of parasitic diseases that elude diagnosis using conventional methods. ( J Mol

Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a major challenge in health care, yet the limited heterogeneity within this group hinders molecular investigations of related outbreaks. Pulsed-field gel electrophoresis (PFGE) has been the gold standard approach but is impractical for many clinical laboratories and is often replaced with PCR-based methods. Regardless, both approaches can prove problematic for identifying subclonal outbreaks. Here, we explore the use of whole-genome sequencing for clinical laboratory investigations of MRSA molecular epidemiology. We examine the relationships of 44 MRSA isolates collected over a period of 3 years by using whole-genome sequencing and two PCR-based methods, multilocus variablenumber tandem-repeat analysis (MLVA) and spa typing. We find that MLVA offers higher resolution than spa typing, as it resolved 17 versus 12 discrete isolate groups, respectively. In contrast, whole-genome sequencing reproducibly cataloged genomic variants (131,424 different single nucleotide polymorphisms and indels across the strain collection) that uniquely identified each MRSA clone, recapitulating those groups but enabling higher-resolution phylogenetic inferences of the epidemiological relationships. Importantly, whole-genome sequencing detected a significant number of variants, thereby distinguishing between groups that were considered identical by both spa typing (minimum, 1,124 polymorphisms) and MLVA (minimum, 193 polymorphisms); this suggests that these more conventional approaches can lead to false-positive identification of outbreaks due to inappropriate grouping of genetically distinct strains. An analysis of the distribution of variants across the MRSA genome reveals 47 mutational hot spots (comprising ϳ2.5% of the genome) that account for 23.5% of the observed polymorphisms, and the use of this selected data set successfully recapitulates most epidemiological relationships in this pathogen group.

BackgroundInvasive Mucorales infections (IMI) lead to significant morbidity and mortality in immunocompromised hosts. The role of season and climatic conditions in case clustering of IMI remain poorly understood.MethodsFollowing detection of a cluster of sinopulmonary IMIs in patients with hematologic malignancies, we reviewed center-based medical records of all patients with IMIs and other invasive fungal infections (IFIs) between January of 2012 and August of 2015 to assess for case clustering in relation to seasonality.ResultsA cluster of 7 patients were identified with sinopulmonary IMIs (Rhizopus microsporus/azygosporus, 6; Rhizomucor pusillus, 1) during a 3 month period between June and August of 2014. All patients died or were discharged to hospice. The cluster was managed with institution of standardized posaconazole prophylaxis to high-risk patients and patient use of N-95 masks when outside of protected areas on the inpatient service. Review of an earlier study period identified 11 patients with IMIs of varying species over the preceding 29 months without evidence of clustering. There were 9 total IMIs in the later study period (12 month post-initial cluster) with 5 additional cases in the summer months, again suggesting seasonal clustering. Extensive environmental sampling did not reveal a source of mold. Using local climatological data abstracted from National Centers for Environmental Information the clusters appeared to be associated with high temperatures and low precipitation.ConclusionsSinopulmonary Mucorales clusters at our center had a seasonal variation which appeared to be related to temperature and precipitation. Given the significant mortality associated with IMIs, local climatic conditions may need to be considered when considering center specific fungal prevention and prophylaxis strategies for high-risk patients.