Comprehensive evaluation of complex polymicrobial specimens using next generation sequencing and standard microbiological culture.

Optimal clinical decision-making depends on identification of clinically relevant organisms present in a sample. Standard microbiological culture may fail to identify unusual or fastidious organisms and can misrepresent relative abundance of sample constituents. Culture-independent methods have improved our ability to deconvolute polymicrobial patient samples. We used next-generation 16S rRNA gene sequencing (NGS16S) to determine how often cultivatable organisms in complex polymicrobial samples are not reported by standard culture. Twenty consecutive bronchoalveolar lavage (BAL) samples were plated to standard and additional media; bacteria were identified by NGS16S analysis of DNA extracted directly from samples or from washed culture plates. 96% of organisms identified were cultivable, but only 21% were reported by standard culture, indicating that standard work-up provides an incomplete assessment of microbial constituents. Direct NGS16S correlated well with standard culture, identifying the same predominant organism in 50% of samples. When predominant organisms differed, NGS16S most often detected anaerobes, whose growth is unsupported by standard culture conditions for this specimen. NGS16S identified more organisms per sample and allowed identification of fastidious organisms, while culture was better at capturing organisms when bacterial load was low, and allowed incidental recovery of non-bacterial pathogens. Molecular and culture-based methods together detect more organisms than either method alone.

Clinical Next Generation Sequencing Outperforms Standard Microbiological Culture for Characterizing Polymicrobial Samples.

BACKGROUND: Humans suffer from infections caused by single species or more complex polymicrobial communities. Identification of infectious bacteria commonly employs microbiological culture, which depends upon the in vitro propagation and isolation of viable organisms. In contrast, detection of bacterial DNA using next generation sequencing (NGS) allows culture-independent microbial profiling, potentially providing important new insights into the microbiota in clinical specimens. METHODS: NGS 16S rRNA gene sequencing (NGS16S) was compared with culture using (a) synthetic polymicrobial samples for which the identity and abundance of organisms present were precisely defined and (b) primary clinical specimens. RESULTS: Complex mixtures of at least 20 organisms were well resolved by NGS16S with excellent reproducibility. In mixed bacterial suspensions (107 total genomes), we observed linear detection of a target organism over a 4-log concentration range (500-3 × 106 genomes). NGS16S analysis more accurately recapitulated the known composition of synthetic samples than standard microbiological culture using nonselective media, which distorted the relative abundance of organisms and frequently failed to identify low-abundance pathogens. However, extended quantitative culture using selective media for each of the component species recovered the expected organisms at the proper abundance, validating NGS16S results. In an analysis of sputa from cystic fibrosis patients, NGS16S identified more clinically relevant pathogens than standard culture. CONCLUSIONS: 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.

Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology.

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.

Characterization of a multidrug-resistant, novel Bacteroides genomospecies.

Metronidazole- and carbapenem-resistant Bacteroides fragilis are rare in the United States. We isolated a multidrug-resistant anaerobe from the bloodstream and intraabdominal abscesses of a patient who had traveled to India. Whole-genome sequencing identified the organism as a novel Bacteroides genomospecies. Physicians should be aware of the possibility for concomitant carbapenem- and metronidazole-resistant Bacteroides infections.

Performance comparison of Illumina and ion torrent next-generation sequencing platforms for 16S rRNA-based bacterial community profiling.

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.

Whole genome sequencing indicates Corynebacterium jeikeium comprises 4 separate genomospecies and identifies a dominant genomospecies among clinical isolates.

Corynebacterium jeikeium is an opportunistic pathogen which has been noted for significant genomic diversity. The population structure within this species remains poorly understood. Here, we explore the relationships among 15 clinical isolates of C. jeikeium (reference strains K411 and ATCC 43734, and 13 primary isolates collected over a period of 7 years) through genetic, genomic, and phenotypic studies. We report a high degree of divergence among strains based on 16S ribosomal RNA (rRNA) gene and rpoB gene sequence analysis, supporting the presence of genetically distinct subgroups. Whole genome sequencing indicates genomic-level dissimilarity among subgroups, which qualify as four separate and distinct Corynebacterium species based on an average nucleotide identity (ANIb) threshold of <95%. Functional distinctions in antibiotic susceptibilities and metabolic profiles characterize two of these genomospecies, allowing their differentiation from others through routine laboratory testing. The remaining genomospecies can be classified through a biphasic approach integrating phenotypic testing and rpoB gene sequencing. The genomospecies predominantly recovered from patient specimens does not include either of the existing C. jeikeium reference strains, implying that studies of this pathogen would benefit from examination of representatives from the primary disease-causing group. The clinically dominant genomospecies also has the smallest genome size and gene repertoire, suggesting the possibility of increased virulence relative to the other genomospecies. The ability to classify isolates to one of the four C. jeikeium genomospecies in a clinical context provides diagnostic information for tailoring antimicrobial therapy and may aid in identification of species-specific disease associations.

Whole-genome sequencing for high-resolution investigation of methicillin-resistant Staphylococcus aureus epidemiology and genome plasticity.

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 variable-number 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 ∼ 2.5% 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.

Coinfection of Fusobacterium nucleatum and Actinomyces israelii in mastoiditis diagnosed by next-generation DNA sequencing.

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.

Molecular diagnosis of Actinomadura madurae infection by 16S rRNA deep sequencing.

Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms.

Regulation of phenotypic heterogeneity permits Salmonella evasion of the host caspase-1 inflammatory response.

Sensing and adapting to the environment is one strategy by which bacteria attempt to maximize fitness in an unpredictable world; another is the stochastic generation of phenotypically distinct subgroups within a genetically clonal population. In culture, Salmonella Typhimurium populations are bistable for the expression of flagellin. We report that YdiV controls this expression pattern by preventing transcription of the sigma factor that recruits RNA polymerase to the flagellin promoter. Bistability ensues when the sigma factor is repressed in a subpopulation of cells, resulting in two phenotypes: flagellin expressors and flagellin nonexpressors. Although the ability to swim is presumably a critical survival trait, flagellin activates eukaryotic defense pathways, and Salmonella restrict the production of flagellin during systemic infection. Salmonella mutants lacking YdiV are unable to fully repress flagellin at systemic sites, rendering them vulnerable to caspase-1 mediated colonization restriction. Thus, a regulatory mechanism producing bistability also impacts Salmonella virulence.

Humanized nonobese diabetic-scid IL2rgammanull mice are susceptible to lethal Salmonella Typhi infection.

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rgamma(null) mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.

Adaptive Immune Responses during Salmonella Infection.

The interaction betweenSalmonella and its host is complex and dynamic: the host mounts an immune defense against the pathogen, which in turn acts to reduce, evade, or exploit these responses to successfully colonize the host. Although the exact mechanisms mediating protective immunity are poorly understood, it is known that T cells are a critical component of immunity to Salmonella infection, and a robust T-cell response is required for both clearance of primary infection and resistance to subsequent challenge. B-cell functions, including but not limited to antibody production, are also required for generation of protective immunity. Additionally, interactions among host cells are essential. For example, antigen-presenting cells (including B cells) express cytokines that participate in CD4+ T cell activation and differentiation. Differentiated CD4+ T cells secrete cytokines that have both autocrine and paracrine functions, including recruitment and activation of phagocytes, and stimulation of B cell isotype class switching and affinity maturation. Multiple bacterium-directed mechanisms, including altered antigen expression and bioavailability and interference with antigen-presenting cell activation and function, combine to modify Salmonella's "pathogenic signature" in order to minimize its susceptibility to host immune surveillance. Therefore, a more complete understanding of adaptive immune responses may provide insights into pathogenic bacterial functions. Continued identification of adaptive immune targets will guide rational vaccine development, provide insights into host functions required to resist Salmonella infection, and correspondingly provide valuable reagents for defining the critical pathogenic capabilities of Salmonella that contribute to their success in causing acute and chronic infections.

Salmonella typhimurium coordinately regulates FliC location and reduces dendritic cell activation and antigen presentation to CD4+ T cells.

During infection, Salmonella transitions from an extracellular-phase (STEX, growth outside host cells) to an intracellular-phase (STIN, growth inside host cells): changes in gene expression mediate survival in the phagosome and modifies LPS and outer membrane protein expression, including altered production of FliC, an Ag recognized by immune CD4+ T cells. Previously, we demonstrated that systemic STIN bacteria repress FliC below the activation threshold of FliC-specific T cells. In this study, we tested the hypothesis that changes in FliC compartmentalization and bacterial responses triggered during the transition from STEX to STIN combine to reduce the ability of APCs to present FliC to CD4+ T cells. Approximately 50% of the Salmonella-specific CD4+ T cells from Salmonella-immune mice were FliC specific and produced IFN-gamma, demonstrating the potent immunogenicity of FliC. FliC expressed by STEX bacteria was efficiently presented by splenic APCs to FliC-specific CD4+ T cells in vitro. However, STIN bacteria, except when lysed, expressed FliC within a protected intracellular compartment and evaded stimulation of FliC-specific T cells. The combination of STIN-mediated responses that reduced FliC bioavailability were overcome by dendritic cells (DCs), which presented intracellular FliC within heat-killed bacteria; however, this ability was abrogated by live bacterial infection. Furthermore, STIN bacteria, unlike STEX, limited DC activation as measured by increased MHC class II, CD86, TNF-alpha, and IL-12 expression. These data indicate that STIN bacteria restrict FliC bioavailability by Ag compartmentalization, and together with STIN bacterial responses, limit DC maturation and cytokine production. Together, these mechanisms may restrain DC-mediated activation of FliC-specific CD4+ T cells.

In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted.

FliC is a natural antigen recognized by the innate and adaptive immune systems during Salmonella infection in mice and humans; however, the regulatory mechanisms governing its expression in vivo are incompletely understood. Here, we use flow cytometry to quantify fliC gene expression in single bacteria. In vitro, fliC transcription was not uniformly positive; a viable fliC-negative subpopulation was also identified. Intracellular Salmonella repressed transcription of fliC and its positive regulator fliA, but constitutively transcribed the master regulator flhD; fliC repression required ClpXP protease, known to degrade FlhD. In orally infected mice, fliC transcription was anatomically restricted: Salmonella transcribed fliC in the Peyer's Patches (PP) but not in the mesenteric lymph nodes and spleen. The intracellularly transcribed pagC promoter was upregulated by Salmonella in all tissues, defining the infected PP as a unique environment that initiates expression of intracellularly induced genes and yet permits transcription of fliC. Because a single bacterium can escape the GI tract to colonize deeper tissues, heterogeneous gene expression may have important implications for Salmonella pathogenesis: FliC-positive bacteria in the PP could stimulate inflammation and facilitate the priming of FliC-specific immune responses, while FliC-negative bacteria escape host detection in the gut and spread to systemic sites of replication.

CD4+-T-cell responses generated during murine Salmonella enterica serovar Typhimurium infection are directed towards multiple epitopes within the natural antigen FliC.

The flagellar filament protein FliC is a natural antigen recognized by memory CD4+ T cells recovered from Salmonella enterica serovar Typhimurium-infected humans and mice. To further investigate T-cell responses to FliC, we derived FliC-specific CD4+-T-cell clones from mice of two different haplotypes following oral S. enterica serovar Typhimurium infection. Using C-terminal truncations of MalE-FliC recombinant fusion proteins, we mapped antigenic activity to four different regions of FliC; three of the four epitope-containing regions were present in both FliC and the alternate flagellin subunit FljB. We determined that two novel FliC epitopes were also present in flagellins from several gram-negative enteric bacterial species: E(k)-restricted FliC 80-94 (amino acids 80 to 94) and A(b)-restricted FliC 455-469. Further mapping confirmed the presence of two previously identified FliC epitopes: A(k)-restricted FliC 339-350 and A(b)-restricted FliC 428-442. Therefore, like the recognition site of the innate immune receptor Toll-like receptor 5, three of four FliC epitopes recognized by CD4+ T cells colocalize in the D0/D1 domains of FliC. Salmonella-infected macrophages and dendritic cells stimulated epitope-specific CD4+-T-cell proliferation; infected dendritic cells also activated T cells to produce gamma interferon. These data demonstrate that Salmonella infection generates murine CD4+-T-cell responses to multiple epitopes in the natural antigen FliC and that recognition of infected phagocytes by FliC-specific CD4+ T cells triggers effector functions known to be essential for protective immunity. Together, these data suggest that FliC-specific CD4+ T cells may contribute to cell-mediated host defenses against Salmonella.

FliC-specific CD4+ T cell responses are restricted by bacterial regulation of antigen expression.

Salmonella typhimurium, a facultatively intracellular pathogen, regulates expression of virulence factors in response to distinct environments encountered during the course of infection. We tested the hypothesis that the transition from extra- to intracellular environments during Salmonella infection triggers changes in Ag expression that impose both temporal and spatial limitations on the host T cell response. CD4(+) T cells recovered from Salmonella immune mice were propagated in vitro using Ag derived from bacteria grown in conditions designed to emulate extra- or intracellular environments in vivo. Extracellular phase bacteria supported a dominant T cell response to the flagellar subunit protein FliC, whereas intracellular phase bacteria were unable to support expansion of FliC-specific T cells from populations known to contain T cells with reactivity to this Ag. This result was attributed to bacterial regulation of FliC expression: transcription and protein levels were repressed in bacteria growing in the spleens of infected mice. Furthermore, Salmonella-infected splenocytes taken directly ex vivo stimulated FliC-specific T cell clones only when intracellular FliC expression was artificially up-regulated. Although it has been suggested that a microanatomical separation of immune T cells and infected APC exists in vivo, we demonstrate that intracellular Salmonella can repress FliC expression below the T cell activation threshold. This potentially provides a mechanism for intracellular Salmonella at systemic sites to avoid detection by Ag-specific T cells primed at intestinal sites early in infection.

CD4+ T cells and toll-like receptors recognize Salmonella antigens expressed in bacterial surface organelles.

A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition.

Laboratory implementation of human papillomavirus testing.

Human papillomavirus testing is becoming an integral component of cervical cancer screening. Market forces will require most laboratories that perform Papanicolaou tests to develop a system for handling human papillomavirus testing also. Data and information are presented that may facilitate laboratories when addressing the following issues in the process of developing a human papillomavirus testing service: Which methodology is the best fit for the laboratory? Is it better to develop an in-house testing service or to send it out? How do I get started? What are the financial and economic issues, and how should they be managed?