Contribution of the patient microbiome to surgical site infection and antibiotic prophylaxis failure in spine surgery.

Despite modern antiseptic techniques, surgical site infection (SSI) remains a leading complication of surgery. However, the origins of SSI and the high rates of antimicrobial resistance observed in these infections are poorly understood. Using instrumented spine surgery as a model of clean (class I) skin incision, we prospectively sampled preoperative microbiomes and postoperative SSI isolates in a cohort of 204 patients. Combining multiple forms of genomic analysis, we correlated the identity, anatomic distribution, and antimicrobial resistance profiles of SSI pathogens with those of preoperative strains obtained from the patient skin microbiome. We found that 86% of SSIs, comprising a broad range of bacterial species, originated endogenously from preoperative strains, with no evidence of common source infection among a superset of 1610 patients. Most SSI isolates (59%) were resistant to the prophylactic antibiotic administered during surgery, and their resistance phenotypes correlated with the patient's preoperative resistome (P = 0.0002). These findings indicate the need for SSI prevention strategies tailored to the preoperative microbiome and resistome present in individual patients.

Nuclear factor kappa B-dependent persistence of Salmonella Typhi and Paratyphi in human macrophages.

Salmonella serovars Typhi and Paratyphi cause a prolonged illness known as enteric fever, whereas other serovars cause acute gastroenteritis. Mechanisms responsible for the divergent clinical manifestations of nontyphoidal and enteric fever Salmonella infections have remained elusive. Here, we show that S. Typhi and S. Paratyphi A can persist within human macrophages, whereas S. Typhimurium rapidly induces apoptotic macrophage cell death that is dependent on Salmonella pathogenicity island 2 (SPI2). S. Typhi and S. Paratyphi A lack 12 specific SPI2 effectors with pro-apoptotic functions, including nine that target nuclear factor κB (NF-κB). Pharmacologic inhibition of NF-κB or heterologous expression of the SPI2 effectors GogA or GtgA restores apoptosis of S. Typhi-infected macrophages. In addition, the absence of the SPI2 effector SarA results in deficient signal transducer and activator of transcription 1 (STAT1) activation and interleukin 12 production, leading to impaired TH1 responses in macrophages and humanized mice. The absence of specific nontyphoidal SPI2 effectors may allow S. Typhi and S. Paratyphi A to cause chronic infections. IMPORTANCE: Salmonella enterica is a common cause of gastrointestinal infections worldwide. The serovars Salmonella Typhi and Salmonella Paratyphi A cause a distinctive systemic illness called enteric fever, whose pathogenesis is incompletely understood. Here, we show that enteric fever Salmonella serovars lack 12 specific virulence factors possessed by nontyphoidal Salmonella serovars, which allow the enteric fever serovars to persist within human macrophages. We propose that this fundamental difference in the interaction of Salmonella with human macrophages is responsible for the chronicity of typhoid and paratyphoid fever, suggesting that targeting the nuclear factor κB (NF-κB) complex responsible for macrophage survival could facilitate the clearance of persistent bacterial infections.

The Epistemic Value of Gain of Function Experiments.

The phrase "gain of function" (GOF) has recently acquired a negative connotation in experimental biology by its association with risky science. Whereas much of the discussion on the relative merits of GOF-type experiments has focused on their risk-benefit equation, relatively little has been said about their epistemic value. In this article, we recount how GOF experiments were critical for establishing DNA as the genetic material, the identification of cellular receptors, and the role of oncogenes in cancer research. Today, many of the products of the biomedical revolution such as synthetic insulin, growth factors, and monoclonal antibodies are the result of GOF experiments where cells were given the new function of synthesizing medically important products. GOF experiments and complementary loss of function experiments are epistemically powerful tools for establishing causality in biology.

Availability of Ferritin-Bound Iron to Enterobacteriaceae.

The sequestration of iron in case of infection, termed nutritional immunity, is an established strategy of host defense. However, the interaction between pathogens and the mammalian iron storage protein ferritin is hitherto not completely understood. To better characterize the function of ferritin in Gram-negative infections, we incubated iron-starved cultures of Salmonella Typhimurium and knockout mutant strains defective for major iron uptake pathways or Escherichia coli with horse spleen ferritin or ionic iron as the sole iron source. Additionally, we added bovine superoxide dismutase and protease inhibitors to the growth medium to assess the effect of superoxide and bacterial proteases, respectively, on Salmonella proliferation and reductive iron release. Compared to free ionic iron, ferritin-bound iron was less available to Salmonella, but was still sufficient to significantly enhance the growth of the bacteria. In the absence of various iron acquisition genes, the availability of ferritin iron further decreased. Supplementation with superoxide dismutase significantly reduced the growth of the ΔentC knockout strain with holoferritin as the sole iron source in comparison with ionic ferrous iron. In contrast, this difference was not observed in the wildtype strain, suggesting that superoxide dismutase undermines bacterial iron uptake from ferritin by siderophore-independent mechanisms. Ferritin seems to diminish iron availability for bacteria in comparison to ionic iron, and its iron sequestering effect could possibly be enhanced by host superoxide dismutase activity.

Analysis of Salmonella Typhi Pathogenesis in a Humanized Mouse Model.

Efforts to understand molecular mechanisms of pathogenesis of the human-restricted pathogen Salmonella enterica serovar Typhi, the causative agent of typhoid fever, have been hampered by the lack of a tractable small animal model. This obstacle has been surmounted by a humanized mouse model in which genetically modified mice are engrafted with purified CD34+ stem cells from human umbilical cord blood, designated CD34+ Hu-NSG (formerly hu-SRC-SCID) mice. We have shown that these mice develop a lethal systemic infection with S. Typhi that is dependent on the presence of engrafted human hematopoietic cells. Immunological and pathological features of human typhoid are recapitulated in this model, which has been successfully employed for the identification of bacterial genetic determinants of S. Typhi virulence. Here we describe the methods used to infect CD34+ Hu-NSG mice with S. Typhi in humanized mice and to construct and analyze a transposon-directed insertion site sequencing S. Typhi library, and provide general considerations for the use of humanized mice for the study of a human-restricted pathogen.

Cyclopropane Fatty Acids Are Important for Salmonella enterica Serovar Typhimurium Virulence.

A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in the cfa mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

Direct and Indirect Inhibition of Salmonella Peptide Deformylase by Nitric Oxide.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that elicits nitric oxide (NO·) production by host macrophages. NO· is a potent antimicrobial mediator with diverse targets, including protein thiols and metal centers. The mobilization of zinc from metalloproteins by NO· increases the availability of free intracellular zinc, which is detrimental to bacterial cells, but the precise mechanism of zinc cytotoxicity is uncertain. Here, we show that excess zinc results in the mismetallation of the essential iron-containing enzyme peptide deformylase (PDF), thereby diminishing its activity. PDF mismetallation is observed in zinc-treated bacteria lacking the zinc exporters ZntA and ZitB and is also observed during nitrosative stress, suggesting that NO·-mediated zinc mobilization results in PDF mismetallation. However, NO· also inhibits PDF directly by S-nitrosylating the metal-binding Cys90 residue. These observations identify PDF as an essential bacterial protein that is subject to both direct and indirect inactivation by NO·, providing a novel mechanism of zinc toxicity and NO·-mediated antibacterial activity.IMPORTANCE We have previously shown that the host-derived antimicrobial mediator nitric oxide (NO·) mobilizes zinc from bacterial metalloproteins. The present study demonstrates that NO· inactivates the essential iron-containing enzyme peptide deformylase, both by promoting its mismetallation by zinc and by directly modifying its metal-binding site. We explain how free intracellular zinc is detrimental for cells and reveal a new mechanism of NO·-mediated bacterial growth inhibition that is distinct from previously known targets.

Long-Term Carriage of Medicopsis romeroi, an Agent of Black-Grain Mycetoma, Presenting as Phaeohyphomycosis in a Renal Transplant Patient.

Medicopsis species are rare fungal pathogens that frequently resist common antifungal therapies and are difficult to identify morphologically as conidia are produced in pycnidia, a key feature of coelomycetes. Immunocompromised patients are at risk of these infections, even after remote exposure, and typically present with phaeohyphomycoses without dissemination. We present the case of a renal transplant recipient 6.5 years post-transplant who developed a slowly progressive soft tissue infection mimicking a synovial cyst. A cultured isolate was identified as Medicopsis romeroi by sequencing of multiple ribosomal loci. The patient responded well to debridement and posaconazole therapy. Solid-organ transplant patients are at risk of opportunistic fungal infection long after transplant, and molecular methods are often required for definitive identification.

Genome-wide Analysis of Salmonella enterica serovar Typhi in Humanized Mice Reveals Key Virulence Features.

Salmonella enterica serovar Typhi causes typhoid fever only in humans. Murine infection with S. Typhimurium is used as a typhoid model, but its relevance to human typhoid is limited. Non-obese diabetic-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID) are susceptible to lethal S. Typhi infection. In this study, we use a high-density S. Typhi transposon library in hu-SRC-SCID mice to identify virulence loci using transposon-directed insertion site sequencing (TraDIS). Vi capsule, lipopolysaccharide (LPS), and aromatic amino acid biosynthesis were essential for virulence, along with the siderophore salmochelin. However, in contrast to the murine S. Typhimurium model, neither the PhoPQ two-component system nor the SPI-2 pathogenicity island was required for lethal S. Typhi infection, nor was the CdtB typhoid toxin. These observations highlight major differences in the pathogenesis of typhoid and non-typhoidal Salmonella infections and demonstrate the utility of humanized mice for understanding the pathogenesis of a human-specific pathogen.

Reactive nitrogen species in host-bacterial interactions.

Reactive nitrogen species play diverse and essential roles in host-pathogen interactions. Here, we review selected recent discoveries regarding nitric oxide (NO) in host defense and the pathogenesis of infection, mechanisms of bacterial NO resistance, production of NO by human macrophages, NO-based antimicrobial therapeutics and NO interactions with the gut microbiota.

Dopamine Is a Siderophore-Like Iron Chelator That Promotes Salmonella enterica Serovar Typhimurium Virulence in Mice.

We have recently shown that the catecholamine dopamine regulates cellular iron homeostasis in macrophages. As iron is an essential nutrient for microbes, and intracellular iron availability affects the growth of intracellular bacteria, we studied whether dopamine administration impacts the course of Salmonella infections. Dopamine was found to promote the growth of Salmonella both in culture and within bone marrow-derived macrophages, which was dependent on increased bacterial iron acquisition. Dopamine administration to mice infected with Salmonella enterica serovar Typhimurium resulted in significantly increased bacterial burdens in liver and spleen, as well as reduced survival. The promotion of bacterial growth by dopamine was independent of the siderophore-binding host peptide lipocalin-2. Rather, dopamine enhancement of iron uptake requires both the histidine sensor kinase QseC and bacterial iron transporters, in particular SitABCD, and may also involve the increased expression of bacterial iron uptake genes. Deletion or pharmacological blockade of QseC reduced but did not abolish the growth-promoting effects of dopamine. Dopamine also modulated systemic iron homeostasis by increasing hepcidin expression and depleting macrophages of the iron exporter ferroportin, which enhanced intracellular bacterial growth. Salmonella lacking all central iron uptake pathways failed to benefit from dopamine treatment. These observations are potentially relevant to critically ill patients, in whom the pharmacological administration of catecholamines to improve circulatory performance may exacerbate the course of infection with siderophilic bacteria.IMPORTANCE Here we show that dopamine increases bacterial iron incorporation and promotes Salmonella Typhimurium growth both in vitro and in vivo These observations suggest the potential hazards of pharmacological catecholamine administration in patients with bacterial sepsis but also suggest that the inhibition of bacterial iron acquisition might provide a useful approach to antimicrobial therapy.

Nitric Oxide Disrupts Zinc Homeostasis in Salmonella enterica Serovar Typhimurium.

Nitric oxide (NO·) produced by mammalian cells exerts antimicrobial actions that result primarily from the modification of protein thiols (S-nitrosylation) and metal centers. A comprehensive approach was used to identify novel targets of NO· in Salmonella enterica serovar Typhimurium (S. Typhimurium). Newly identified targets include zinc metalloproteins required for DNA replication and repair (DnaG, PriA, and TopA), protein synthesis (AlaS and RpmE), and various metabolic activities (ClpX, GloB, MetE, PepA, and QueC). The cytotoxic actions of free zinc are mitigated by the ZntA and ZitB zinc efflux transporters, which are required for S. Typhimurium resistance to zinc overload and nitrosative stress in vitro Zinc efflux also ameliorates NO·-dependent zinc mobilization following internalization by activated macrophages and is required for virulence in NO·-producing mice, demonstrating that host-derived NO· causes zinc stress in intracellular bacteria.IMPORTANCE Nitric oxide (NO·) is produced by macrophages in response to inflammatory stimuli and restricts the growth of intracellular bacteria. Mechanisms of NO·-dependent antimicrobial actions are incompletely understood. Here, we show that zinc metalloproteins are important targets of NO· in Salmonella, including the DNA replication proteins DnaG and PriA, which were hypothesized to be NO· targets in earlier studies. Like iron, zinc is a cofactor for several essential proteins but is toxic at elevated concentrations. This study demonstrates that NO· mobilizes free zinc in Salmonella and that specific efflux transporters ameliorate the cytotoxic effects of free zinc during infection.

Host Nitric Oxide Disrupts Microbial Cell-to-Cell Communication to Inhibit Staphylococcal Virulence.

Staphylococcus aureus is a commensal bacterium that can asymptomatically colonize its host but also causes invasive infections. Quorum sensing regulates S. aureus virulence and the transition from a commensal to a pathogenic organism. However, little is known about how host innate immunity affects interbacterial communication. We show that nitric oxide suppresses staphylococcal virulence by targeting the Agr quorum sensing system. Nitric oxide-mediated inhibition occurs through direct modification of cysteine residues C55, C123, and C199 of the AgrA transcription factor. Cysteine modification decreases AgrA promoter occupancy as well as transcription of the agr operon and quorum sensing-activated toxin genes. In a staphylococcal pneumonia model, mice lacking inducible nitric oxide synthase develop more severe disease with heightened mortality and proinflammatory cytokine responses. In addition, staphylococcal α-toxin production increases in the absence of nitric oxide or nitric oxide-sensitive AgrA cysteine residues. Our findings demonstrate an anti-virulence mechanism for nitric oxide in innate immunity.

Genetic and Dietary Iron Overload Differentially Affect the Course of Salmonella Typhimurium Infection.

Genetic and dietary forms of iron overload have distinctive clinical and pathophysiological features. HFE-associated hereditary hemochromatosis is characterized by overwhelming intestinal iron absorption, parenchymal iron deposition, and macrophage iron depletion. In contrast, excessive dietary iron intake results in iron deposition in macrophages. However, the functional consequences of genetic and dietary iron overload for the control of microbes are incompletely understood. Using Hfe+/+ and Hfe-/- mice in combination with oral iron overload in a model of Salmonella enterica serovar Typhimurium infection, we found animals of either genotype to induce hepcidin antimicrobial peptide expression and hypoferremia following systemic infection in an Hfe-independent manner. As predicted, Hfe-/- mice, a model of hereditary hemochromatosis, displayed reduced spleen iron content, which translated into improved control of Salmonella replication. Salmonella adapted to the iron-poor microenvironment in the spleens of Hfe-/- mice by inducing the expression of its siderophore iron-uptake machinery. Dietary iron loading resulted in higher bacterial numbers in both WT and Hfe-/- mice, although Hfe deficiency still resulted in better pathogen control and improved survival. This suggests that Hfe deficiency may exert protective effects in addition to the control of iron availability for intracellular bacteria. Our data show that a dynamic adaptation of iron metabolism in both immune cells and microbes shapes the host-pathogen interaction in the setting of systemic Salmonella infection. Moreover, Hfe-associated iron overload and dietary iron excess result in different outcomes in infection, indicating that tissue and cellular iron distribution determines the susceptibility to infection with specific pathogens.

Moonshot Science-Risks and Benefits.

Ever since the successful Apollo 11 Moon landing in 1969, a "moonshot" has come to signify a bold effort to achieve a seemingly impossible task. The Obama administration recently called for a moonshot to cure cancer, an initiative that has elicited mixed responses from researchers who welcome additional funding but worry about raising expectations. We suggest that a successful moonshot requires a sufficient understanding of the basic science underlying a problem in question so that efforts can be focused on engineering a solution. Current gaps in our basic knowledge of cancer biology make the cancer moonshot a uniquely challenging endeavor. Nevertheless, history has shown that intensive research efforts have frequently yielded conceptual and technological breakthroughs with unanticipated benefits for society. We expect that this effort will be no different.

Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content.

UNLABELLED: The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments. IMPORTANCE: In order to cause disease, pathogenic bacteria must rapidly sense and respond to antimicrobial pressures encountered within the host. Prominent among these stresses, and of particular relevance to intracellular pathogens such as Salmonella, are nutritional restriction and the enzymatic generation of reactive oxygen and nitrogen species. The conserved transcriptional regulator DksA controls adaptive responses to nutritional limitation, as well as to oxidative and nitrosative stress. Here, we demonstrate that each of these functions contributes to bacterial pathogenesis. Our observations highlight the importance of metabolic adaptation in bacterial pathogenesis and show the mechanism by which DksA orthologues are optimized to sense the levels of oxidative and nitrosative stress encountered in their natural habitats. An improved understanding of the conserved processes used by bacteria to sense, respond to, and limit host defense will inform the development of novel strategies to treat infections caused by pathogenic, potentially multidrug-resistant bacteria.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

BACKGROUND: Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. MATERIALS AND METHODS: Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. RESULTS: The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. CONCLUSIONS: The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen.

Lipocalin-2 ensures host defense against Salmonella Typhimurium by controlling macrophage iron homeostasis and immune response.

Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages.

Iron Regulatory Proteins Mediate Host Resistance to Salmonella Infection.

Macrophages are essential for systemic iron recycling, and also control iron availability to pathogens. Iron metabolism in mammalian cells is orchestrated posttranscriptionally by iron-regulatory proteins (IRP)-1 and -2. Here, we generated mice with selective and combined ablation of both IRPs in macrophages to investigate the role of IRPs in controlling iron availability. These animals are hyperferritinemic but otherwise display normal clinical iron parameters. However, mutant mice rapidly succumb to systemic infection with Salmonella Typhimurium, a pathogenic bacterium that multiplies within macrophages, with increased bacterial burdens in liver and spleen. Ex vivo infection experiments indicate that IRP function restricts bacterial access to iron via the EntC and Feo bacterial iron-acquisition systems. Further, IRPs contain Salmonella by promoting the induction of lipocalin 2, a host antimicrobial factor that inhibits bacterial uptake of iron-laden siderophores, and by suppressing the ferritin iron pool. This work reveals the importance of the IRPs in innate immunity.

Human Diphyllobothrium nihonkaiense infection in Washington State.

A patient in Washington State harbored a fish tapeworm most likely acquired from eating raw salmon. Diphyllobothrium nihonkaiense was identified by cox1 sequence analysis. Although this is the first documented human D. nihonkaiense infection in the United States, the parasite may have been present earlier but misidentified as Diphyllobothrium latum.