Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Whole genome sequencing of Borrelia burgdorferi isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto ( Bbss ) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ∻800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext).

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

Macrolide and Clindamycin Resistance in Group a Streptococci Isolated From Children With Pharyngitis.

Group A streptococcus (GAS) is responsible for 15%-30% of cases of acute pharyngitis in children. Macrolides such as azithromycin have become popular for treating GAS pharyngitis. We report macrolide resistance rates in a primary care setting in our geographic area over the past 5 years and discuss the implications of resistance in making treatment decisions. Throat swabs were collected from children with pharyngitis from May 2011 to May 2015 in a primary care setting in Madison, Wisconsin. Susceptibility testing was performed for erythromycin and clindamycin using the Kirby-Bauer disk diffusion method. GAS was identified on 143 throat cultures. Overall, 15% of GAS isolates demonstrated nonsusceptibility for both clindamycin and erythromycin. Inducible resistance (positive D-test) was detected in 17 isolates (12%). The rate of detection of nonsusceptibility in each year of the study did not change over time. Azithromycin should only be used for patients with pharyngitis and substantial manifestations of penicillin hypersensitivity and when used, susceptibility testing should always be performed.

Principles and Pitfalls: a Guide to Death Certification.

Death certificates serve the critical functions of providing documentation for legal/administrative purposes and vital statistics for epidemiologic/health policy purposes. In order to satisfy these functions, it is important that death certificates be filled out completely, accurately, and promptly. The high error rate in death certification has been documented in multiple prior studies, as has the effectiveness of educational training interventions at mitigating errors. The following guide to death certification is intended to illustrate some basic principles and common pitfalls in electronic death registration with the goal of improving death certification accuracy.

Canine blastomycosis in Wisconsin: a survey of small-animal veterinary practices.

The disease burden and impact of canine blastomycosis in Wisconsin is uncertain. We surveyed small-animal veterinary practices to obtain estimates of disease incidence, determine patient outcomes, and investigate variation in diagnostic and treatment strategies used by veterinarians. Veterinarians representing small-animal practices in Wisconsin were contacted by mail with the option to complete a paper or online questionnaire. Questionnaires were returned from 68 of 443 veterinary practices (15%) that estimated diagnosing 239 cases of canine blastomycosis annually, with an overall mortality of 36%. Annual incidence rates of canine blastomycosis were calculated for 43 individual veterinary clinics and differed significantly between clinics in endemic and nonendemic counties (P = 0.01), with the mean in endemic counties being 204/100,000/yr and nonendemic counties being 72/100,000/yr. Veterinarians reported an increase in canine blastomycosis cases from April through August. A wide variety of methods were used for diagnosis, ranging from clinical signs alone to antigen testing and "in-house" cytology. Of note, fungal culture was used rarely for diagnosis. In addition, veterinarians at these 68 clinics estimated diagnosing 36 cases of feline blastomycosis annually. The incidence of canine blastomycosis is high but quite variable among veterinary practices in Wisconsin. Diagnosis is based frequently on clinical signs exclusively due, in part, to the perceived high cost of laboratory tests. Similarly, the mortality associated with blastomycosis is likely negatively impacted because some dog owners defer therapy due to the cost of antifungal drugs.

Actionable diagnosis of neuroleptospirosis by next-generation sequencing.

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Multilocus sequence typing of Borrelia burgdorferi suggests existence of lineages with differential pathogenic properties in humans.

The clinical manifestations of Lyme disease, caused by Borrelia burgdorferi, vary considerably in different patients, possibly due to infection by strains with varying pathogenicity. Both rRNA intergenic spacer and ospC typing methods have proven to be useful tools for categorizing B. burgdorferi strains that vary in their tendency to disseminate in humans. Neither method, however, is suitable for inferring intraspecific relationships among strains that are important for understanding the evolution of pathogenicity and the geographic spread of disease. In this study, multilocus sequence typing (MLST) was employed to investigate the population structure of B. burgdorferi recovered from human Lyme disease patients. A total of 146 clinical isolates from patients in New York and Wisconsin were divided into 53 sequence types (STs). A goeBURST analysis, that also included previously published STs from the northeastern and upper Midwestern US and adjoining areas of Canada, identified 11 major and 3 minor clonal complexes, as well as 14 singletons. The data revealed that patients from New York and Wisconsin were infected with two distinct, but genetically and phylogenetically closely related, populations of B. burgdorferi. Importantly, the data suggest the existence of B. burgdorferi lineages with differential capabilities for dissemination in humans. Interestingly, the data also indicate that MLST is better able to predict the outcome of localized or disseminated infection than is ospC typing.

Variation in clinical phenotype of human infection among genetic groups of Blastomyces dermatitidis.

BACKGROUND: Blastomyces dermatitidis, the etiologic agent of blastomycosis, has 2 genetic groups and shows varied clinical presentation, ranging from silent infections to fulminant respiratory disease and dissemination. The objective of this study was to determine whether clinical phenotype and outcomes vary based on the infecting organism's genetic group. METHODS: We used microsatellites to genotype 227 clinical isolates of B. dermatitidis from Wisconsin patients. For each isolate, corresponding clinical disease characteristics and patient demographic information were abstracted from electronic health records and Wisconsin Division of Health reportable disease forms and questionnaires. RESULTS: In univariate analysis, group 1 isolates were more likely to be associated with pulmonary-only infections (P < .0001) and constitutional symptoms such as fever (P < .0001). In contrast, group 2 isolates were more likely to be associated with disseminated disease (P < .0001), older patient age (P < .0001), and comorbidities (P = .0019). In multivariate analysis, disease onset to diagnosis of >1 month (P < .0001), older age at diagnosis (P < .0001), and current smoking status (P = .0001) remained predictors for group 2 infections. CONCLUSIONS: This study identified previously unknown associations between clinical phenotype of human infection and genetic groups of B. dermatitidis and provides a framework for further investigations of the genetic basis for virulence in B. dermatitidis.

Rapid response team-triggered procalcitonin measurement predicts infectious intensive care unit transfers*.

OBJECTIVE: Determine if procalcitonin at the time of initial rapid response team activation identifies patients who are likely to need subsequent intensive care unit transfer. DESIGN: Prospective observational cohort study. SETTING: Urban, tertiary care hospital with rapid response team activation through an electronic modified early warning score. PATIENTS: One hundred nineteen oncology and 100 consecutive non-oncology patients after initial rapid response team visit precipitated by an elevated electronic modified early warning score were recruited. Rapid response team activations by request of nursing or for other reasons were not studied. Five oncology patients seen by a rapid response team for complications of interleukin-2 therapeutic infusions were subsequently excluded. INTERVENTIONS: Residual serum from the next ordered clinical test (within 12 hrs) was retrieved, frozen, and stored for procalcitonin determination. A second sample 12-24 hrs after the initial specimen was also retrieved if available and if the patient had not yet been transferred to the intensive care unit. MEASUREMENTS AND MAIN RESULTS: Seventy-three patients (33%) were transferred to the intensive care unit. Rapid response team activations that did not result in intensive care unit transfer had significantly lower procalcitonin levels (median 0.28 ng/mL [interquartile range 0.09-1.24]) than those that resulted in intensive care unit transfer (median 0.51 ng/mL [interquartile range 0.11-1.97], p = .0001) but the area under the receiver operating curve was only 0.656. The change in procalcitonin level in patients with intensive care unit transfers was very heterogeneous but was significantly increased compared to the change in patients not transferred to the intensive care unit. Procalcitonin levels for intensive care unit transfers for probable or definite infection were 2.28 ng/mL [interquartile range 0.68-8.05], and were significantly greater than rapid response team visits that did not result in transfer (p = .0001). The difference between infectious and noninfectious intensive care unit transfers (0.95 ng/mL [interquartile range 0.26-1.89]) was also significant (p = .03). The procalcitonin levels of patients with noninfectious intensive care unit transfers were also different than the levels of patients who never transferred (p = .04). CONCLUSIONS: Preliminary results suggest procalcitonin levels in patients at the time of initial visit by a rapid response team correlate with the need for subsequent intensive care unit transfer, particularly for infectious reasons.

Legionella cardiaca sp. nov., isolated from a case of native valve endocarditis in a human heart.

A Gram-negative, rod-shaped bacterium, designated H63(T), was isolated from aortic valve tissue of a patient with native valve endocarditis. 16S rRNA gene sequencing revealed that H63(T) belongs to the genus Legionella, with its closest neighbours being the type strains of Legionella brunensis (98.8% similarity), L. londiniensis (97.0%), L. jordanis (96.8%), L. erythra (96.2%), L. dresdenensis (96.0%) and L. rubrilucens, L. feeleii, L. pneumophila and L. birminghamensis (95.7%). DNA-DNA hybridization studies yielded values of <70% relatedness between strain H63(T) and its nearest neighbours in terms of 16S rRNA gene sequence similarity, indicating that the strain represents a novel species. Phylogenetic analysis of the 16S rRNA, macrophage infectivity potentiator (mip) and RNase P (rnpB) genes confirmed that H63(T) represents a distinct species, with L. brunensis being its closest sister taxon. Fatty acid composition and biochemical traits, such as the inability to ferment glucose and reduce nitrate, supported the affiliation of H63(T) to the genus Legionella. H63(T) was distinguishable from its neighbours based on it being positive for hippurate hydrolysis. H63(T) was further differentiated by its inability to grow on BCYE agar at 17 °C, its poor growth on low-iron medium and the absence of sliding motility. Also, H63(T) did not react with antisera generated from type strains of Legionella species. H63(T) replicated within macrophages. It also grew in mouse lungs, inducing histopathological evidence of pneumonia and dissemination to the spleen. Together, these results confirm that H63(T) represents a novel, pathogenic Legionella species, for which the name Legionella cardiaca sp. nov. is proposed. The type strain is H63(T) ( = ATCC BAA-2315(T)  = DSM 25049(T)  = JCM 17854(T)).

Placental and fetal findings in intrauterine Candida lusitaniae infection following in vitro fertilization and embryo transfer.

Intrauterine infection with non- albicans Candida species is rare but can be catastrophic to the fetus. A subset of intrauterine infections with non- albicans Candida species has occurred in women who have undergone in vitro fertilization and embryo transfer (IVF-ET). We report a case of a 33-year-old healthy woman, pregnant with triplets by in vitro fertilization, who experienced preterm premature rupture of membranes of fetus A at 16 weeks' gestation and subsequently developed oligohydramnios in all 3 fetuses. Following elective pregnancy termination, microscopic examination and molecular analysis demonstrated Candida lusitaniae chorioamnionitis and pneumonia in all 3 fetuses associated with granulomatous inflammation. Our case is only the 2nd report of C. lusitaniae chorioamnionitis and should raise awareness that C. lusitaniae intrauterine infection is associated with IVF-ET. We also show here that C. lusitaniae can cause granulomatous intraplacental inflammation and intrauterine pneumonia.

Native valve endocarditis due to a novel strain of Legionella.

Legionellae are Gram-negative bacteria which are capable of causing disease, most commonly in the form of pneumonia. We describe a case of native valve endocarditis caused by a Legionella strain which by genotypic (16S rRNA and mip gene sequencing) and phenotypic analyses is unlike previously described strains of Legionella.

Population genetic structure of clinical and environmental isolates of Blastomyces dermatitidis, based on 27 polymorphic microsatellite markers.

Blastomyces dermatitidis, a thermally dimorphic fungus, is the etiologic agent of North American blastomycosis. Clinical presentation is varied, ranging from silent infections to fulminant respiratory disease and dissemination to skin and other sites. Exploration of the population genetic structure of B. dermatitidis would improve our knowledge regarding variation in virulence phenotypes, geographic distribution, and difference in host specificity. The objective of this study was to develop and test a panel of microsatellite markers to delineate the population genetic structure within a group of clinical and environmental isolates of B. dermatitidis. We developed 27 microsatellite markers and genotyped B. dermatitidis isolates from various hosts and environmental sources (n=112). Assembly of a neighbor-joining tree of allele-sharing distance revealed two genetically distinct groups, separated by a deep node. Bayesian admixture analysis showed that two populations were statistically supported. Principal coordinate analysis also reinforced support for two genetic groups, with the primary axis explaining 61.41% of the genetic variability. Group 1 isolates average 1.8 alleles/locus, whereas group 2 isolates are highly polymorphic, averaging 8.2 alleles/locus. In this data set, alleles at three loci are unshared between the two groups and appear diagnostic. The mating type of individual isolates was determined by PCR. Both mating type-specific genes, the HMG and α-box domains, were represented in each of the genetic groups, with slightly more isolates having the HMG allele. One interpretation of this study is that the species currently designated B. dermatitidis includes a cryptic subspecies or perhaps a separate species.

Non-rural point source blastomycosis outbreak near a yard waste collection site.

BACKGROUND: Blastomycosis is a potentially fatal infection caused by the fungus Blastomyces dermatitidis. During January 1 through March 5, 2006, twenty-one laboratory confirmed cases of blastomycosis were reported among residents of an endemic area in north-central Wisconsin; a striking increase compared with previous years. The objective of the study was to determine if an observed increase in blastomycosis among residents of an urban area in north-central Wisconsin was caused by a point-source exposure and to identify its source. METHODS: We compared epidemiologic features, and signs and symptoms of B. dermatitidis infection among 46 historic (1999-2005) and 21 possible outbreak case patients. In addition, a case-control study was conducted to compare risk factors of the outbreak case patients with those of 64 age, gender, and geographically-matched control subjects. We conducted site inspections, evaluated meteorological data, genetically compared outbreak and non-outbreak isolates, and attempted environmental detection of B. dermatitidis using polymerase chain reaction, in vitro isolation, and in vivo isolation by tail vein injection of mice. RESULTS: The unusual risk profile of this outbreak included: residence within non-rural city limits with limited time spent outdoors and an equivalent gender ratio and young median age among case patients consistent with common source rather than unrelated exposures. Thirteen of fourteen outbreak-associated clinical isolates of B. dermatitidis clustered in the same genetic group by PCR-RFLP analysis. Inspections near the cluster center suggested a yard waste collection site as the probable exposure source. B. dermatitidis nucleic acid was detected in one of 19 environmental samples. Environmental and meteorological conditions and material management practices were identified that may have facilitated growth and dispersal of B. dermatitidis conidia near this residential area. CONCLUSIONS: Results of our investigation of this large non-rural outbreak of blastomycosis suggest bioaerosol hazards may exist near yard waste collection and composting facilities, especially where pine tree litter is present, in B. dermatitidis endemic areas.

Virulence genes and genotypic associations in nasal carriage, community-associated methicillin-susceptible and methicillin-resistant USA400 Staphylococcus aureus isolates.

It is not well understood why strains of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), a major cause of skin and soft tissue infections, became successful so quickly, overtaking the place of methicillin-sensitive S. aureus (MSSA) in many communities. To evaluate the genetic basis of differences in their virulence traits, 293 S. aureus isolates consisting of three cohorts, genotypically defined clinical CA-MRSA (n = 77), clinical MSSA (n = 103), and nasal carriage MSSA (n = 113), collected over a 19-year period in two Midwestern states in the United States, were (i) extensively genotyped and (ii) screened for 40 known virulence genes which included those for enterotoxins, leukocidins, hemolysins, and surface proteins and several newly identified putative toxin genes from the USA400 lineage of CA-MRSA. Genotypically, nasal carriage and clinical MSSA isolates were much more diverse than was the CA-MRSA group, which was found to be of USA400 lineage only. Virulence gene profiles of the three groups showed that CA-MRSA strains harbored significantly higher percentages (≥95%; P value, <0.05) of the sea, sec, sec4, seg2, seh, sek, sel, sel2, ear, ssl1, lpl10, lukSF-PV, lukD, lukE, and clfA genes than did the carriage and the clinical MSSA group (range, 0% to 58%). Genes of the enterotoxin gene cluster, seg, sei, sem, sen, and seo, were present in the clinical and carriage isolates but not in the CA-MRSA group. These results suggest that the presence of additional virulence factors in USA400 CA-MRSA strains compared to the nasal carriage and clinical MSSA strains probably contributed to their enhanced virulence.

Evolution of northeastern and midwestern Borrelia burgdorferi, United States.

The per capita incidence of human Lyme disease in the northeastern United States is more than twice that in the Midwest. However, the prevalence of Borrelia burgdorferi, the bacterium that causes Lyme disease, in the tick vector is nearly identical in the 2 regions. The disparity in human Lyme disease incidence may result from a disparity in the human invasiveness of the bacteria in the Northeast and Midwest caused by fundamentally different evolutionary histories. B. burgdorferi populations in the Northeast and Midwest are geographically isolated, enabling evolutionary divergence in human invasiveness. However, we found that B. burgdorferi populations in the Northeast and Midwest shared a recent common ancestor, which suggests that substantial evolutionary divergence in human invasiveness has not occurred. We propose that differences in either animal ecology or human behavior are the root cause of the differences in human incidence between the 2 regions.

Expanded clinical presentation of community-acquired methicillin-resistant Staphylococcus aureus pneumonia.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has been documented to cause community-acquired pneumonias (CAP), notable for necrotizing features. The frequency of occurrence, risk factors, and optimal treatment of CA-MRSA CAP are unclear. METHODS: This was a retrospective analysis of patients admitted to Northwestern Memorial Hospital from January 2005 to April 2007 with initial clinical presentation of pneumonia and respiratory or blood culture positive for CA-MRSA. Definition of CA-MRSA was based on sensitivity to trimethoprim/sulfamethoxazole and clindamycin. RESULTS: Fifteen patients with CA-MRSA CAP were identified during the 28-month period. Only one of the 14 patients tested had evidence of preceding influenza, and no seasonal pattern was seen. Seven patients were never admitted to the ICU. Eight of 14 with chest CT scans had evidence of lung necrosis. Nine of 15 had evidence of pleural effusions early in their hospital course, and five of nine required at least one pleural drainage procedure. Seven of 15 were immunocompromised (three HIV, one acute lymphocytic leukemia [ALL], one high-dose steroids, and two immunoglobulin deficiency) with an additional three patients with diabetes. Mortality was only 13% (two of 15); both deaths occurred in patients with severe immunocompromise (ALL post chemotherapy and AIDS). Fourteen of 15 patients were treated with antimicrobials that inhibit exotoxin production (clindamycin or linezolid). CONCLUSIONS: CA-MRSA pneumonia is not necessarily a post-influenza infection. Despite necrotizing features in many, the mortality of CA-MRSA pneumonia in our series is lower than previously reported, and patients do not routinely require ICU care. Treatment with antibiotics that inhibit exotoxin production and/or nontoxigenic strains may explain this improved outcome.