Laboratory diagnosis of bacterial meningitis by direct detection, serotyping and Next Generation Sequencing: How 10 years of testing in New York State has evolved to improve laboratory diagnosis and public health.

Since 2005, the Wadsworth Center (WC) has provided molecular testing on cerebrospinal fluid (CSF) and whole blood specimens in close collaboration with epidemiologists in New York State and New York City. In this study, we analyzed 10 years of data to demonstrate the significant value of utilizing molecular methods to assess patient specimens for etiologic agents of bacterial meningitis. A comprehensive molecular testing algorithm to detect and serotype/serogroup bacterial agents known to cause bacterial meningitis (Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae and Streptococcus agalactiae) has evolved, and retrospective specimen testing has been essential for each improvement. Over a ten-year span from 2010 to 2019 the WC received 831 specimens from 634 patients with suspected bacterial meningitis. Real-time PCR was positive for at least one of the agents in 223 (27%) specimens from 183 patients (29%). Of the 223 positives, 146 (66%) were further characterized by real-time PCR into serogroup/serotype. Additionally, examination of 131 paired specimens of CSF and whole blood from the same patients found better detection in CSF, but whole blood is a useful alternative for diagnosis when CSF is not available. For specimens initially PCR-negative, 16S rDNA Sanger sequencing was requested by the submitter for 146 cases resulting in the identification of bacterial agents in an additional 24 (16%) specimens. In a retrospective study, Next Generation Sequencing (NGS) was evaluated for the detection of pathogens in 53 previously tested PCR-negative CSF specimens and identified bacteria in 14 (26%) specimens. This molecular testing algorithm has provided clinicians a diagnosis when culture is negative with the potential to guide therapy. It has also aided public health in determining when antibiotic prophylaxis was needed, augmented surveillance data to yield a fuller picture of community prevalence, and highlighted gaps in the spectrum of agents that cause bacterial meningitis.

Improvements to the Success of Outbreak Investigations of Legionnaires' Disease: 40 Years of Testing and Investigation in New York State.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.

First 12 Months of Life for Infants in New York City, New York, With Possible Congenital Zika Virus Exposure.

BACKGROUND: Our goal was to characterize the epidemiology and clinical significance of congenital Zika virus (ZIKV) exposure by prospectively following a cohort of infants with possible congenital exposure through their first year of life. METHODS: We included infants born in New York City between 2016 and 2017 who had or were born to a woman who had laboratory evidence of ZIKV infection during pregnancy. We conducted provider/patient interviews and reviewed medical records to collect information about the pregnant women and, for infants, clinical and neurodevelopmental status at birth and 2, 6, and 12 months of age. RESULTS: Of the 404 infants who met inclusion criteria, most (385 [95.3%]) appeared well, whereas 19 (4.7%) had a possible ZIKV-associated birth defect. Seven had congenital ZIKV syndrome, and 12 were microcephalic without other abnormalities. Although infants with congenital ZIKV syndrome manifested clinical and neurodevelopmental sequelae during their first year of life, all 12 infants with isolated microcephaly were normocephalic and appeared well by 2 months of age. Laboratory evidence of ZIKV was detected for 22 of the infants, including 7 (31.8%) with a birth defect. Among 148 infants without a birth defect and negative/no laboratory results on ZIKV testing, and for whom information was available at 1 year, 4 presented with a developmental delay. CONCLUSIONS: Among infants with possible congenital ZIKV exposure, a small proportion had possible ZIKV-associated findings at birth or at follow-up, or laboratory evidence of ZIKV. Identifying and monitoring infants with possible ZIKV exposure requires extensive efforts by providers and public health departments. Longitudinal studies using standardized clinical and developmental assessments are needed for infants after possible congenital ZIKV exposure.

Laboratory Analysis of an Outbreak of Candida auris in New York from 2016 to 2018: Impact and Lessons Learned.

Candida auris is a multidrug-resistant yeast which has emerged in health care facilities worldwide; however, little is known about identification methods, patient colonization, environmental survival, spread, and drug resistance. Colonization on both biotic (patients) and abiotic (health care objects) surfaces, along with travel, appear to be the major factors for the spread of this pathogen across the globe. In this investigation, we present laboratory findings from an ongoing C. auris outbreak in New York (NY) from August 2016 through 2018. A total of 540 clinical isolates, 11,035 patient surveillance specimens, and 3,672 environmental surveillance samples were analyzed. Laboratory methods included matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast isolate identification, real-time PCR for rapid surveillance sample screening, culture on selective/nonselective media for recovery of C. auris and other yeasts from surveillance samples, antifungal susceptibility testing to determine the C. auris resistance profile, and Sanger sequencing of the internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal gene for C. auris genotyping. Results included (a) identification and confirmation of C. auris in 413 clinical isolates and 931 patient surveillance isolates as well as identification of 277 clinical cases and 350 colonized cases from 151 health care facilities, including 59 hospitals, 92 nursing homes, 1 long-term acute care hospital (LTACH), and 2 hospices, (b) successful utilization of an in-house developed C. auris real-time PCR assay for the rapid screening of patient and environmental surveillance samples, (c) demonstration of relatively heavier colonization of C. auris in nares than in the axilla/groin, and (d) predominance of the South Asia clade I with intrinsic resistance to fluconazole and elevated MIC to voriconazole (81%), amphotericin B (61%), flucytosine (5FC) (3%), and echinocandins (1%). These findings reflect greater regional prevalence and incidence of C. auris and the deployment of better detection tools in an unprecedented outbreak.

Zika Virus Infection Among Pregnant Women and Their Neonates in New York City, January 2016-June 2017.

OBJECTIVE: To describe and compare differences in the epidemiologic, clinical, and laboratory characteristics of pregnant women with confirmed or probable Zika virus infection and to compare the risk of having a neonate with laboratory evidence of Zika virus infection with that of having a neonate without evidence of Zika virus infection by maternal characteristics. METHODS: We conducted a retrospective cohort study of women with Zika virus infection who completed pregnancy in New York City from January 1, 2016 to June 30, 2017. Confirmed Zika virus infection was defined as 1) nucleic acid amplification test-detected Zika virus, or 2) a nonnegative enzyme-linked immunosorbent assay test result and a plaque-reduction neutralization test result positive for Zika virus but negative for dengue virus, or 3) delivery of a neonate with laboratory evidence of Zika virus infection. Probable infection was defined as a nonnegative enzyme-linked immunosorbent assay test result and a positive plaque-reduction neutralization test result for Zika virus and dengue virus. RESULTS: We identified 390 women with confirmed (28%) or probable (72%) Zika virus infection. Fever, rash, arthralgia, or conjunctivitis was reported by 31% of women and were more common among women with confirmed than with probable infection (43% vs 26%, P=.001). Of 366 neonates born to these women, 295 (81%) were tested for Zika virus and 22 (7%) had laboratory-diagnosed congenital Zika virus infection. The relative risk (RR) for having a neonate with laboratory evidence of Zika virus infection was greater among women with fever (RR 4.8, 95% CI 2.1-10.7), tingling (RR 4.8, CI 1.7-13.7), or numbness (RR 6.9, CI 2.6-18.2) during pregnancy or the periconception period. However, the RR did not differ whether the mother had confirmed or probable Zika virus infection (RR 1.6, CI 0.7-4.1). CONCLUSION: In New York City, a greater proportion of women had probable Zika virus infection than confirmed infection. Women with some symptoms during pregnancy or periconceptionally were more likely to have a neonate with laboratory evidence of Zika virus infection. Neonates born to women with confirmed or probable Zika virus infection should be tested for Zika virus infection.

Epidemiological Characteristics and Laboratory Findings of Zika Virus Cases in New York City, January 1, 2016-June 30, 2017.

BACKGROUND: An outbreak of Zika virus (ZIKV) began in May 2015 in Brazil and rapidly spread throughout the Americas; New York City (NYC) has a diverse population with ∼1.8 million residents who were born in ZIKV-affected areas. Before July 24, 2017, the Centers for Disease Control and Prevention (CDC) ZIKV testing recommendations included nucleic acid amplification-based tests for serum and urine specimens collected ≤14 days of illness onset or last potential exposure, and ZIKV immunoglobulin M (IgM) assay when ZIKV RNA is not detected or for specimens collected within 2-12 weeks of illness onset or last potential exposure, followed by a plaque reduction neutralization test (PRNT). However, the New York public health laboratories and commercial laboratories tested specimens collected beyond these time frames. METHODS: We analyzed 1080 noncongenital ZIKV cases in NYC residents who met the Council for State and Territorial Epidemiologist's ZIKV case definitions. RESULTS: Among cases, 98% were travel associated, 1% were sexually transmitted, and 1% had unknown exposures; 412 (38%) cases were pregnant women. Of 672 patients with ZIKV RNA detected in serum or urine specimens, 48 (7%) tested positive >14 days after either symptom onset or last potential exposure date (range 15-99 days). Of 390 patients diagnosed based on serology alone (i.e., not tested or not detectable for ZIKV RNA), 60 (15%) had a positive ZIKV IgM and PRNT >12 weeks after symptom onset or last potential exposure date (range 85-273 days). CONCLUSION: Our findings correspond with CDC's updated guidance to test symptomatic pregnant women up to 12 weeks past onset of symptoms. ZIKV IgM antibody testing may also be warranted for pregnant women regardless of symptoms if their exposure occurred during their pregnancy or periconception period. Providers should understand the scope of diagnostic testing and its limitations to appropriately counsel patients, especially pregnant women.

Investigational Testing for Zika Virus among U.S. Blood Donors.

BACKGROUND: Because of the potential severe clinical consequences of Zika virus (ZIKV) infection, the large numbers of asymptomatic travelers returning from ZIKV-active areas, the detection of ZIKV nucleic acid in blood, and reports of transmission of ZIKV through transfusion, in 2016 the Food and Drug Administration released recommendations for individual-unit nucleic acid testing to minimize the risk of transmission of ZIKV through blood transfusions. METHODS: The American Red Cross implemented investigational screening of donated blood for ZIKV RNA by means of transcription-mediated amplification (TMA). Confirmatory testing of reactive donations involved repeat TMA, TMA testing in exploratory minipools, real-time reverse-transcriptase polymerase chain reaction, IgM serologic testing, and red-cell TMA. Viral loads in plasma and red cells were estimated by means of end-point TMA. The costs of interdicting a donation that was confirmed to be positive were calculated for the 15-month period between June 2016 and September 2017. RESULTS: Of the 4,325,889 donations that were screened, 393,713 (9%) were initially tested in 24,611 minipools, and no reactive donations were found. Of the 3,932,176 donations that were subsequently tested individually, 160 were initially reactive and 9 were confirmed positive (a 1:480,654 confirmed-positive rate overall; positive predictive value, 5.6%; specificity, 99.997%). Six (67%) of the confirmed-positive donations were reactive on repeat TMA, of which 4 were IgM-negative; of these 4, all 3 that could be tested were reactive on minipool TMA. Two confirmed-positive donors had infections that had been transmitted locally (in Florida), 6 had traveled to ZIKV-active areas, and 1 had received an experimental ZIKV vaccine. ZIKV RNA levels in red cells ranged from 40 to 800,000 copies per milliliter and were detected up to 154 days after donation, as compared with 80 days of detection in plasma at levels of 12 to 20,000 copies per milliliter. On the basis of industry-reported costs of testing and the yield of the tests in our study, the cost of identifying 8 mosquito-borne ZIKV infections through individual-unit nucleic acid testing was $5.3 million per ZIKV RNA-positive donation. CONCLUSIONS: Screening of U.S. blood donations for ZIKV by individual-donation TMA was costly and had a low yield. Among the 9 confirmed ZIKV-positive donations, only 4 were IgM-negative; of these donations, all 3 that were tested were reactive on minipool TMA. (Funded by the American Red Cross and Grifols Diagnostic Solutions.).

Development of Zika Virus Serological Testing Strategies in New York State.

The recent outbreak of Zika virus (ZIKV) in the Americas has challenged diagnostic laboratory testing strategies. At the Wadsworth Center, ZIKV serological testing was performed for over 10,000 specimens, using a combination of an enzyme-linked immunosorbent assay (ELISA) for IgM antibodies (Abs) to ZIKV, a polyvalent microsphere immunoassay (MIA) to detect Abs broadly reactive with flaviviruses, and a plaque reduction neutralization test (PRNT) for further testing. Overall, 42% of patients showed serological evidence of flavivirus infection (primarily past dengue virus [DENV] infection), while 7% possessed IgM Abs to ZIKV and/or DENV. ZIKV IgM Abs typically arose within 3 to 4 days, with only one instance of duration beyond 100 days after reported symptoms. PRNT analysis of 826 IgM-positive specimens showed 7% positive neutralization to ZIKV alone, 9% to DENV alone, and 85% to both ZIKV and DENV. Thus, the extensive Ab cross-reactivity among flaviviruses significantly reduced the value of performing PRNT analysis, especially when a traditional paired serum algorithm with viral neutralization titering was used. Nevertheless, the finding of a negative ZIKV result by PRNT was invaluable for reassuring both physicians and patients. The MIA detected both IgM and IgG, which enabled us to identify patients who presented without IgM anti-ZIKV Abs but still had ZIKV-specific neutralizing Abs. On the basis of these results, a new algorithm, which included an IgM Ab capture (MAC)-ELISA to detect recent infection, a flavivirus MIA to identify patients no longer producing IgM, and a single-dilution PRNT for ZIKV exclusion and occasional discrimination of ZIKV and DENV, was implemented.

Comprehensive Whole-Genome Sequencing and Reporting of Drug Resistance Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State.

Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterize Mycobacterium tuberculosis and other M. tuberculosis complex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.

Zika Virus Testing Considerations: Lessons Learned from the First 80 Real-Time Reverse Transcription-PCR-Positive Cases Diagnosed in New York State.

The performance and interpretation of laboratory tests for Zika virus (ZKV) continue to be evaluated. Serology is cross-reactive, laborious, and frequently difficult to interpret, and serum was initially solely recommended for molecular diagnosis. ZKV testing was initiated in January 2016 in New York State for symptomatic patients, pregnant women, their infants, and patients with Guillain-Barré syndrome who had traveled to areas with ZKV transmission. Subsequently, eligibility was expanded to pregnant women with sexual partners with similar travel histories. Serum and urine collected within 4 weeks of symptom onset or within 6 weeks of travel were tested with real-time reverse transcription-PCR (RT-PCR) assays targeting the ZKV envelope and NS2B genes. In this review of lessons learned from the first 80 positive cases in NYS, ZKV RNA was detected in urine only in 50 patients, in serum only in 19 patients, and in both samples concurrently in 11 patients, with average viral loads in urine a log higher than those in serum. Among 93 positive samples from the 80 patients, 41 were positive on both gene assays, 52 were positive on the envelope only, and none were positive on the NS2B only. Of the 80 infected patients, test results for 74 (93%) would have defined their infection status as not detected or equivocal if the requirement for positive results from two assay targets (two-target-positive requirement) in the initial federal guidance to public health laboratories was enforced, if urine was not tested, or if the extended eligibility time for molecular testing was not implemented. These changes facilitated more extensive molecular diagnosis of ZKV, reducing reliance on time-consuming and potentially inconclusive serology.

Lyme disease: a case report of a 17-year-old male with fatal Lyme carditis.

Lyme disease is a systemic infection commonly found in the northeastern, mid-Atlantic, and north-central regions of the United States. Of the many systemic manifestations of Lyme disease, cardiac involvement is uncommon and rarely causes mortality. We describe a case of a 17-year-old adolescent who died unexpectedly after a 3-week viral-like syndrome. Postmortem examination was remarkable for diffuse pancarditis characterized by extensive infiltrates of lymphocytes and focal interstitial fibrosis. In the cardiac tissue, Borrelia burgdorferi was identified via special stains, immunohistochemistry, and polymerase chain reaction. The findings support B. burgdorferi as the causative agent for his fulminant carditis and that the patient suffered fatal Lyme carditis. Usually, Lyme carditis is associated with conduction disturbances and is a treatable condition. Nevertheless, few cases of mortality have been reported in the literature. Here, we report a rare example of fatal Lyme carditis in an unsuspected patient.

Dioxygen and nitric oxide scavenging by Treponema denticola flavodiiron protein: a mechanistic paradigm for catalysis.

Flavodiiron proteins (FDPs) contain a unique active site consisting of a non-heme diiron carboxylate site proximal to a flavin mononucleotide (FMN). FDPs serve as the terminal components for reductive scavenging of dioxygen (to water) or nitric oxide (to nitrous oxide), which combats oxidative or nitrosative stress in many bacteria. Characterizations of FDPs from spirochetes or from any oral microbes have not been previously reported. Here, we report characterization of an FDP from the anaerobic spirochete, Treponema (T.) denticola, which is associated with chronic periodontitis. The isolated T. denticola FDP exhibited efficient four-electron dioxygen reductase activity and lower but significant anaerobic nitric oxide reductase activity. A mutant T. denticola strain containing the inactivated FDP-encoding gene was significantly more air-sensitive than the wild-type strain. Single turnover reactions of the four-electron-reduced FDP (FMNH2-Fe(II)Fe(II)) (FDPred) with O2 monitored on the milliseconds to seconds time scale indicated initial rapid formation of a spectral feature consistent with a cis-µ-1,2-peroxo-diferric intermediate, which triggered two-electron oxidation of FMNH2. Reaction of FDPred with NO showed apparent cooperativity between binding of the first and second NO to the diferrous site. The resulting diferrous dinitrosyl complex triggered two-electron oxidation of the FMNH2. Our cumulative results on this and other FDPs indicate that smooth two-electron FMNH2 oxidation triggered by the FDPred/substrate complex and overall four-electron oxidation of FDPred to FDPox constitutes a mechanistic paradigm for both dioxygen and nitric oxide reductase activities of FDPs. Four-electron reductive O2 scavenging by FDPs could contribute to oxidative stress protection in many other oral bacteria.

Rapid whole-genome sequencing for surveillance of Salmonella enterica serovar enteritidis.

For Salmonella enterica serovar Enteritidis, 85% of isolates can be classified into 5 pulsed-field gel electrophoresis (PFGE) types. However, PFGE has limited discriminatory power for outbreak detection. Although whole-genome sequencing has been found to improve discrimination of outbreak clusters, whether this procedure can be used in real-time in a public health laboratory is not known. Therefore, we conducted a retrospective and prospective analysis. The retrospective study investigated isolates from 1 confirmed outbreak. Additional cases could be attributed to the outbreak strain on the basis of whole-genome data. The prospective study included 58 isolates obtained in 2012, including isolates from 1 epidemiologically defined outbreak. Whole-genome sequencing identified additional isolates that could be attributed to the outbreak, but which differed from the outbreak-associated PFGE type. Additional putative outbreak clusters were detected in the retrospective and prospective analyses. This study demonstrates the practicality of implementing this approach for outbreak surveillance in a state public health laboratory.

Enhanced identification and characterization of non-O157 Shiga toxin-producing Escherichia coli: a six-year study.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are emerging pathogens with the potential to cause serious illness and impact public health due to diagnostic challenges. Between 2005 and 2010, the Wadsworth Center (WC), the public health laboratory of the New York State (NYS) Department of Health, requested that Shiga toxin enzyme immunoassay (EIA)-positive stool enrichment broths and/or stool specimens be submitted by clinical and commercial reference laboratories testing NYS patient specimens. A total of 798 EIA-positive specimens were received for confirmation and serotyping, and additionally a subset of STEC was assessed for the presence of six virulence genes (stx1, stx2, eaeA, hlyA, nleA, and nleB) by real-time polymerase chain reaction. We confirmed 591 specimens as STEC, 164 (28%) as O157 STEC, and 427 (72%) as non-O157 STEC. Of the non-O157 STEC serogroups identified, over 70% were O103, O26, O111, O45, O121, or O145. During this time period, WC identified and characterized a total of 1282 STEC received as E. coli isolates, stool specimens, or EIA broths. Overall, the STEC testing identified 59% as O157 STEC and 41% as non-O157 STEC; however, out of 600 isolates submitted to the WC as E. coli cultures, 543 (90%) were identified as O157 STEC. This report summarizes a 6-year study utilizing enhanced STEC testing that resulted in increased identification and characterization of non-O157 STEC in NYS. Continued utilization of enhanced STEC testing may lead to effective and timely outbreak response and improve monitoring of trends in STEC disease epidemiology.

Treponema denticola superoxide reductase: in vivo role, in vitro reactivities, and a novel [Fe(Cys)(4)] site.

In vitro and in vivo results are presented demonstrating that superoxide reductase (SOR) from the air-sensitive oral spirochete, Treponema denticola (Td), is a principal enzymatic scavenger of superoxide in this organism. This SOR contains the characteristic non-heme [Fe(His)(4)Cys] active sites. No other metal-binding domain has been annotated for Td SOR. However, we found that Td SOR also accommodates a [Fe(Cys)(4)] site whose spectroscopic and redox properties resemble those in so-called 2Fe-SORs. Spectroscopic comparisons of the wild type and engineered Cys → Ser variants indicate that three of the Cys ligands correspond to those in [Fe(Cys)(4)] sites of "canonical" 2Fe-SORs, whereas the fourth Cys ligand residue has no counterpart in canonical 2Fe-SORs or in any other known [Fe(Cys)(4)] protein. Structural modeling is consistent with iron ligation of the "noncanonical" Cys residue across subunit interfaces of the Td SOR homodimer. The Td SOR was isolated with only a small percentage of [Fe(Cys)(4)] sites. However, quantitative formation of stable [Fe(Cys)(4)] sites was readily achieved by exposing the as-isolated protein to an iron salt, a disulfide reducing agent and air. The disulfide/dithiol status and iron occupancy of the Td SOR [Fe(Cys)(4)] sites could, thus, reflect intracellular redox status, particularly during periods of oxidative stress.

Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens.

An analysis of 16S rRNA gene sequences from archived clinical reference specimens has identified two novel Neisseria species. For each species, two strains from independent sources were identified. Amongst species with validly published names, the closest species to the newly identified organisms were Neisseria canis, N. dentiae, N. zoodegmatis, N. animaloris and N. weaveri. DNA-DNA hybridization studies demonstrated that the newly identified isolates represent species that are distinct from these nearest neighbours. Analysis of partial 23S rRNA gene sequences for the newly identified strains and their nearest neighbours provided additional support for the species designation. Bayesian analysis of 16S rRNA gene sequences suggested that the newly identified isolates belong to distinct but related species of the genus Neisseria, and are members of a clade that includes N. dentiae, N. bacilliformis and N. canis. The predominant cellular fatty acids [16 : 0, summed feature 3 (16 : 1ω7c and/or iso-15 : 0 2-OH) and 18 : 1ω7c], as well as biochemical and morphological analyses further support the designation of Neisseria wadsworthii sp. nov. (type strain 9715(T) =DSM 22247(T) =CIP 109934(T)) and Neisseria shayeganii sp. nov. (type strain 871(T) =DSM 22246(T) =CIP 109933(T)).

Cryo-electron tomography elucidates the molecular architecture of Treponema pallidum, the syphilis spirochete.

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.

The flat-ribbon configuration of the periplasmic flagella of Borrelia burgdorferi and its relationship to motility and morphology.

Electron cryotomography was used to analyze the structure of the Lyme disease spirochete, Borrelia burgdorferi. This methodology offers a new means for studying the native architecture of bacteria by eliminating the chemical fixing, dehydration, and staining steps of conventional electron microscopy. Using electron cryotomography, we noted that membrane blebs formed at the ends of the cells. These blebs may be precursors to vesicles that are released from cells grown in vivo and in vitro. We found that the periplasmic space of B. burgdorferi was quite narrow (16.0 nm) compared to those of Escherichia coli and Pseudomonas aeruginosa. However, in the vicinity of the periplasmic flagella, this space was considerably wider (42.3 nm). In contrast to previous results, the periplasmic flagella did not form a bundle but rather formed a tight-fitting ribbon that wraps around the protoplasmic cell cylinder in a right-handed sense. We show how the ribbon configuration of the assembled periplasmic flagella is more advantageous than a bundle for both swimming and forming the flat-wave morphology. Previous results indicate that B. burgdorferi motility is dependent on the rotation of the periplasmic flagella in generating backward-moving waves along the length of the cell. This swimming requires that the rotation of the flagella exerts force on the cell cylinder. Accordingly, a ribbon is more beneficial than a bundle, as this configuration allows each periplasmic flagellum to have direct contact with the cell cylinder in order to exert that force, and it minimizes interference between the rotating filaments.

Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples.

Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colony-forming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3% (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.

Native cellular architecture of Treponema denticola revealed by cryo-electron tomography.

Using cryo-electron tomography, we are developing a refined description of native cellular structures in the pathogenic spirochete Treponema denticola. Tightly organized bundles of periplasmic flagella were readily observed in intact plunge-frozen cells. The periplasmic space was measured in both wild-type and aflagellate strains, and found to widen by less than the diameter of flagella when the latter are present. This suggests that a structural change occurs in the peptidoglycan layer to accommodate the presence of the flagella. In dividing cells, the flagellar filaments were found to bridge the cytoplasmic cylinder constriction site. Cytoplasmic filaments, adjacent to the inner membrane, run parallel to the tightly organized flagellar filaments. The cytoplasmic filaments may be anchored by a narrow plate-like structure. The tapering of the cell ends was conserved between cells, with a patella-shaped structure observed in the periplasm at the tip of each cytoplasmic cylinder. Several incompletely characterized structures have been observed in the periplasm between dividing cells, including a cable-like structure linking two cytoplasmic cylinders and complex foil-shaped structures.