Arnold G. Steigerwalt

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

ABSTRACT The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov., and to remove the family Bartonellaceae from the order Rickettsiales.

DNA hybridization data (hydroxyapatite method, 50 to 70 degrees C) indicate that Rickettsia prowazekii, the type species of the type genus of the family Rickettsiaceae, is substantially less closely related to Rochalimaea species than was previously thought. The levels of relatedness of Rickettsia prowazekii to Rochalimaea species and to Bartonella bacilliformis under optimal conditions for DNA reassociation were 0 to 14%, with 25.5% or greater divergence in related sequences. When stringent reassociation criteria were used, the levels of relatedness were 0 to 2%. The genera Bartonella and Rochalimaea are currently classified in different families (the Bartonellaceae and the Rickettsiaceae) in the order Rickettsiales. On the basis of DNA relatedness data, previous 16S rRNA sequence data, guanine-plus-cytosine contents, and phenotypic characteristics, neither Bartonella bacilliformis nor Rochalimaea species are closely related to other organisms currently classified in the order Rickettsiales. In fact, the closest relative of these organisms is Brucella abortus. It is therefore proposed that the family Bartonellaceae should be removed from the order Rickettsiales. Previous 16S rRNA sequence data and DNA hybridization data revealed high levels of relatedness between Bartonella bacilliformis and the four Rochalimaea species, indicating that these species are members of a single genus. It is proposed that the genus Rochalimaea should be united with the genus Bartonella in the family Bartonellaceae. The name Bartonella is retained as the genus name since it has nomenclatural priority over the name Rochalimaea. This means that new combinations for the Rochalimaea species must be created. Proposals are therefore made for the creation of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov.

Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies

DNA relatedness was determined among 303 strains of Leptospira and Leptonema. Included in the analysis were reference strains from 228 well-characterized and recognized serovars. The study included 268 serovars from 29 named and one or more unnamed serogroups. The strains clustered into 17 DNA hybridization groups, representing 12 previously described species (292 strains) and five new genomospecies (11 strains). The largest groups included Leptospira interrogans (91 strains from 82 serovars), Leptospira santarosai (65 strains from 59 serovars), Leptospira borgpetersenii (49 strains from 43 serovars), Leptospira kirschneri (29 strains from 26 serovars) and Leptospira noguchii (20 strains from 20 serovars). The new genomospecies include Leptospira genomospecies 1 (two strains, serovars pinagchang and sichuan), Leptospira genomospecies 2 (six strains, serovars lushui, manhao 3, manzhuang, nanding, mengla and yunnan), Leptospira genomospecies 3 (one strain, serovar holland), Leptospira genomospecies 4 (one strain, serovar hualin) and Leptospira genomospecies 5 (one strain, serovar saopaulo). With the exception of Ballum, all serogroups with greater than one serovar studied were genetically heterogeneous. Phenotypic tests, including optimal growth temperature, lipase activity and growth inhibition by copper sulfate or 2,6-diaminopurine, were of little use in differentiating DNA relatedness groups. The name Leptospira alexanderi sp. nov. is proposed for Leptospira genomospecies 2 (type strain L 60T = ATCC 700520T, serovar manhao 3).

Edwardsiella ictaluri sp. nov., the Causative Agent of Enteric Septicemia of Catfish

We characterized 13 cultures of the enteric bacterium causing enteric septicemia of catfish by studying their biochemical reactions, deoxyribonucleic hybridizations, and deoxyribonucleic acid guanine-plus-cytosine contents. We confirmed that this bacterium is a new species, which is most closely related to Edwardsiella tarda of the family Enterobacteriaceae. Five strains of the bacterium causing enteric septicemia of catfish were 80% or more related to the type strain, SECFDL GA 77–52 (= CDC 1976–78 = ATCC 33202), in 60°C deoxyribonucleic acid homology reactions. Species level relatedness among the 13 strains which we studied was demonstrated by the more than 80% relatedness in 75°C reactions. The bacterium causing enteric septicemia of catfish was most closely related to E. tarda (56 to 62%) in 60°C reactions. The guanine-plus-cytosine was 53 mol%, as determined by buoyant density centrifugation. We propose the name Edwardsiella ictaluri sp. nov. for the bacterium causing enteric septicemia of catfish.

Deoxyribonucleic Acid Relatedness between Serogroups and Serovars in the Family Leptospiraceae with Proposals for Seven New Leptospira Species

Deoxyribonucleic acid hybridization (hydroxyapatite method, 55 and 70°C) was used to characterize 38 serovars from 22 named serogroups of Leptospira interrogans and Leptospira biflexa, 6 serovars from 4 new unnamed serogroups of Leptospira interrogans, and single serovars of the proposed species Leptospira parva and Leptonema illini. Deoxyribonucleic acid relatedness confirmed the validity of Leptospira parva and Leptonema illini. The well-accepted species Leptospira interrogans and Leptospira biflexa, as currently defined, were extremely heterogeneous. Relatedness results revealed at least five new species among the parasitic serovars formerly included in Leptospira interrogans and two new species among the saprophytic serovars formerly included in Leptospira biflexa. Serogrouping did not equate with species identification, as serovars from several different subserogroups belonged to different species. The new species named in this paper are Leptospira, noguchii, Leptospira weilii, Leptospira santarosai, Leptospira borgpetersenii, Leptospira meyeri, Leptospira wolbachii, and Leptospira inadai.

Accuracy of Phenotypic and Genotypic Testing for Identification of Streptococcus pneumoniae and Description of Streptococcus pseudopneumoniae sp. nov.

ABSTRACT We have identified an unusual group of viridans group streptococci that resemble Streptococcus pneumoniae. DNA-DNA homology studies suggested that a subset of these isolates represent a novel species that may be included in the S. oralis-S. mitis group of viridans group streptococci. We suggest that this novel species be termed Streptococcus pseudopneumoniae. A combination of phenotypic and genetic reactions allows its identification. S. pseudopneumoniae strains do not have pneumococcal capsules, are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO2 but are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres, are not soluble in bile, and are positive by the GenProbe AccuProbe Pneumococcus test. The bile solubility test is more specific than the optochin test for identification of S. pneumoniae. Genetic tests for pneumolysin (ply) and manganese-dependent superoxide dismutase (sodA) and identification tests with a commercial probe, AccuProbe Pneumococcus, do not discriminate between the new species and S. pneumoniae.

Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.

Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest.

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (>82 % relatedness at 55 degrees C and >76 % relatedness at 70 degrees C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a well-supported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were non-haemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120(T) (=ATCC BAA-1595(T) =BEIR NR 9579(T) =CCUG 56148(T)). L. marthii has not been associated with human or animal disease at this time.

Phenotypic and genotypic characterization of atypical Lactococcus garvieae strains isolated from water buffalos with subclinical mastitis and confirmation of L. garvieae as a senior subjective synonym of Enterococcus seriolicida.

During a survey of bacterial agents that cause subclinical mastitis in water buffalos, we isolated several strains of gram-positive cocci that appeared to be enterococci except that they grew very slowly at 45 degrees C and grew slowly in broth containing 6.5% NaCl. On the basis of the results of conventional physiologic tests, these strains were identified as Enterococcus durans. However, none of the strains reacted with the AccuProbe Enterococcus genetic probe. The whole-cell protein profiles of these organisms were compared with the profiles of Enterococcus and Lactococcus reference strains. apart from minor quantitative differences, the mastitis isolates had indistinguishable protein profiles that were similar to the profiles of the Lactococcus garvieae and Enterococcus seriolicida type strains. The results of DNA relatedness studies performed by using the hydroxyapatite method at 55 and 70 degrees C indicated that all of the mastitis isolates were related to the type strain of L. garvieae at the species level, despite the fact that they exhibited several uncommon phenotypic characteristics (growth at 45 degrees C, growth in broth containing 6.5% NaCl, and failure to produce acid from mannitol and sucrose). The high levels of DNA relatedness between strains of L. garvieae is a senior synonym of E. seriolicida, L. garvieae should be retained as the species name and strain ATCC 43921 should remain the type strain of this species.

Ten New Species of Legionella

Ten new Legionella species were characterized on the basis of biochemical reactions, antigens, cellular fatty acids, isoprenoid quinones, and deoxyribonucleic acid relatedness. Nine of the new species were isolated from the environment, and one, Legionella hackeliae, was isolated from a bronchial biopsy specimen obtained from a patient with pneumonia. The species all exhibited the following biochemical reactions typical of the legionellae: growth on buffered cysteine-yeast extract agar, but not on blood agar; growth requirement for cysteine; gram negative; nitrate negative; urease negative; nonfermentative; catalase positive; production of a brown pigment on tyrosine-containing yeast extract agar; liquefaction of gelatin; and motility. Legionella spiritensis was weakly positive for hydrolysis of hippurate; the other species were hippurate negative. Legionella cherrii, Legionella steigerwaltii, and Legionella parisiensis exhibited bluish white autofluorescence. Legionella rubrilucens and Legionella erythra exhibited red autofluorescence. The other species, L. spiritensis, L. hackeliae, Legionella maceachernii, Legionella jamestowniensis, and Legionella santicrucis did not autofluoresce bluish white or red. All species had cellular fatty acid contents qualitatively similar to those of previously described legionellae and had major amounts of ubiquinones with more than 10 isoprene units in the side chains. Each new species was serologically distinct from previously described Legionella species. As determined by the hydroxyapatite method at 60°C, two strains of L. maceachernii were 100% related, and four strains of L. cherrii were 94 to 99% related. The other new species were represented by single strains. The levels of relatedness of the new species to each other and to previously described legionellae ranged from 1 to 67%. L. maceachernii, L. jamestowniensis, and L. hackeliae were less than 25% related to other species. L. rubrilucens and L. erythra, and two red-autofluorescing species, were about 60% interrelated. L. spiritensis (a non-autofluorescing species) was 34% related to L. rubrilucens. L. santicrucis was 64% related to Legionella sainthelensi. The three bluish white-autofluorescing species, L. parisiensis, L. cherrii, and L. steigerwaltii, were most closely related to other bluish white-autofluorescing species, especially Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and “Legionella anisa” (35 to 67%).