Implicating exudate macrophages and Ly-6C(high) monocytes in CCR2-dependent lung fibrosis following gene-targeted alveolar injury.

The alveolar epithelium is characteristically abnormal in fibrotic lung disease, and we recently established a direct link between injury to the type II alveolar epithelial cell (AEC) and the accumulation of interstitial collagen. The mechanisms by which damage to the epithelium induces lung scarring remain poorly understood. It is particularly controversial whether an insult to the type II AEC initiates an inflammatory response that is required for the development of fibrosis. To explore whether local inflammation occurs following a targeted epithelial insult and contributes to lung fibrosis, we administered diphtheria toxin to transgenic mice with type II AEC-restricted expression of the diphtheria toxin receptor. We used immunophenotyping techniques and diphtheria toxin receptor-expressing, chemokine receptor-2-deficient (CCR2(-/-)) mice to determine the participation of lung leukocyte subsets in pulmonary fibrogenesis. Our results demonstrate that targeted type II AEC injury induces an inflammatory response that is enriched for CD11b(+) nonresident exudate macrophages (ExM) and their precursors, Ly-6C(high) monocytes. CCR2 deficiency abrogates the accumulation of both cell populations and protects mice from fibrosis, weight loss, and death. Further analyses revealed that the ExM are alternatively activated and that ExM and Ly-6C(high) monocytes express mRNA for IL-13, TGF-ß, and the collagen genes, COL1A1 and COLIIIA1. Furthermore, the accumulated ExM and Ly-6C(high) monocytes contain intracellular collagen, as detected by immunostaining. Together, these results implicate CCR2 and the accumulation of ExM and Ly-6C(high) monocytes as critical determinants of pulmonary fibrosis induced by selective type II AEC injury.

Evaluation of methods for identification and determination of the taxonomic status of strains belonging to the Streptococcus porcinus-Streptococcus pseudoporcinus complex isolated from animal, human, and dairy sources.

Ninety-seven animal, human, and dairy Streptococcus porcinus or Streptococcus pseudoporcinus isolates in the CDC Streptococcus strain collection were evaluated on the basis of DNA-DNA reassociation, 16S rRNA and rpoB gene sequencing, conventional biochemical and Rapid ID 32 Strep identification methods, and antimicrobial susceptibility testing to determine their taxonomic status, characteristics for species differentiation, antimicrobial susceptibility, and relevance of clinical source. Nineteen of the 97 isolates (1 human, 18 swine) were identified as S. porcinus. The remaining 72 human isolates and 6 dairy isolates were identified as S. pseudoporcinus. The use of 16S rRNA or rpoB gene sequencing was required to differentiate S. porcinus from S. pseudoporcinus. The human and dairy S. pseudoporcinus isolates were biochemically distinct from each other as well as distinct by 16S rRNA and rpoB gene sequencing. Therefore, we propose the subspecies denominations S. pseudoporcinus subsp. hominis subsp. nov. for the human isolates and S. pseudoporcinus subsp. lactis subsp. nov. for the dairy isolates. Most strains were susceptible to the antimicrobials tested, with the exception of tetracycline. Two strains of each species were also resistant to clindamycin and erythromycin and carried the erm(A) (S. pseudoporcinus) or the erm(B) (S. porcinus) gene. S. porcinus was identified from a single human isolate recovered from a wound in an abattoir worker. S. pseudoporcinus was primarily isolated from the genitourinary tract of women but was also associated with blood, placental, and wound infections. Isolates reacting with group B antiserum and demonstrating wide beta-hemolysis should be suspected of being S. pseudoporcinus and not S. agalactiae.

PAI-1 promotes the accumulation of exudate macrophages and worsens pulmonary fibrosis following type II alveolar epithelial cell injury.

Fibrotic disorders of the lung are associated with perturbations in the plasminogen activation system. Specifically, plasminogen activator inhibitor-1 (PAI-1) expression is increased relative to the plasminogen activators. A direct role for this imbalance in modulating the severity of lung scarring following injury has been substantiated in the bleomycin model of pulmonary fibrosis. However, it remains unclear whether derangements in the plasminogen activation system contribute more generally to the pathogenesis of lung fibrosis beyond bleomycin injury. To answer this question, we employed an alternative model of lung scarring, in which type II alveolar epithelial cells (AECs) are specifically injured by administering diphtheria toxin (DT) to mice genetically engineered to express the human DT receptor (DTR) off the surfactant protein C promoter. This targeted AEC injury results in the diffuse accumulation of interstitial collagen. In the present study, we found that this targeted type II cell insult also increases PAI-1 expression in the alveolar compartment. We identified AECs and lung macrophages to be sources of PAI-1 production. To determine whether this elevated PAI-1 concentration was directly related to the severity of fibrosis, DTR(+) mice were crossed into a PAI-1-deficient background (DTR(+) : PAI-1(-/-) ). DT administration to DTR(+) : PAI-1(-/-) animals caused significantly less fibrosis than was measured in DTR(+) mice with intact PAI-1 production. PAI-1 deficiency also abrogated the accumulation of CD11b(+) exudate macrophages that were found to express PAI-1 and type-1 collagen. These observations substantiate the critical function of PAI-1 in pulmonary fibrosis pathogenesis and provide new insight into a potential mechanism by which this pro-fibrotic molecule influences collagen accumulation. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Legionella nagasakiensis sp. nov., isolated from water samples and from a patient with pneumonia.

A novel Legionella species was identified based on analysis of 16S rRNA and mip (macrophage infectivity potentiator) gene sequences, cellular fatty acids, isoprenoid quinones, biochemical reactions, antigens and quantitative DNA-DNA hybridization. Strain CDC-1796-JAP-E(T) was isolated from well water at the Nagasaki Municipal Medical Center, Japan. Two strains, CDC-3041-AUS-E and CDC-3558-AUS-E, were isolated from water samples during an outbreak of legionellosis in South Australia. Strain CDC-5427-OH-H was isolated from a 66-year-old female patient diagnosed with Legionnaires' disease in the US. Cells from these four strains were gram-negative, non-fluorescent, rod-shaped, and positive for alkaline phosphatase, esterase, leucine arylamidase, catalase, gelatinase, ß-lactamase and tyrosine browning assay. Phylogenetic analysis of 16S rRNA and mip genes revealed that the four strains formed a distinct cluster within the genus Legionella. The bacteria contained branched-chain fatty acids and quinones that are typical of members of the genus Legionella. Slide agglutination tests demonstrated no cross-reaction with 52 previously described members of the Legionellaceae. DNA-DNA hybridization studies indicated that DNAs from the four strains were highly related (78-84 %) but they showed 29 % relatedness to Legionella oakridgensis ATCC 33761(T) and less than 10 % to strains of other Legionella species tested. These characterizations suggest that the isolates represent a novel species, for which the name Legionella nagasakiensis sp. nov. is proposed; the type strain is CDC-1796-JAP-E(T) ( = ATCC BAA-1557(T) = JCM 15315(T)).

Investigation of an apparent outbreak of Rhodococcus equi bacteremia.

During January to April 2007, hospital staff reported 3 patients with Rhodococcus equi bloodstream infections. Isolates were analyzed at the Centers for Disease Control and Prevention, Atlanta, GA, to confirm identification and to assess strain relatedness; 2 were R. equi but genetically distinct, and 1 was identified as Gordonia polyisoprenivorans. Rapid reference laboratory support prevented an unnecessary outbreak investigation.

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.

Designation of the provisional new enterococcus species CDC PNS-E2 as Enterococcus sanguinicola sp. nov., isolated from human blood, and identification of a strain previously named Enterococcus CDC PNS-E1 as Enterococcus italicus Fortina, Ricci, Mora, and Manachini 2004.

We have previously characterized two new enterococcal species (provisionally designated CDC PNS-E1 and CDC PNS-E2) recovered from clinically significant specimens associated with invasive infections in humans. In the present report we provide additional data and propose formal denominations for isolates of these two species of Enterococcus. Results of 16S rRNA gene sequencing, sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of whole-cell protein profiles, and DNA-DNA reassociation experiments indicated that the blood isolate ATCC BAA-780 (SS 1728; CDC PNS-E1) corresponds to Enterococcus italicus, whose species epithet was proposed to designate isolates from artisanal Italian cheese. Strain ATCC BAA-781 (CCUG 47861; SS 1729; CDC PNS-E2), a vancomycin-resistant isolate recovered from the blood of a patient in the United States, was found to be highly related at the species level to another blood isolate (SS 1743; CCUG 47884) from Sweden, and for these we propose the designation Enterococcus sanguinicola sp. nov.

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital.

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.

Streptococcus ictaluri sp. nov., isolated from Channel Catfish Ictalurus punctatus broodstock.

A streptococcal-like organism was associated with diseased Channel Catfish Ictalurus punctatus broodstock on four commercial aquaculture operations in the Mississippi Delta. Conventional biochemical testing, 16S rRNA gene sequence analysis and DNA-DNA hybridization distinguished the isolates from these fish from previously published Streptococcus species. Comparative 16S rRNA gene sequencing studies revealed that the isolates were phylogenetically most similar to Streptococcus iniae, Streptococcus uberis and Streptococcus parauberis with divergence ranging from 2.0 to 2.3 %. Streptococcus pyogenes, Streptococcus urinalis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus canis were included in the analysis and showed even greater differences (2.5-3.2 % divergence). DNA relatedness was 22 % or less to the most phylogenetically related species at the optimal temperature. These data suggest that the isolates represent a novel species of Streptococcus for which the name Streptococcus ictaluri sp. nov. is proposed. The type strain is 707-05(T) (=SO2-1108(T)=ATCC BAA-1300(T)=CCUG 52536(T)).

Identification of "Haematobacter," a new genus of aerobic Gram-negative rods isolated from clinical specimens, and reclassification of Rhodobacter massiliensis as "Haematobacter massiliensis comb. nov.".

Twelve strains of gram-negative, nonfermenting rods recovered mainly from septicemic patients were studied using conventional and molecular methods. The phenotypic profiles of these strains most closely resembled Psychrobacter phenylpyruvicus. They produced catalase, oxidase, urease, and H(2)S (lead acetate paper) but did not produce indole, reduce nitrate or nitrite, or hydrolyze gelatin or esculin. No acid production was observed in a King's oxidation-fermentation base containing d-glucose, d-xylose, d-mannitol, sucrose, lactose, or maltose. All strains were nonmotile and nonpigmented. Most strains produced green discoloration on blood agar. All strains grew at 25 degrees C and 35 degrees C and most grew on MacConkey agar. They shared a common cellular fatty acid (CFA) profile characterized by large amounts (56% to 90%) of 18:1omega7c and the presence of 3-OH-10:0, 16:1omega7c, 16:0, and 19:0cycomega8c that overall was most similar to that of Rhodobacter species but was quite distinct from that of P. phenylpyruvicus. The MICs for most beta-lactams, fluoroquinolones, aminoglycosides, and carbapenems were low. MICs for aztreonam and piperacillin were higher, with MICs for some strains of > 64 mg/liter and > 128 mg/liter, respectively. Polyphasic analysis of these strains, including morphological, biochemical, CFA composition, DNA-DNA hybridization, 16S rRNA gene sequencing, and percent guanine-plus-cytosine (G+C) content analysis, demonstrated that these strains and Rhodobacter massiliensis represent a new genus, "Haematobacter" (proposed name), with the species H. missouriensis (type strain H1892(T) = CCUG 52307(T) = CIP 109176(T)) and H. massiliensis comb. nov. (type strain Framboise(T) = CCUG 47968(T) = CIP 107725(T)) and an unnamed genomospecies.

Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing.

The genus Leptospira is classified into 13 named species and 4 genomospecies based upon DNA-DNA reassociation studies. Phenotypic tests are unable to distinguish between species of Leptospira, and there is a need for a simplified molecular approach to the identification of leptospires. 16S rRNA gene sequences are potentially useful for species identification of Leptospira, but there are a large number of sequences of various lengths and quality in the public databases. 16S rRNA gene sequences of near full length and bidirectional high redundancy were determined for all type strains of the species of the Leptospiraceae. Three clades were identified within the genus Leptospira, composed of pathogenic species, nonpathogenic species, and another clade of undetermined pathogenicity with intermediate 16S rRNA gene sequence relatedness. All type strains could be identified by 16S rRNA gene sequences, but within both pathogenic and nonpathogenic clades as few as two or three base pairs separated some species. Sequences within the nonpathogenic clade were more similar, and in most cases < or =10 bp distinguished these species. These sequences provide a reference standard for identification of Leptospira species and confirm previously established relationships within the genus. 16S rRNA gene sequencing is a powerful method for identification in the clinical laboratory and offers a simplified approach to the identification of Leptospira species.

Enterococcus caccae sp. nov., isolated from human stools.

The National Antimicrobial Resistance Monitoring System Laboratory at the Centers for Disease Control and Prevention (CDC) isolated two enterococcus-like strains that were referred to the CDC Streptococcus Laboratory for further identification. The isolates were recovered from human stool samples collected on different occasions from the same individual in Portland (OR, USA) in July 2000. Conventional physiological tests distinguished these strains from all known species of enterococci. Analyses of whole-cell-protein electrophoretic profiles showed the same unique profile for the two isolates, being most similar those of Enterococcus moraviensis and Enterococcus haemoperoxidus albeit not close enough to allow conclusive inclusion in any enterococcal species. Both isolates gave positive results in tests using the AccuProbe Enterococcus genetic probe, and Lancefield extracts reacted with CDC group D antiserum. Comparative 16S rRNA gene sequencing studies also revealed that these strains were closely related to the species E. moraviensis (99.6 % identity). The results of DNA-DNA relatedness experiments confirmed that these strains represented a single novel taxon. The highest level of DNA-DNA relatedness found between the novel taxon and any of the currently recognized species of Enterococcus was 32 %, for both E. moraviensis and E. haemoperoxidus. On the basis of this evidence, it is proposed that these stool isolates constitute a novel species, for which the name Enterococcus caccae sp. nov. is proposed. The type strain is 2215-02(T) (=SS-1777(T)=ATCC BAA-1240(T)=CCUG 51564(T)).

Native valve endocarditis due to Gordonia polyisoprenivorans: case report and review of literature of bloodstream infections caused by Gordonia species.

We report the first case of endocarditis caused by Gordonia polyisoprenivorans and concisely review the English literature regarding bloodstream infections caused by Gordonia species.

Characterization of clinical isolates previously identified as Oerskovia turbata: proposal of Cellulosimicrobium funkei sp. nov. and emended description of the genus Cellulosimicrobium.

Taxonomic studies were performed on 13 clinical isolates (ten of which were epidemiologically related) that had been previously identified as Oerskovia turbata. Comparative phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolates are closely related to Cellulosimicrobium cellulans with sequence similarity values ranging from 99.5 to 99.8 %. Chemotaxonomic results (fatty acid profiles and menaquinones) supported the inclusion of these isolates in the genus Cellulosimicrobium. The DNA G+C content was 74.5 mol%. The results of DNA-DNA reassociation, whole-cell sugars (with galactose as the characteristic whole sugar) and phenotypic properties, including antimicrobial resistance, indicated that these isolates are representatives of a novel species of the genus Cellulosimicrobium. The name Cellulosimicrobium funkei sp. nov. is proposed for the novel strains, with strain W6122T (=ATCC BAA-886T = DSM 16025T = CCUG 50705T) as the type strain. The definition of this novel Cellulosimicrobium species will assist in the understanding of the epidemiology and clinical significance of these micro-organisms.

Leptospira broomii sp. nov., isolated from humans with leptospirosis.

Isolates of Leptospira from two human cases of leptospirosis in Denmark and France were studied using DNA-DNA relatedness, G + C content, 16S rRNA gene sequence data and pulsed-field gel electrophoresis. These isolates differed from previously described species of Leptospira and are defined as Leptospira broomii sp. nov. The type strain is 5399T (= ATCC BAA-1107T = KIT 5399T).

Leptospirosis during dengue outbreak, Bangladesh.

We collected acute-phase serum samples from febrile patients at 2 major hospitals in Dhaka, Bangladesh, during an outbreak of dengue fever in 2001. A total of 18% of dengue-negative patients tested positive for leptospirosis. The case-fatality rate among leptospirosis patients (5%) was higher than among dengue fever patients (1.2%).

Phenotypic and genetic characterization of clinical isolates of CDC coryneform group A-3: proposal of a new species of Cellulomonas, Cellulomonas denverensis sp. nov.

CDC coryneform group A-3 bacteria are rare human pathogens. In this study, six group A-3 isolates (two from blood, one from cerebrospinal fluid, and one each from homograft valve, lip wound, and pilonidal cyst) were compared to the type strains of phenotypically related organisms, Cellulomonas fimi, Cellulomonas hominis, Oerskovia turbata, and Sanguibacter suarezii, and characterized by phenotypic, chemotaxonomic, and genotypic studies. DNA-DNA reassociation analysis identified two genomic groups, and phylogenetic analysis of the 16S rRNA gene sequence identified the taxonomic positions of these groups to genus level. Two groups were defined, and both were more closely related to Cellulomonas species: one group of three strains, for which we propose the new species Cellulomonas denverensis sp. nov., with the type strain W6929 (ATCC BAA-788(T) or DSM 15764(T)), was related to C. hominis ATCC 51964(T) (98.5% 16S rRNA gene sequence similarity), and the second group of three strains was related to C. hominis ATCC 51964(T) (99.8 to 99.9% 16S rRNA gene sequence similarity). The definition of this new Cellulomonas species and the confirmation of three strains as C. hominis serve to further clarify the complex taxonomy of CDC coryneform group A-3 bacteria and will assist in our understanding of the epidemiology and clinical significance of these microorganisms.

Detection of pathogenic leptospires by real-time quantitative PCR.

Definitive diagnosis of leptospirosis has traditionally depended upon the isolation of leptospires from clinical specimens or the demonstration of seroconversion in paired acute and convalescent serum samples. Both of these approaches require expertise not routinely available in clinical laboratories and usually result in delayed diagnosis. Conventional PCR assays have been developed, but all have limitations which have restricted their widespread use. In order to overcome these limitations, a real-time PCR assay was developed using a 423 bp target on the lipL32 gene, which is conserved among pathogenic serovars of LEPTOSPIRA: Reactions were monitored by SYBR green fluorescence and melting curve analysis. Representative serovars from 16 species of Leptospira and over 40 species of other bacteria and fungi were tested. Positive results were obtained with all pathogenic leptospiral serovars, with the exception of Leptospira fainei serovar Hurstbridge. The analytical sensitivity of this assay was 3 genome equivalents per reaction; approximately 10 genome equivalents were detectable in human urine. Leptospiral DNA was amplified from blood containing EDTA or citrate anticoagulants, but heparin, sodium polyanetholesulfonate and saponin were inhibitory. The assay successfully detected leptospiral DNA from serum and urine samples of patients with leptospirosis. This assay has the potential to facilitate rapid, sensitive diagnosis of acute leptospirosis.