Nocardia aobensis Sp. Nov., isolated from patients in Japan

Five clinical isolates, strains IFM 0137, 0372(T), 0496, 0556, and 0952, were provisionally assigned to the genus Nocardia based on morphological criteria. Nearly complete 16S rDNA sequences were determined for these strains. These data showed that they are most similar to that of Nocardia africana, Nocardia cerradoensis and Nocardia veterana. However, DNA-DNA relatedness data showed that the five strains were of a single species and were distinguishable from N. africana, N. cerradoensis and N. veterana. Therefore, these strains represent a new species within the genus Nocardia. The designation of these five strains is Nocardia aobensis sp. nov. The type strain is IFM 0372(T) (=NBRC 100429(T)=JCM 12352(T)=DSM 44805(T)).     

Nocardia beijingensis,is a Pathogenic Bacterium to Humans: The First Infectious Cases in Thailand and Japan

Nocardia beijingensis, a recently established new species, is an isolate from soil in China. During our taxonomic studies on 450 nocardial clinical isolates in Thailand and Japan, 17 strains from Thailand and 1 strain from Japan were found to have a similar physiological characteristic to those of N. beijingensis, such as a drug susceptibility pattern to three antimicrobial agents. Our phylogenetic studies on these 18 strains by 16S rRNA gene sequence analysis confirmed that these strains belong to N. beijingensis species. Phylogenetically, these newly isolated N. beijingensis strains were found to be classified into two distinct clades: one is a Japanese clade and other is a Chinese clade, including a reference strain and 17 Thai strains. This is the first report of human infection due to N. beijingensis strains, and we propose that the bacterium be categorized as an opportunistic infectious group regardless of its original isolation from soil.     

Nocardia caviae: a Report of 13 New Isolations with Clinical Correlation

Thirteen isolates of Nocardia caviae from 12 different clinical sources were received and identified over a 5(1/2)-year period by the Mycology Division of the Center for Disease Control. The results of morphological, biochemical, and physiological studies on these isolates were compared with those obtained with four reference cultures of N. caviae received from the Institute of Microbiology, Rutgers University. Comparison showed that N. caviae isolates form a homogeneous group that is usually easily distinguished from N. asteroides, N. brasiliensis, and other pathogenic aerobic actinomycetes. The clinical sources included nine human and two animal infections and one human isolate apparently not associated with disease. Previous reports of N. caviae infections in man have been limited to rare cases of actinomycotic mycetoma. Among the human infections reported in this series are one case of mycetoma, one case of "mycotic" keratitis, one case of skin abscess, two cases of osteomyelitis, and four cases of serious pulmonary infection caused by N. caviae.     

Reliability of the detection of meningococcal gamma-glutamyl transpeptidase as an identification marker for Neisseria meningitidis

Detection of gamma-glutamyl transpeptidase (GGT; ggt ) activity is one of the useful methods for a specific identification of Neisseria meningitidis. However, we previously happened to isolate a ggt -deficient N. meningitidis strain (NIID113) from a healthy carrier. In this study, in order to re-examine the reliability of the marker, we again investigated the GGT activity of 245 N. meningitidis human isolates and identified two other GGT-defective N. meningitidis isolates besides NIID113. The isolation frequency (1.2%) of ggt mutants among human isolates strongly confirmed the 98.8% reliability of GGT activity as the identification marker for N. meningitidis.     

Isolation and characterization of a new variant of black pigmented asaccharolytic Bacteroides

Abstract Several strains of asaccharolytic black pigmented Bacteroides species (both oral and rumen isolates) were studied. Ultrastructural and biochemical characteristics in addition to agglutination tests showed, that the isolate ES 54B was different from the reference strains of B. gingivalis and B. asaccharolyticus . The strain ES 54B, isolated from human periapical osteitis, appears to represent a new species.     

Comparative sensitivities of Sabin and Mahoney poliovirus type 1 prototype strains and two recent isolates to low concentrations of glutaraldehyde.

Significant intratypic differences in the glutaraldehyde (GTA) sensitivity of echovirus isolates have been shown. While exploring ways to optimize the study of GTA sensitivity of enteroviruses, we also observed intratypic differences in poliovirus type 1 isolates collected in France. A suspension procedure was used for assessing the virucidal effect of GTA at low concentrations (< or = 0.10%) against purified viruses. Two recent isolates of poliovirus type 1 tested were first fully characterized by the PCR restriction fragment length polymorphism (RFLP) test. The RFLP pattern of clinical isolate 5617 was similar to that of poliovirus type 1 LS-c, 2ab (Sabin strain), confirming the vaccine origin of strain 5617. The RFLP pattern of strain 5915 recovered from sewage was different from that of the Mahoney strain, suggesting a genetic variation in this wild isolate. We then analyzed under the same controlled conditions the GTA sensitivities of both isolates and their respective prototype strains. The wild Mahoney and 5915 strains exhibited significantly lower sensitivities to GTA than did the vaccine Sabin and 5617 strains. The inactivation rates of clinical isolates 5617 and 5915 were very similar to those of their corresponding reference Sabin and Mahoney strains. Both the conformational structure of the capsid of each strain and the amino acid constitution of structural polypeptides could be involved in the variations observed. The relevance of our comparative sensitivity studies to standardization of virucidal tests is discussed.     

In vitro culture and biochemical characterization of six trypanosome isolates from Peru and Brazil

Six trypanosomatids isolated from different geographical areas from South America (Peru and Brazil) and different vectors and reservoir hosts (the triatomine Panstrongylus chinai [TP1], Triatoma infestans [TP2], Rhodnius ecuadorensis [TP3], R. prolixus [TB1], Didelphys marsupialis [TB2]), and one from a human asymptomatic patient [TB3], were characterized using lectin agglutination, isoenzyme profile, in vitro culture final metabolite patterns, and compared with a reference strain (Trypanosoma cruzi, Maracay strain [TC]). The different isolates were cultured in vitro in Grace's medium supplemented with 10% inactivated bovine foetal serum. According to our results and the statistical study, the isolate obtained from R. ecuadorensis should be designed as a Trypanosoma rangeli sp., showing all other isolates strong similarities to T. cruzi. Between them, two clusters could be identified, strongly correlating with the geographical origin. Cluster I grouped isolates from Peru and T. cruzi reference strain, and cluster II grouped the three Brazilian isolates.     

Characterization of Bilophila wadsworthia Isolates Using PCR Fingerprinting

Bilophila wadsworthia, an under-appreciated anaerobic organism, was originally described in 1989. Ninety-nine Bilophila wadsworthia isolates, recovered form environmental and clinical specimens in Germany and in Southern California, were examined in this study. Many isolates were recovered in mixed culture with facultative aerobic and other anaerobic bacteria. All isolates were identified by standard laboratory procedures, including gas–liquid chromatography (GLC). A PCR fingerprint assay was established to compare the profiles of clinical and environmental isolates to the type strain (ATCC 49260) and to an environmental (sewage) reference strain (DSM 11045, RZATAU) for intra-species differences. Two primers, one universal primer, M13 core, and one tDNA primer, T3B, were used individually to analyse the strains. Homogeneous PCR fingerprint profiles were found for the majority of strains using the M13 core primer; two PCR groups were determined with T3B, one matching the type strain and one matching the environmental reference strain (DSM 11045, RZATAU). Two urease negative strains, WAL 11470 (blood isolate from California) and TÜB 754 (intra-abdominal isolate from Germany) formed unique PCR fingerprint profiles with each of these primers. These results were confirmed by PCR fingerprinting using the T3A primer. These latter results suggest a possible genetic diversity in B. wadsworthia.     

Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes

Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec's isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba.     

Analysis of regions determining resistence to fluoroquinolones in genes gyrA and parC in clinical isolates of Mycoplasma hominis

Fifteen strains of M. hominis isolated from patients with urogenital inflammations were analyzed. Variations in the quinolone resistance-determining regions (QRDR) have been found in fluoroquinolone-resistant M. hominis clinical isolates in comparison with the reference PG21 strain. In one isolate, parC had Asn substitute at position 91.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

Comparative adherence to human A549 cells, plant fibronectin-like protein, and polystyrene surfaces of four Pseudomonas fluorescens strains from different ecological origin

The main objective of this study was to compare the adherence properties of four Pseudomonas fluorescens isolates from different ecological niches (human tissue, rhizosphere, drinking water, and cow milk). The substrates used to test P. fluorescens adherence were as follows: cultured human respiratory epithelial cells A549, immobilized plant fibronectin-like protein, and polystyrene. For all the experiments, bacteria were grown at 27 degrees C. The adherence assay to human cells was performed at 37 degrees C, whereas adherence to fibronectin and polystyrene was done at 27 degrees C. The four strains tested adhered to A549 cells but showed different adherence patterns. At 3 h, the milk isolate showed an aggregative adherence phenotype, whereas the three other isolates showed a diffuse adherence pattern. With a longer incubation time of 24 h, the aggregative pattern of the milk isolate disappeared, the adherence of the clinical strain increased, the adherence of the water isolate decreased, and morphological changes in A549 cells were observed with the clinical, water, and soil isolates. The four strains tested formed biofilms on polystyrene dishes. The clinical and milk isolates were the more efficient colonizers of polystyrene surfaces and also the more adherent to immobilized plant fibronectin-like protein. There was no relation between bacterial surface hydrophobicity and P. fluorescens adherence to the substrates tested. The main conclusions of these results are that P. fluorescens is an adherent bacterium, that no clear correlation exists between adherence and ecological habitat, and that P. fluorescens can adhere well to substrates not present in its natural environment.     

In Vitro Activity of Rifampicin and Verapamil Combination in Multidrug-Resistant Mycobacterium tuberculosis

The aim of the present study was to evaluate the effect of the combination of rifampicin (RIF) and verapamil (VP) against the Mycobacterium tuberculosis H₃₇Rv reference strain and six multidrug-resistant (MDR) M. tuberculosis clinical isolates by determining Time-Kill Curves and the ability to efflux drug by fluorometry. The RIF+VP combination showed synergism in one MDR clinical isolate. For the other five MDR clinical isolates, the drug combination showed no interaction. The MDR clinical isolate had lower ethidium bromide (EtBr) accumulation when exposed to the RIF+VP combination, compared with RIF and VP exposure alone. The other MDR clinical isolates showed no significant difference in EtBr accumulation. These results suggest greater efflux action in one of the MDR clinical isolates compared with the M. tuberculosis H₃₇Rv reference strain. The other five MDR isolates may have additional mechanisms of drug resistance to RIF. The use of the RIF+VP combination made one MDR bacillus more susceptible to RIF probably by inhibiting efflux pumps, and this combination therapy, in some cases, may contribute to a reduction of resistance to RIF in M. tuberculosis.     

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.     

Molecular identification of Korean Trichinella isolates

Muscle larvae of Trichinella isolates from two outbreaks in Korea were analyzed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiple-PCR. All of the muscle larvae showed a band similar to that of T. spiralis larvae of the reference strain. The two Korean Trichinella isolates (isolate code ISS623 and ISS1078) might be classifiable to Trichinella spiralis.     

A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.     

Isolation of free-living dinitrogen-fixing bacteria and their activity in compost containing de-inking paper sludge

Knowledge of the microbiology of dinitrogen (N2)-fixing bacteria in compost rich in de-inking paper sludge (DPS) is limited. Dinitrogen (N2)-fixing bacteria from DPS composts were isolated and studied for their N2-fixing activity in vitro and in vivo. Two Gram-negative N2-fixing isolates were identified as Pseudomonas. At 20 degrees C, both isolates revealed that N2-fixing activity was higher than that of three arctic Pseudomonas strains. Their N2-fixing activity was found to occur between 18 and 25 degrees C, a pattern that was similar to the reference isolate Azotobacter ATCC 7486. Composts successfully showed N2-fixing activity after carbohydrate amendments both with and without inoculation of a N2-fixing isolate. These results suggest that DPS composts support N2-fixing bacteria and that N2-fixing activity is dependent on a usable carbohydrate source.     

Proteolytic activity of rumen fungi belonging to the genera Neocallimastix and Piromyces

Proteolytic activity of two rumen fungal isolates Neocallimastix sp. strain N1 and Piromyces sp. strain P1 was examined. Proteases are active between pH 6.5 and 9.0 with maximum at 7.9 for isolate N1 and between 6.5 and 10.5 with maximum at 8.8 for isolate P1. Proteolytic activity increased as temperature increased until 50°C and a sudden decrease at 60°C was observed in both isolates. EDTA, 1,10-phenanthroline, p -chloromercuribenzoate (PCMB), merthiolate and phenylmethylsulphonyl fluoride (PMSF) were effective proteolytic inhibitors against both isolates.     

Comparative Study of Six Strains of Naegleria with Special Reference to Nonpathogenic Variants of Naegleria fowleri*

SYNOPSIS. Naegleria fowleri strains HB-1 and KUL, pathogenic for humans, Naegleria gruberi strain 1518/1e, and 3 strains (Vm1, LvH₁, and LvH₂) of Naegleria isolated from a body of water polluted with thermal effluents were compared in an attempt at specific identifications of the latter strains. The 3 environmental isolates were morphologically almost identical with N. fowleri and had almost the same temperature tolerance, although at 37 and 42 C the growth rates of LvH₁ and LvH₂ were higher than those of the human pathogen, N. fowleri, and of isolate Vm1, which was pathogenic for mice. Serologic examinations by indirect fluorescent antibody method revealed a very close relationship of the new isolates with the human pathogens. While Vm1 was indistinguishable from N. fowleri, LvH₁ and LvH₂ were not, when cross-absorbed antisera were used. Of all the strains examined, only the 2 LvH isolates were not inhibited by amphotericin B, while only N. gruberi was not inhibited by fumagillin. The cytopathic effect in Vero cell cultures suggested that the LvH strains could have a certain degree of virulence, although this was not confirmed by intranasal and intracerebral inoculations of mice. The cytopathic effects of the human pathogens and of the isolate pathogenic for mice were related to their virulence for mice. It is concluded that there exists an intermediate form between N. gruberi and N. fowleri, with a strong relationship to the latter species. We refer to such strains as nonpathogenic variants of N. fowleri. Further research is needed to reveal their place in the taxonomy.     

猪Ⅱ型圆环病毒四川分离株鉴定及其ORF2基因序列分析

猪圆环病毒(Porcine circovirus,PCV),属于圆环病毒科(Circoviridae),圆环病毒属(Circovirus),血清型为PCV-1和PCV-2[1]。PCV-1首先由Tis-cher[2]于1974年从PK-15猪肾传代细胞系中分离获得,PCV-2首先由Clark[3]报道了是断奶仔猪多系统衰竭综合征(PMWS)的主要病原,随即相继报道了PCV-2与PDNSS(猪皮炎与肾炎综合症)、NP(增生性坏死性肿炎)、PRDC(猪呼吸道综合征)、繁殖障碍、先天性颤抖、肠炎等疾病亦有重要关联[4,5];它常与猪呼吸与繁殖综合征病毒(PRRSV)或猪细小病毒(PPV)并发感染或继发细菌感染[6]。我国自从2000年郎…