Molecular epidemiologic analysis of a Pneumocystis pneumonia outbreak among renal transplant patients.

Between 18 November and 3 December 2011, five renal transplant patients at the Department of Nephrology, Toho University Omori Medical Centre, Tokyo, were diagnosed with Pneumocystis pneumonia (PCP). We used molecular epidemiologic methods to determine whether the patients were infected with the same strain of Pneumocystis jirovecii. DNA extracted from the residual bronchoalveolar lavage fluid from the five outbreak cases and from another 20 cases of PCP between 2007 and 2014 were used for multilocus sequence typing to compare the genetic similarity of the P. jirovecii. DNA base sequencing by the Sanger method showed some regions where two bases overlapped and could not be defined. A next-generation sequencer was used to analyse the types and ratios of these overlapping bases. DNA base sequences of P. jirovecii in the bronchoalveolar lavage fluid from four of the five PCP patients in the 2011 outbreak and from another two renal transplant patients who developed PCP in 2013 were highly homologous. The Sanger method revealed 14 genomic regions where two differing DNA bases overlapped and could not be identified. Analyses of the overlapping bases by a next-generation sequencer revealed that the differing types of base were present in almost identical ratios. There is a strong possibility that the PCP outbreak at the Toho University Omori Medical Centre was caused by the same strain of P. jirovecii. Two different types of base present in some regions may be due to P. jirovecii's being a diploid species.

[Murine IgE antibody to recognize human major allergen, Mal f4].

To elucidate the specificity of murine IgE antibody against Malassezia furfur, the immunoblot patterns of IgE in sera obtained from mice inoculated repeatedly in the nasal cavity with M. f. cells, or injected intraperitoneally with M. f. cells mixed with Al (OH)3 gel were compared with those of IgE antibody detected in sera from patients with AD. Most of the murine IgE anti-Malassezia antibodies shared some antigenic bands with IgE in sera from patients with AD. The IgE antibody of murine also recognized Mal f4, one of the major allergens detected in patients with AD.

[Recent progress in molecular diagnosis of deep-seated mycoses].

Diagnosing deep-seated mycoses continues to be a major challenge for the clinician. The non-culture-dependent laboratory assay with high sensitivity and specificity are needed for rapid diagnosis of deep-seated mycoses. Future clinical mycology laboratories will increasingly utilize DNA-based methods for the recognition of pathogenic fungi in patient specimens and for the identification of fungal isolates. Over the last ten years, increasing numbers of papers were published which document the molecular biological methods feasible to detect fungus-specific DNA sequence in clinical specimens. The polymerase chain reaction(PCR) and internal probes are central to these procedures. More recently, the non-isotopic in situ technique has been applied in the detection of pathogenic fungi. These methods have the potential to improve diagnostic accuracy and hasten the institution of specific antifungal therapy. This article will review some of the recent advances in molecular diagnosis of fungal infections.

[Assimilation test of Malassezia furfur isolated from the environment].

The lipophilic yeasts Malassezia species are the causative agents of tinea versicolor and known also to be a member of normal skin flora. They are commonly isolated from the skin of humans and animals, but not from the environment. This is the first report of the isolation of Malassezia sp. from the environment (a hospital floor). The results of assimilation tests of lipids and karyotyping showed that these isolates were M. furfur. They assimilated not only lipids including floor wax and car wax but also some ointments (except antifungal agents) used clinically. The results suggest that we need to take care when using such ointments to treat skin diseases.

[Taxonomy of genus Malassezia: progress and problems].

The genus Malassezia is composed of lipophilic basidiomycetous yeasts which were recently shown to consist of seven species, one lipid-independent species, M. pachydermatis and six lipid-dependent species, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae. Based on this classification, we will be able to analyze pathogenicity or relationship between Malassezia-related diseases and each species.

Molecular diagnosis and epidemiology of fungal infections.

A variety of methods are utilized for DNA strain subtyping of Candida spp. because no 'gold standard' exists. Random amplified polymorphic DNA (RAPD) or restriction enzyme analysis (REA) are useful to determine the source of an outbreak, but more reproducible and discriminatory methods such as Southern hybridization and pulsed field gel electrophoresis (PFGE) may be required. When applied to some nosocomial Candida infections, multiple strains and species have been identified. Microevolution of yeast species occurs and epidemiologically related isolates may show minor pattern differences, creating uncertainty as to whether they are distinct strains. Approximately 1000 isolates of Aspergillus fumigatus from environmental and clinical sources were typed by REA probed with an A. fumigatus-specific retrotransposon-like sequence. Patients with no symptom of aspergillosis may carry several strains, whereas patients with pulmonary aspergillosis may carry one or two strains; nocosomial transmission of aspergillosis was proven in 39% of the patients studied; any given environmental strain can be infectious; the environmental population of A. fumigatus is extremely diverse and no specific niche was found in the hospital. A PCR assay was designed to target conserved 18S-ribosomal DNA (rDNA) sequences shared by most fungi and a 687 bp product was amplified from 25 medically important fungal species. Studies with blood, cerebrospinal fluid and sputum specimens from patients with mycoses indicated that the PCR assay is more sensitive in diagnosing invasive fungal infections than blood culture methods. More specific identification is obtainable with genus/species-specif c probes designed from within the PCR-amplified sequences for C. albicans, C. krusei, C. lusitaniae, Pneumocystis carinii, Cryptococcus neoformans, Aspergillus/Penicillium spp. and C. glabrata/Saccharomyces cerevisiae. A. fumigatus and A. niger were differentiated by denaturing gradient gel electrophoresis. In situ hybridization (ISH) detected a 648 bp fragment of the 18S rDNA of C. neoformans and a 568 bp fragment of the alkaline proteinase gene of A. fumigatus in tissues from experimentally infected animals. In ISH, the entire process can be automated, making this procedure rapid and easy. The difficulty in establishing a diagnosis of invasive candidiasis has prompted the quest for a clinically useful PCR test for candidaemia. The universal fungal oligonucleotide primer pair, ITS3 and ITS4, amplifies portions of the 5.8S ad 28S rDNA subunits, and the ITS2 region. Although rRNA genes are highly conserved, the ITS regions are distinctive. DNA probes were designed from ITS2 that were specific for 16 different Candida species. Simple, rapid sample preparation was suitable for PCR analysis of BacT/Alert blood culture bottles. Sample preparation, PCR, and EIA detection of the amplicon from five different Candida species was accomplished in 7 h, 2.5 days sooner than by conventional culture methods. As well as saving time, minor yeast species among a major species, or among bacteria, were simultaneously detected. PCR-EIA using a microtitration plate format had sensitivity 10-times greater than that obtained with ethidium bromide-stained agarose gels. Taqman combines in one step PCR, probe hybridization, and fluorescent signal generation. Taqman PCR had sensitivity equivalent to PCR-EIA and required only 5 h, including sample preparation.

[Genetic diagnosis of aspergillosis].

Aspergillosis is an opportunistic infection caused by pathogenic Aspergillus species (spp.) and is a major hazard for immunocompromised patients and even for non-immunocompromised individuals. Clinical diagnosis of aspergillosis, especially invasive pulmonary aspergillosis (IPA) is difficult and is largely presumptive, typically based on spiking fevers not responding to antibiotics in a patient with the risk factors. It is well known that Aspergillus spp. can be only infrequently cultured from clinical specimens, and that the cultural examination is laborious and time-consuming. Moreover, positive culture from bronchoalveolar lavage or sputa is indicative, but not proof of infection. The criterion for diagnosis of pulmonary infection by aspergilli requires repeated isolation of the same species of Aspergillus from respiratory specimens. There have been some successful attempts using serological assays to detect circulating antibodies to Aspergillus spp. in the noninvasive form of the disease, but these are generally negative in an acute phase IPA patient. A currently available serodiagnostic kit, Pastrex Aspergillus is limited in clinical usefulness because of low sensitivity and specificity in spite of being simple and rapid. Contamination of clinical specimens with various saprophytic filamentous fungi other than aspergilli also often give false positive. Diagnostic methods using such molecular biological techniques, as polymerase chain reaction (PCR) have recently been employed to identify DNA from a number of pathogens when diagnostic means are limited. PCR is known as the most sensitive and specific technique by which to detect a specific DNA sequence. In this paper we have reviewed new genetic methods of diagnosing aspergillosis including PCR and in situ hybridization.

Karyotyping by PFGE of clinical isolates of Sporothrix schenckii.

From October 1991 to December 1992 we had eight patients with sporotrichosis at Tsukuba University Hospital in Japan. With 8 strains isolated from these patients, PFGE (pulsed-field gel electrophoresis) analyses were carried out to examine whether the karyotype of S. schenckii is distinguished by our method and whether this molecular approach is a useful means of biotyping of S. schenckii strains. Chromosomes were separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The strains had six to eight chromosomes and a total genome size was approx. 28 Mbp. Although these karyotypes of all the isolates looked closely similar to each other, they were grouped into three types.

Isolation of thermosensitive mutants of Yersinia enterocolitica by transposon insertion.

During bacterial infection, pathogens are exposed to a variety of stimuli, e.g., sudden temperature increase on entering mammalian host or oxidative stress associated with exposure to phagocytes. Yersinia enterocolitica, which is a facultative intracellular bacteria, responds to macrophage phagocytosis by the production of a set of stress proteins; which are also induced by heat shock (Yamamoto et al., 1994, Microbiol. Immunol. 38, 295-300). To examine the role of bacterial stress proteins in the adaptation to environmental changes encountered during infectious processes, we have isolated stress-sensitive mutants from Y. enterocolitica in which mini-Tn10 transposon insertions allow bacterial growth at 28 degrees C but prevent growth at an elevated temperature, 39 degrees C. Eight independent insertions were obtained and preliminarily characterized by Southern blot hybridization and morphological analysis.

Specific detection of Aspergillus and Penicillium species from respiratory specimens by polymerase chain reaction (PCR).

A polymerase chain reaction (PCR) method capable of detecting both Aspergillus fumigatus infections, pulmonary non-fumigatus Aspergillus species (spp.) and Penicillium spp. from clinical specimens was established. The primer pair was designed on the basis of the sequence of the 18S-ribosomal RNA gene of A. fumigatus and P. notatum. A 385 bp product was successfully amplified by this PCR method from all of 12 medically important Aspergillus spp. and Penicillium spp. (38 strains), but not from human, calf, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), any of 14 medically important yeastlike fungal species tested (32 strains) including Candida albicans, several non-albicans Candida and Saccharomyces cerevisiae, Cryptococcus neoformans, Mucor spp. or Pneumocystis carinii. This specificity was subsequently confirmed by Southern hybridization analysis. The established PCR can detect such a small amount as 1 pg of A. fumigatus DNA by staining the PCR product with ethidium bromide. With sputum specimens from clinically diagnosed aspergilloma patients, this PCR technique was demonstrated to be a more sensitive diagnostic method for Aspergillus infections than conventional culture techniques.

Detection of a wide range of medically important fungi by the polymerase chain reaction.

A polymerase chain reaction (PCR) method was developed that was capable of detecting a wide range of medically important fungi from clinical specimens. The primer pair was designed in conserved sequences of 18S-ribosomal RNA genes shared by most fungi. The lower limit of detection of this PCR technique was 1 pg of Candida albicans genomic DNA by ethidium bromide staining and 100 fg after Southern analysis. A 687-bp product was amplified successfully by PCR from all 78 strains of 25 medically important fungal species studies, including Candida spp., Hansenula spp., Saccharomyces cerevisiae, Cryptococcus neoformans, Trichosporon beigelii, Malassezia furfur, Pneumocystis carinii, Aspergillus spp., and Penicillium spp., but not from any strains of Mucor spp., Escherichia coli, or methicillin-resistant Staphylococcus aureus (MRSA), calf thymus or human placenta. This specificity was subsequently confirmed by Southern analysis. PCR analysis of blood specimens collected from mice systemically infected with C. albicans and clinical samples including blood, cerebrospinal fluid and sputum appeared to be a more sensitive diagnostic method for invasive fungal infections than a conventional blood culture technique.

Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity.

Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed.

[Pneumonia caused by Moraxella subgenus Moraxella sp].

Moraxella subgenus Moraxella sp. was isolated in pure culture from the sputum of a 43-year-old male with pneumonia and congestive heart failure due to idiopathic dilated cardiomyopathy. In this case, we concluded that the patient's bacterial pneumonia was caused by M. (M.) sp. based on a Gram stain of the sputum smear and bacterial findings, increased WBC count, and elevated CRP. A chest X-ray revealed right middle, and left upper and middle lobe infiltrates. This Moraxella strain produced a BRO-type beta-lactamase, a carbenicillinase-type enzyme.

cDNA cloning of an aspartic proteinase secreted by Candida albicans.

cDNA of an aspartic proteinase secreted by Candida albicans No. 114 was isolated using the polymerase chain reaction (PCR). The primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA and compared with the structures of Saccharomyces cerevisiae proteinase A and vacuolar aspartyl proteinase of C. albicans. The mature aspartic proteinase consisted of 341 amino acid residues, and was 17.6 and 15.3% identical with the proteinase A and the aspartyl proteinase, respectively. Two active aspartic acid sites and the amino acids near those sites were conserved in the aspartic proteinase. We also showed that there is another gene of aspartic proteinase than that of strain ATCC10231 reported by Hube et al (J. Med. Vet. Mycol. 29 (1991)) in the same C. albicans genome, both in that strain and in No. 114.

Comparison of secretory acid proteinases from Candida tropicalis, C. parapsilosis and C. albicans.

Acid proteinases secreted by Candida tropicalis and C. parapsilosis were newly isolated. Their physico-chemical and enzymatic properties of molecular weight, pH stability, isoelectric points, specific activity, and N-terminal amino acid sequences were determined and compared with those of a C. albicans acid proteinase. The two acid proteinases secreted by C. parapsilosis were found to be new enzymes in their molecular weights. The acid proteinases from C. tropicalis and C. parapsilosis showed lower activity at neutral pH, less resistance to neutral and alkaline pH than that from C. albicans, and a half or a third of the specific activity of the C. albicans enzyme. These differences seemed to be associated with the difference of pathogenesis between Candida species. Of the 31 N-terminal amino acids, the enzymes of these three Candida species revealed 12 homologous amino acids.

Salmonella typhimurium has two homologous but different umuDC operons: cloning of a new umuDC-like operon (samAB) present in a 60-megadalton cryptic plasmid of S. typhimurium.

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The DNA which can restore UV mutability to a umuD44 strain and to a umuC122::Tn5 strain of E. coli has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the cloned DNA potentially encoded proteins with calculated molecular weights of 15,523 and 47,726 and was an analog of the E. coli umuDC operon. We have termed this cloned DNA the samAB (for Salmonella mutagenesis) operon and tentatively referred to the umuDC operon of S. typhimurium LT2 (C. M. Smith, W. H. Koch, S. B. Franklin, P. L. Foster, T. A. Cebula, and E. Eisenstadt, J. Bacteriol. 172:4964-4978, 1990; S. M. Thomas, H. M. Crowne, S. C. Pidsley, and S. G. Sedgwick, J. Bacteriol. 172:4979-4987, 1990) as the umuDCST operon. The samAB operon is 40% diverged from the umuDCST operon at the nucleotide level. Among five umuDC-like operons so far sequenced, i.e., the samAB, umuDCST, mucAB, impAB, and E. coli umuDC operons, the samAB operon shows the highest similarity to the impAB operon of TP110 plasmid while the umuDCST operon shows the highest similarity to the E. coli umuDC operon. Southern hybridization experiments indicated that (i) S. typhimurium LT2 and TA1538 had both the samAB and the umuDCST operons and (ii) the samAB operon was located in a 60-MDa cryptic plasmid. The umuDCST operon is present in the chromosome. The presence of the two homologous but different umuDC operons may be involved in the poor mutability of S. typhimurium by UV and chemical mutagens.

Lipopolysaccharide alteration mediated by the virulence plasmid of Salmonella.

Lipopolysaccharide (LPS) of Salmonella dublin, S. enteritidis, S. typhimurium, S. choleraesuis and their derivative strains was analysed to investigate the correlation between LPS and virulence plasmid of Salmonella. All wild-type strains had smooth type LPS, i.e. LPS with long O-specific polysaccharide. The virulence plasmid-cured strain of S. dublin, C524, exhibited a shorter O-specific chain than its parent strain, 5240. No distinct ladder bands were observed at the high molecular weight region on the SDS-PAGE gel for C524 LPS. By chemical analysis the number of O-repeating unit of C524 LPS was shown to be approximately one. The chain length of O-specific polysaccharide was restored by reintroduction of the virulence plasmid. The alteration of LPS by curing and reintroduction of the virulence plasmid was not observed when other wild-type strains of S. dublin were used. In the case of S. enteritidis, S. typhimurium, and S. choleraesuis, alteration of neither chemical composition nor electrophoretical profile of LPS was detected by curing and reintroduction of the virulence plasmids. Those results suggest that certain factor for regulation of the chain length of O-specific polysaccharide is encoded on the virulence plasmid of S. dublin.

Mutation of Escherichia coli capable of expressing gene(s) for beta-lactamase production of Citrobacter freundii.

A mutation in a chromosomal gene of Escherichia coli, designated reb, acted in trans to increase the expression of the cloned beta-lactamase gene of Citrobacter freundii. The reb gene was located around 99 min. Deletion mutants in the cloned gene(s) which had lost the regulatory region for induction were also constructed.