MP1 homologue-based multilocus sequence system for typing the pathogenic fungus Penicillium marneffei: a novel approach using lineage-specific genes

A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of Penicillium marneffei, the most important thermal dimorphic fungus causing respiratory, skin, and systemic mycosis in Southeast Asia. The sequences of 11 housekeeping genes were identical among 10 strains of P. marneffei, but those of MP1 and its 13 homologues, a novel superfamily of mannoproteins in the subdivision Pezizomycotina of Ascomycetes, mostly species of Penicillium and Aspergillus, showed significant variations. Therefore, a multilocus sequence typing (MLST) system for P. marneffei was constructed using MP1 (549 bp) and the four of its homologues (MPLP4 [337 bp], MPLP7 [347 bp], MPLP10 [546 bp], and MPLP13 [422 bp]) that showed the greatest variations. Among the 2,201 bp of the five loci, 183 polymorphic sites were observed in 44 strains of P. marneffei. The median number of alleles at each locus was five (range, 5 [MPLP4, MPLP7, and MPLP13] to 15 [MPLP10]). Four of the five genes had nonsynonymous substitution/synonymous substitution (d(n)/d(s)) ratios of >1. A total of 35 different sequence types (STs) were assigned to the 44 P. marneffei isolates, with 28 of the 35 STs identified only once. The discriminatory power was 0.9884. MP1 and its homologues were better than housekeeping genes for MLST in P. marneffei. Due to their more rapid evolutionary rates, lineage-specific genes may be better candidates than housekeeping genes for sequence-based typing, especially in microbes that evolve slowly or have evolved recently.     

Comparative sequence analysis of a polymorphic region of the spike glycoprotein S1 subunit of enteric bovine coronavirus isolates

Summary Complementary oligonucleotide primers which flank a 1146-nucleotide gene fragment (S1B: nt 1185 to 2333) encompassing a polymorphic region (nt 1368 to 1776) of the S1 subunit of bovine coronavirus spike glycoprotein were used for enzymatic amplification by PCR. We chose four clinical isolates, recovered from cases of epidemic diarrhea in neonatal calves in Québec dairy herds between 1987–1990, to specifically amplify and analyze their sequences in the selected genomic area. Nucleotide sequence analysis of the four clinical isolates indicated that their S1B gene fragments were highly conserved. We also compared the S1B gene sequences of the Québec BCV isolates to the published corresponding sequences from BCV-L9 [37], BCV-MEB [1], and BCV-F15 [3] reference strains. A high degree of similarity was demonstrated for all viruses, no deletions or insertions were observed, and the only variations that were identified consisted of nucleotide substitutions. The differing nucleotides and amino acids (aa) were not distributed randomly over the entire sequence but rather were clustered in the polymorphic region. Of these, four sporadic aa changes were located in antigenic domain II (aa residues 517 to 720) of S1. This correlates with varied antigenicity observed among the BCV Québec isolates when reacting with MAbs directed against the S glycoprotein of the Mebus strain. The other mutations seem to be fixed in all Québec isolates.     

Molecular Typing of Penicillium marneffei Isolates from Thailand by NotI Macrorestriction and Pulsed-Field Gel Electrophoresis

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.     

Analysis of polymorphic microsatellite markers for typing Penicillium marneffei isolates

Penicillium marneffei is an emerging opportunistic dimorphic fungal pathogen that is endemic in Southeast Asia. A typing method based on the analysis of size polymorphisms in microsatellite loci was investigated. Three loci available from the GenBank database were identified to harbor microsatellites. PCR primers flanking the microsatellite repeats were designed with one primer in the set fluorescently labeled. PCR products were then sized by automated capillary electrophoresis. As expected for a haploid fungus, a single band was observed for each microsatellite locus for all isolates. Polymorphic microsatellite marker (PMM) analysis detected a total of 22 different allelic types for 35 isolates of P. marneffei with a high discriminatory power (D = 0.956). Microsatellites I, II, and III detected 14, 10, and 7 alleles, respectively. The reproducibility of length polymorphisms was confirmed by using different DNA preparations from the same isolate or by repeated runs from the same DNA preparation. PMM profiles for eight isolates passaged in vitro for 7 to 8 weeks were identical to the original culture, demonstrating short-term stability and reproducibility. PCR products were not observed for other dimorphic fungi or human DNA. Comparison of allelic frequencies in isolates obtained from China and Thailand identified distinct allele combinations, suggesting the potential geographic isolation of populations. Due to the high discriminatory power, reproducibility, and potential for high throughput, PMM analysis may provide a good typing method for epidemiologic and surveillance investigations of P. marneffei.     

Extensive allelic variation in Cryptococcus neoformans.

The orotidine monophosphate pyrophosphorylase (OMPPase) gene locus of the DNA of 13 Cryptococcus neoformans var. neoformans strains, including 10 recent clinical isolates, was studied by using restriction fragment length polymorphisms and nucleotide sequence analysis. The OMPPase locus (URA5) is highly polymorphic, and at least six alleles were identified. The nucleotide sequences of some alleles differed by up to 5%. The majority of the nucleotide polymorphisms in the protein-coding region occurred at the third codon position and were silent. The low frequency of replacement nucleotide substitutions relative to silent nucleotide substitutions implied that there is strong selection against amino acid changes in OMPPase. The allelic variation suggested that there is extensive genomic diversity among C. neoformans clinical isolates from one geographic area. The various alleles are potentially useful markers in the study of the population structure, epidemiology, and pathogenesis of C. neoformans strains.     

Human cytomegalovirus a sequence and UL144 variability in strains from infected children

Human cytomegalovirus (HCMV) displays genetic polymorphisms. This variability may contribute to strain-specific tissue tropism and disease expression in HCMV-infected humans. To determine strain variability in a sequence and UL144 gene regions, 51 low-passage isolates from 44 HCMV-infected children were studied. Isolates were obtained from 28 healthy children attending child care centers in Iowa and from 16 congenitally infected infants born in Texas. Isolates demonstrated substantial nucleotide variation in each gene region. Phylogenetic analysis of a sequence variability allowed 39 isolates to be grouped into six clades. The largest clade contained 16 isolates with > or = 95% nucleotide homology. Forty-eight of the 49 HCMV isolates yielding UL144 amplicons was grouped according to the clades described a few years ago [Lurain et al. (1999) Journal of Virology 73:10040-10050]. No linkage was observed among a sequence, UL144, and glycoprotein B (gB; UL55) polymorphisms. Four Texas and 11 Iowa isolates displayed > or = 95% sequence homology for a sequence and UL144 regions and possessed identical gB genotypes. No relationship between UL144 polymorphisms and outcome of congenital HCMV infection was observed. These data indicate that HCMV strains circulating among young children have UL144 polymorphisms similar to those of HCMV strains excreted by immunocompromised adults. Identification of conserved nucleotide sequences among Iowa and Texas children suggests genetic stability and biologic importance of these gene regions.     

Characterization of human symptomatic rotavirus isolates MP409 and MP480 having ‘long’ RNA electropherotype and subgroup I specificity, highly related to the P6[1],G8 type bovine rotavirus A5, from Mysore, India

Summary.  In an epidemiological study of symptomatic human rotaviruses in Mysore, India during 1993 and 1994, isolates MP409 and MP480 were isolated from two children suffering from severe, acute dehydrating diarrhea. Both isolates exhibited ‘long’ RNA pattern and subgroup I specificity suggesting the likelihood of their animal origin. Both isolates did not react with monoclonal antibodies (MAbs) specific for serotypes G1 to G6 as well as G10. To determine the genetic origin of these isolates, complete nucleotide sequences of genes encoding the outer capsid proteins VP4 and VP7, nonstructural proteins NSP1 and NSP3 and viral enterotoxin protein NSP4 from MP409 and partial sequences of genes from MP480 were determined. Comparison of the 5′ and 3′ terminal sequences of 250 nucleotides revealed complete identity of the gene sequences in both strains suggesting that MP409 and MP480 are two different isolates of a single strain. Comparison of the nucleotide and deduced amino acid sequences of VP4, VP7, NSP1 and NSP3 of MP409 with published sequences of strains belonging to different serotypes revealed that both outer capsid proteins VP4 and VP7 and NSP1 are highly related to the respective proteins from the P6[1], G8 type bovine rotavirus A5 isolated from a calf with diarrhoea in Thailand and that the NSP3 is highly homologous to that of bovine rotaviruses. The NSP4 protein showed greatest sequence identity with NSP4s belonging to the KUN genetic group to which NSP4s from human G2 type strains and bovine rotaviruses belong. MP409 and MP480 likely signify interspecies transmission of P6[1], G8 type strains from cattle to humans and represent the first P6[1] type rotaviruses isolated in humans. These and our previous studies on the asymptomatic neonatal strain I321 are of evolutionary and epidemiological significance in the context of close association of majority of the Indian population with cattle. Received September 29, 1999 Accepted February 4     

Variation in the spacer regions separating tRNA genes in Renibacterium salmoninarum distinguishes recent clinical isolates from the same location

A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles.     

Multilocus sequence typing is a reliable alternative method to DNA fingerprinting for discriminating among strains of Candida albicans

Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for Candida albicans have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of C. albicans, we applied it to a previously well-characterized set of 29 C. albicans isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques.     

Evaluation of internal transcribed spacer region of ribosomal DNA sequence analysis for molecular characterization of Candida albicans and Candida dubliniensis isolates from HIV-infected patients.

Molecular typing systems have been needed to study Candida colonization in HIV-infected patients, particularly for investigating virulence and fluconazole resistance. Three methods--electrophoretic karyotyping (EK), detection of restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNA analysis (RAPD)--have been most frequently used. In this study, comparative sequence analysis of the internal transcribed spacer (ITS) region of rDNA was evaluated for delineation of Candida isolates from 14 HIV-infected patients. EK, ITS sequence analysis, RFLP and RAPD resulted in 11, 10, 9 and 8 DNA genotypes, respectively, from 39 Candida albicans isolates. The 10 genotypes observed using ITS sequence analysis were defined by six variation sites in the sequence. Molecular typing of sequential oral isolates showed the persistence of the same genotype of C. albicans in nine patients, and genotype variation in one patient. EK and RAPD showed that another patient was co-infected by two distinct genotypes and ITS analysis identified one of the two genotypes as Candida dubliniensis. Comparative ITS sequence analysis is a quick and reproducible method that provides clear and objective results, and it also identifies C. dubliniensis. The discriminatory power of this new typing approach could be improved by concomitant analysis of other DNA polymorphic sequences.     

Sequence divergency of the cytadhesin gene of Mycoplasma pneumoniae.

The Mycoplasma pneumoniae cytadhesin P1 genes from two groups of clinical isolates that display restriction fragment length polymorphisms were cloned and sequenced. Within each group the nucleotide sequences were identical, but two major differences were detected between the groups. These two stretches of sequence divergence were located in multiple-copy regions of the P1 gene and resulted in considerable amino acid changes.     

Comparison of protein A gene sequencing with pulsed-field gel electrophoresis and epidemiologic data for molecular typing of methicillin-resistant Staphylococcus aureus

The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.     

Listeria monocytogenes isolates can be classified into two major types according to the sequence of the listeriolysin gene

The nucleotide sequence of a 3.5-kb BamHI fragment from Listeria monocytogenes 12067, a human clinical isolate of serotype 4b, has been determined. The DNA fragment harbors the gene for listeriolysin, part of the gene for a phosphatidylinositol-specific phospholipase C, and part of the gene for a metalloprotease. Comparison of the sequence with corresponding sequences from two other L. monocytogenes isolates revealed a significant number of nucleotide differences. Several of the differences give rise to amino acid substitutions. The most variable region was the examined part of the mpl gene, whereas the lisA gene showed a relatively high degree of conservation, particularly at the amino acid level. To analyze the pattern of sequence variability in the lisA gene, a 160-bp region covering nine nucleotide differences was sequenced from 36 isolates of different origins. This work showed that the strains can be grouped into two major types according to the nucleotide sequences. Oligonucleotide probing of a larger number of L. monocytogenes isolates showed that the observed differences can be used to subdivide the species. The data suggest a correspondence between the sequence type of the lisA gene and flagellar antigens. Assays based on hybridization or the polymerase chain reaction with type-specific oligonucleotides may provide fast and easy alternative methods for strain typing.     

Restriction endonuclease analysis of Penicillium marneffei.

Forty-six isolates of Penicillium marneffei were differentiated into two DNA types on the basis of their restriction fragment length polymorphisms. Of the 22 human isolates of P. marneffei, 16 (72.7%) were type I and 6 (27.3%) were type II. Of the 23 bamboo rat isolates, 20 from Rhizomys sumatrensis were type I and 3 from Cannomys badius were type II. The soil isolate was type II. These data represent the first molecular epidemiological study of this important emerging fungal pathogen.     

Analysis of the diversity of African streak mastreviruses using PCR-generated RFLPs and partial sequence data

Maize streak virus (MSV) is the most economically significant member of a diverse group of African grass-infecting Mastrevirus species in the family Geminiviridae. We designed a single set of degenerate primers which enables the PCR amplification of an approximately 1300 bp DNA fragment spanning both conserved (the RepA gene) and variable (the long intergenic region and MP gene) portions of these viruses' genomes. Using restriction fragment length polymorphism (RFLP) analysis of PCR products obtained from 39 MSV, one SSV, and two PanSV isolates, it was possible to both identify the different virus species, which differ in nucleotide sequence by up to 40%, and to differentiate between MSV isolates sharing up to 99% sequence identity. The reliability of the RFLP data for typing the MSV isolates was verified by the phylogenetic analysis of the partial genomic nucleotide sequences of a representative subset of the MSV isolates. Based on both the RFLP and sequence data, the MSV isolates could be clearly differentiated into the four groups: these were a group of predominantly maize-infecting isolates, and three groups containing grass/wheat-infecting isolates. RFLP analysis also revealed a number of mixed virus infections in which, in certain instances, it was possible to identify individual population members.     

Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci.

Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.     

Identification of a polymorphic nucleotide in oxyR specific for Mycobacterium bovis.

Automated sequence analysis of a 410-bp region of the axyR gene in 105 Mycobacterium tuberculosis complex isolates identified a polymorphic nucleotide that differentiated Mycobacterium bovis isolates from other complex members. All 29 M. bovis isolates sequenced had an adenine residue at nucleotide 285, whereas all 76 other complex isolates had a guanine residue. PCR-restriction fragment length polymorphism analysis of oxyR with restriction endonuclease AluI in an additional 255 complex isolates from widespread intercontinental sources confirmed and extended the unique association of adenine at position 285 with M. bovis isolates.     

Optimization and validation of multilocus sequence typing for Candida albicans

Multilocus sequence typing (MLST) was applied to 75 Candida albicans isolates, including 2 that were expected to be identical, 48 that came from diverse geographical and clinical sources, and 15 that were sequential isolates from two patients. DNA fragments ( approximately 500 bp) of eight genes encoding housekeeping functions were sequenced, including four that have been described before for C. albicans MLST, and four new gene fragments, AAT1a, AAT1b, MPI, and ZWF1. In total, 87 polymorphic sites were found among 50 notionally different isolates, giving 46 unique sequence types, underlining the power of MLST to differentiate isolates for epidemiological studies. Additional typing information was obtained by detecting variations in size at the transcribed spacer region of the 25S rRNA gene and tests for homozygosity at the mating type-like (MTL) locus. The stability of MLST was confirmed in two sets of consecutive isolates from two patients. In each set the isolates were identical or varied by a single nucleotide. Reference strain SC5314 and a derived mutant, CAF2, gave identical MLST types. Heterozygous polymorphisms were found in at least one isolate for all but 16 (18.4%) of the variable nucleotides, and 35 (41%) of the 87 individual sequence changes generated nonsynonymous amino acids. Cloning and restriction digestion of a gene fragment containing heterozygous polymorphisms indicated that the heterozygosity was genuine and not the result of sequencing errors. Our data validate and extend previous MLST results for C. albicans, and we propose an optimized system based on sequencing eight gene fragments for routine MLST with this species.     

抗肌营养不良蛋白基因内MP1P位点多态性分析及其应用

应用银染聚丙烯酰胺凝胶法，对抗肌营养不良蛋白基因３＇非翻译区ＭＰ１Ｐ位点进行了扩增片段长度多态性分析。此位点在中国人中有多态性，共检出３个等位片段，汉族人群中３个等位片段的频率分别为０．１４１、０、７０３、０．１５６，ＰＩＣ为０．４６２，回族人群中的频率分别为０．１８２、０．５９１、０．２２７，ＰＩＣ为０．５６６。此位点与３＇不翻译区的３＇ＣＡ位点存在连锁不平衡性，在连锁分析中可替代３＇ＣＡ作为基