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PubMed2005

Seven novel variants of the staphylococcal chromosomal cassette mec in methicillin-resistant Staphylococcus aureus isolates from Ireland.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in Irish hospitals between 1971 and 2002 were characterized using multilocus sequence typing (MLST) (n = 130) and SCCmec typing (n = 172). Where atypical SCCmec typing results were obtained, PCR amplification of entire SCCmec elements, analysis of amplimer mobility, and nucleotide sequencing were undertaken. MLST revealed that 129/130 isolates had the same genotypes as internationally spread MRSA clones, including ST239, ST247, ST250, ST5, ST22, ST36, and ST8. A novel genotype, ST496, was identified in one isolate. Half of the isolates (86/172) had SCCmec type I, IA, II, III, or IV. The remaining 86 isolates harbored novel SCCmec variants in three distinct genetic backgrounds: (i) 74/86 had genotype ST8 and either one of five novel SCCmec II (IIA, IIB, IIC, IID, and IIE) or one of two novel SCCmec IV (IVE and IVF) variants; (ii) 3/86 had genotype ST239 and a novel SCCmec III variant; (iii) 9/86 had a novel SCCmec I variant associated with ST250. SCCmec IVE and IVF were similar to SCCmec IVc and IVb, respectively, but differed in the region downstream of mecA. The five SCCmec II variants were similar to SCCmec IVb in the region upstream of the ccr complex but otherwise were similar to SCCmec II, except for the following regions: SCCmec IIA and IID had a novel mec complex, A.4 (Delta mecI-IS1182-Delta mecI-mecR1-mecA-IS431mec); SCCmec IIC and IIE had a novel mec complex, A.3 (IS1182-Delta mecI-mecR1-mecA-IS431mec); SCCmec IID and IIE lacked pUB110; SCCmec IIC and IIE lacked a region of DNA between Tn554 and the mec complex; and SCCmec IIB lacked Tn554. This study has demonstrated a hitherto-undescribed degree of diversity within SCCmec.

Bacterial Proteins↗

PubMed2005

High genetic diversity of ciprofloxacin-nonsusceptible isolates of Streptococcus pneumoniae in Poland.

We have analyzed the susceptibility to ciprofloxacin of 697 pneumococcal isolates collected in 1998-2002 in Poland from patients with respiratory tract diseases. Thirty-one ciprofloxacin-nonsusceptible isolates (MICs, > or =4 microg/ml) were identified, of which two were resistant to levofloxacin (MIC, 8 microg/ml). Serotyping, pulsed-field gel electrophoresis, multilocus sequence typing, and the analysis of resistance determinants showed their great genetic diversity.

Anti-Infective Agents↗

PubMed2005

Inducible clindamycin resistance and molecular epidemiologic trends of pediatric community-acquired methicillin-resistant Staphylococcus aureus in Dallas, Texas.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection occurs commonly in children. Clindamycin resistance may be inducible or constitutive, and the rates of inducible resistance in CA-MRSA that could produce clindamycin treatment failures vary worldwide. The double-disk test was performed in 197 erythromycin-resistant and clindamycin-susceptible CA-MRSA strains from children in Dallas, Texas, from 1999 to 2002 to determine inducible clindamycin resistance. Resistance mechanisms were studied by PCR; epidemiologic trends were studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Inducible resistance was demonstrated in 28 (93%+/-6%) of 30 tested isolates in 1999, 21 (64%, +/-11%) of 33 in 2000, 12 (23%+/-7%) of 52 in 2001, and 6 (7%+/-3%) of 82 in 2002. All noninducible strains had the msr(A) gene. Among inducible resistant strains, 31 had erm(B), 24 had erm(C), and 12 had erm(A) genes. Two distinct pulsed types were the most prevalent; one of them was the most common pulsed type in 1999, whereas in 2002 a different pulsed type was prevalent. MLST analyses determined that ST-8 was the most common type, with 76%+/-5% found in 2002. All but one of these clindamycin-susceptible, erythromycin-resistant ST-8 strains showed no induction of clindamycin resistance. We conclude that, among erythromycin-resistant, clindamycin-susceptible CA-MRSA strains isolated from children in Dallas, inducible methylase resistance became less common from 1999 to 2002 (P<0.001). The phenotype of strains was associated with their sequence type. Our results demonstrate a clonal shift in CA-MRSA in Dallas children from 1999 to 2002.

Adolescent↗

PubMed2005

Population structure of Enterococcus faecium causing bacteremia in a Spanish university hospital: setting the scene for a future increase in vancomycin resistance?

Over an 8-year period (1995 to 2002), 86 Enterococcus faecium blood isolates from 84 patients, of which 54 were ampicillin resistant (AREF) and 32 were ampicillin susceptible (ASEF), were studied in a university hospital (1,200 beds; serving a population of 600,000) in Spain, a country characterized by a near-absence of resistance to vancomycin and very high rates of ampicillin resistance among enterococci. Clonal relatedness by pulsed-field gel electrophoresis (PFGE), antibiotic susceptibility, presence of the virulence/epidemicity genes esp(Efm) and hyl(Efm), and identification of purK alleles were studied. A group of isolates was also analyzed by amplified fragment length polymorphism (AFLP) and multilocus sequence typing. Medical charts (30 variables collected) were reviewed for 60/84 patients. ASEF showed high clonal diversity (32 PFGE types, 11 purK alleles, 4 AFLP genogroups), did not harbor putative virulence genes, and had no specific association with hospital acquisition. AREF isolates belonged to a clonal complex (CC) of genetically related strains (purK-1, AFLP genogroup C), occasionally harboring putative virulence traits, and were from patients with particular risk factors. Within this CC, previously associated with vancomycin-resistant E. faecium isolates causing outbreaks worldwide (W. L. Homan et al., J. Clin. Microbiol. 40:1963-1971, 2002), a great genetic diversity of antibiotic resistance and virulence/epidemicity profiles was found. Associations between esp and a >7-day hospital stay and between purK-1, hospital location, and nosocomial acquisition were noted (P < 0.001). These findings reflect the importance of local environmental differences in the evolution of this CC, suggesting that the emergence of vancomycin resistance among AREF strains in Spain may be a question of time.

Ampicillin↗

PubMed2005

Genotypic diversity of methicillin-resistant Staphylococcus aureus isolates in Korean hospitals.

Ninety-six methicillin-resistant Staphylococcus aureus (MRSA) isolates from eight Korean hospitals were analyzed by multilocus sequence typing, SCCmec typing, and spa typing. The predominant genotype was ST5-MRSA-II of clonal complex 5, which was found in 36 isolates from six hospitals, but ST239-MRSA-III was also common. Overall, results showed a notable genotypic diversity of MRSA strains circulating in Korean hospitals.

Bacterial Proteins↗

PubMed2006

Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies.

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with "Minimum SNPs" software. "Minimum SNPs" selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D=1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.

Animals↗

PubMed2003

Genetic diversity of Campylobacter jejuni isolates from farm animals and the farm environment.

The genetic diversity of Campylobacter jejuni isolates from farm animals and their environment was investigated by multilocus sequence typing (MLST). A total of 30 genotypes, defined by allelic profiles (assigned to sequence types [STs]), were found in 112 C. jejuni isolates originating in poultry, cattle, sheep, starlings, and slurry. All but two of these genotypes belonged to one of nine C. jejuni clonal complexes previously identified in isolates from human disease and retail food samples and one clonal complex previously associated with an environmental source. There was some evidence for the association of certain clonal complexes with particular farm animals: isolates belonging to the ST-45 complex predominated among poultry isolates but were absent among sheep isolates, while isolates belonging to the ST-61 and ST-42 complexes were predominant among sheep isolates but were absent from the poultry isolates. In contrast, ST-21 complex isolates were distributed among the different isolation sources. Comparison with MLST data from 91 human disease isolates showed small but significant genetic differentiation between the farm and human isolates; however, representatives of six clonal complexes were found in both samples. These data demonstrate that MLST and the clonal complex model can be used to identify and compare the genotypes of C. jejuni isolates from farm animals and the environment with those from retail food and human disease.

Alleles↗

PubMed2003

Comparative genomic analyses of the vibrio pathogenicity island and cholera toxin prophage regions in nonepidemic serogroup strains of Vibrio cholerae.

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTXphi), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIphi. In order to determine the prevalence of horizontal transfer of VPI and CTXphi among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI(+) strains have also acquired the CTXphi. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTXphi prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTXphi prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXphi independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.

Amino Acid Sequence↗

PubMed2004

Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes.

Oenococcus oeni is the organism of choice for promoting malolactic fermentation in wine. The population biology of O. oeni is poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with the gyrB, pgm, ddl, recP, and mleA genes the genetic diversity and genetic relationships among 18 O. oeni strains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure in O. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity of O. oeni. A low standardized index of association value indicated that the O. oeni genes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method for O. oeni and the first example of the application of MLST to a nonpathogenic food production bacteria.

Alleles↗

PubMed2004

Evolution of the core genome of Pseudomonas syringae, a highly clonal, endemic plant pathogen.

Pseudomonas syringae is a common foliar bacterium responsible for many important plant diseases. We studied the population structure and dynamics of the core genome of P. syringae via multilocus sequencing typing (MLST) of 60 strains, representing 21 pathovars and 2 nonpathogens, isolated from a variety of plant hosts. Seven housekeeping genes, dispersed around the P. syringae genome, were sequenced to obtain 400 to 500 nucleotides per gene. Forty unique sequence types were identified, with most strains falling into one of four major clades. Phylogenetic and maximum-likelihood analyses revealed a remarkable degree of congruence among the seven genes, indicating a common evolutionary history for the seven loci. MLST and population genetic analyses also found a very low level of recombination. Overall, mutation was found to be approximately four times more likely than recombination to change any single nucleotide. A skyline plot was used to study the demographic history of P. syringae. The species was found to have maintained a constant population size over time. Strains were also found to remain genetically homogeneous over many years, and when isolated from sites as widespread as the United States and Japan. An analysis of molecular variance found that host association explains only a small proportion of the total genetic variation in the sample. These analyses reveal that with respect to the core genome, P. syringae is a highly clonal and stable species that is endemic within plant populations, yet the genetic variation seen in these genes only weakly predicts host association.

Base Sequence↗

PubMed2004

Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens.

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.

Animals↗

PubMed2005

Comparison of minisatellite polymorphisms in the Bacillus cereus complex: a simple assay for large-scale screening and identification of strains most closely related to Bacillus anthracis.

Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.

Animals↗

PubMed2005

Phylogenetic characterization of virulence and resistance phenotypes of Pseudomonas syringae.

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.

Adaptation, Physiological↗

PubMed2006

Prevalence, persistence, and molecular characterization of glycopeptide-resistant enterococci in Norwegian poultry and poultry farmers 3 to 8 years after the ban on avoparcin.

Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant Enterococcus faecium (GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn1546-plasmid junction fragment in 93.9% of E. faecium isolates. A putative postsegregation killing (PSK) system linked to Tn1546 was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible (n = 10) and -resistant (n = 10) E. faecium isolates from humans (n = 10) and poultry (n = 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated vanA-pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.

Animal Husbandry↗

PubMed2006

Sources of Campylobacter spp. colonizing housed broiler flocks during rearing.

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.

Animal Husbandry↗

PubMed2006

Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis.

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.

Bacillus anthracis↗

PubMed2006

Genetic diversity and quinolone resistance in Campylobacter jejuni isolates from poultry in Senegal.

We used the multilocus sequence typing (MLST) method to evaluate the genetic diversity of 46 Campylobacter jejuni isolates from chickens and to determine the link between quinolone resistance and sequence type (ST). There were a total of 16 ST genotypes, and the majority of them belonged to seven clonal complexes previously identified by using isolates from human disease. The ST-353 complex was the most common complex, whereas the ST-21, ST-42, ST-52, and ST-257 complexes were less well represented. The resistance phenotype varied for each ST, and the Thr-86-Ile substitution in the GyrA protein was the predominant mechanism of resistance to quinolone. Nine of the 14 isolates having the Thr-86-Ile substitution belonged to the ST-353 complex. MLST showed that the emergence of quinolone resistance is not related to the diffusion of a unique clone and that there is no link between ST genotype and quinolone resistance. Based on silent mutations, different variants of the gyrA gene were shown to exist for the same ST. These data provide useful information for understanding the epidemiology of C. jejuni in Senegal.

Animals↗

PubMed2005

Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus.

In a prior study, we identified seven clinical isolates of an Aspergillus sp. that were slow to sporulate in multiple media and demonstrated decreased in vitro susceptibilities to multiple antifungals, including amphotericin B, itraconazole, voriconazole, and caspofungin. These isolates were initially considered to be variants of Aspergillus fumigatus because of differences in mitochondrial cytochrome b sequences and unique randomly amplified polymorphic DNA PCR patterns (S. A. Balajee, M. Weaver, A. Imhof, J. Gribskov, and K. A. Marr, Antimicrob. Agents Chemother. 48: 1197-1203, 2004). The present study was performed to clarify the taxonomic status of these organisms by phylogenetic analyses based on multilocus sequence typing of five genes (the beta-tubulin gene, the rodlet A gene, the salt-responsive gene, the mitochondrial cytochrome b gene, and the internal transcribed spacer regions). Results revealed that four of the seven variant isolates clustered together in a clade very distant from A. fumigatus and distinct from other members of the A. fumigatus group. This new clade, consisting of four members, was monophyletic with strong bootstrap support when the protein-encoding regions were analyzed, indicating a new species status under the phylogenetic species concept. Phenotype studies revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48 degrees C. Our findings suggest the presence of a previously unrecognized, potentially drug-resistant Aspergillus species that we designate A. lentulus.

Aspergillus↗