Multilocus Sequence Research Articles

Nosocomial spread of multidrug-resistant group B streptococci with reduced penicillin susceptibility belonging to clonal complex 1

Multiple group B Streptococcus (GBS) isolates with reduced penicillin susceptibility (PRGBS) were recovered from several patients, hence a probable nosocomial transmission of PRGBS in a hospital setting was suspected. Ten PRGBS recovered from eight patients in a general hospital were characterized. Sequence analysis of genes for penicillin-binding proteins (PBPs) and quinolone resistance-determining regions (QRDRs) of gyrA, gyrB and parC was performed, and the macrolide resistance genes were detected by PCR. Genetic relatedness among the isolates was examined by PFGE and multilocus sequence typing. All the PRGBS had the key amino acid substitution V405A, together with F395L, R433H, H438Y and G648A in PBP 2X and T567I in PBP 2B. A 23S rRNA methylase gene, erm(B), was also found in all 10 PRGBS strains. PFGE analysis revealed considerable genetic relatedness among the isolates. Isolates of pulsotype I were obtained from four patients in ward A and one patient in ward B, while isolates of pulsotypes II and III were obtained from two patients in ward B and one patient in ward C, respectively. Isolates of pulsotype I were resistant to levofloxacin (MIC >8 mg/L) and had the following amino acid substitutions in the QRDRs: S81L in GyrA, E476K in GyrB and S79Y in ParC. However, pulsotype II strains resistant to levofloxacin (MIC 8 mg/L) had no change in GyrA, but changes in GyrB (E476K) and ParC (S79Y). All 10 PRGBS strains belonged to serotype VI and ST458 (where ST stands for sequence type). This is the first description of the nosocomial spread of multidrug-resistant PRGBS strains belonging to the genetic lineage ST458.

Recent Speciation in Three Closely Related Sympatric Specialists: Inferences Using Multi-Locus Sequence, Post-Mating Isolation and Endosymbiont Data

Shifting between unrelated host plants is relatively rare for phytophagous insects, and distinct host specificity may play crucial roles in reproductive isolation. However, the isolation status and the relationship between parental divergence and post-mating isolation among closely related sympatric specialists are still poorly understood. Here, multi-locus sequence were used to estimate the relationship among three host plant–specific closely related flea beetles, Altica cirsicola, A. fragariae and A. viridicyanea (abbreviated as AC, AF and AV respectively). The tree topologies were inconsistent using different gene or different combinations of gene fragments. The relationship of AF+(AC+AV) was supported, however, by both gene tree and species tree based on concatenated data. Post-mating reproductive data on the results of crossing these three species are best interpreted in the light of a well established phylogeny. Nuclear-induced but not Wolbachia-induced unidirectional cytoplasmic incompatibility, which was detected in AC-AF and AF-AV but not in AC-AV, may also suggest more close genetic affinity between AC and AV. Prevalence of Wolbachia in these three beetles, and the endosymbiont in most individuals of AV and AC sharing a same wsp haplotype may give another evidence of AF+(AC+AV). Our study also suggested that these three flea beetles diverged in a relative short time (0.94 My), which may be the result of shifting between unrelated host plants and distinct host specificity. Incomplete post-mating isolation while almost complete lineage sorting indicated that effective pre-mating isolation among these three species should have evolved.

Bacterial Translocation in Abdominal Trauma and Postoperative Infections

Bacterial translocation (BT) describes the passage of bacteria residing into the gastrointestinal tract, through the intestinal mucosa barrier to sterile tissues such as mesenteric lymph nodes (MLN) and other organs. This phenomenon has not been thoroughly studied in patients with trauma to date, and an association between BT and postoperative infection has not been well established so far. MLNs from 36 patients with abdominal trauma were removed during laparotomy and cultured to detect BT. Postoperative infectious complications in these patients were registered, and both phenotypical and molecular typings (through multilocus sequencing) were carried out for microorganisms isolated from MLN and postoperative infection sites. Associations between clinical variables, BT presence, and postoperative infection development were established. BT was detected in 33% of the patients (n = 12). Postoperative infections were present in 22.2% of the patients (n = 8). A significant statistical difference was found between postoperative infections in patients with BT evidence (41.6%), when compared with patients without BT (12.5%; p = 0.047). Bacteria isolated from infection sites were the same as those cultured in MLN in 40% of the cases (n = 2 of 5), allowing us to establish causality between BT and postoperative infection. There is higher risk of BT in trauma patients, and it is associated with a significant increase of postoperative infections. An abdominal trauma index ≥10 was found to be associated with the development of BT. This is the first study describing BT among patients with abdominal trauma, where causality is confirmed at molecular level.

Colonization of Returning Travelers With CTX‐M‐ProducingEscherichia coli

We previously identified foreign travel as a risk factor for acquiring infections due to CTX-M (active on cefotaxime first isolated in Munich) producing Escherichia coli. The objective of this study was to assess the prevalence of extended-spectrum β-lactamase (ESBL)-producing E coli among stool samples submitted from travelers as compared to non-travelers (a non-traveler had not been outside of Canada for at least 6 months before submitting a stool specimen). Once a travel case was identified, the next stool from a non-traveler (not been outside of Canada for at least 6 months) was included and cultured on the chromID-ESBL selection media. Molecular characterization was done using polymerase chain reaction and sequencing for bla(CTX-Ms), bla(TEMs), bla(SHVs), plasmid-mediated quinolone-resistant determinants, O25-ST131, phylogenetic groups, pulsed-field gel electrophoresis (PFGE), and multilocus sequencing typing. A total of 226 individuals were included; 195 (86%) were negative, and 31 (14%) were positive for ESBL-producing E coli. Notably, travelers were 5.2 (95% CI 2.1-31.1) times more likely than non-travelers to have an ESBL-producing E coli cultured from their stool. The highest rates of ESBL positivity were associated with travel to Africa or the Indian subcontinent. Among the 31 ESBL-producing E coli isolated, 22 produced CTX-M-15, 8 produced CTX-M-14, 1 produced CTX-M-8, 12 were positive for aac(6')-Ib-cr, and 8 belonged to clone ST131. Our study confirms that foreign travel, especially to the Indian subcontinent and Africa, represents a major risk for rectal colonization with CTX-M-producing E coli and contributed to the Worldwide spread of these bacteria.

Multilocus phylogenetics show high intraspecific variability within Fusarium avenaceum.

Fusarium avenaceum is a common soil saprophyte and plant pathogen of a variety of hosts worldwide. This pathogen is often involved in the crown rot and head blight of cereals that affects grain yield and quality. F. avenaceum contaminates grain with enniatins more than any species, and they are often detected at the highest prevalence among fusarial toxins in certain geographic areas. We studied intraspecific variability of F. avenaceum based on partial sequences of elongation factor-1 alpha, enniatin synthase, intergenic spacer of rDNA, arylamine N-acetyltransferase and RNA polymerase II data sets. The phylogenetic analyses incorporated a collection of 63 F. avenaceum isolates of various origin among which 41 were associated with wheat. Analyses of the multilocus sequence (MLS) data indicated a high level of genetic variation within the isolates studied with no significant linkage disequilibrium. Correspondingly, maximum parsimony analyses of both MLS and individual data sets showed lack of clear phylogenetic structure within F. avenaceum in relation to host (wheat) and geographic origin. Lack of host specialization indicates no host selective pressure in driving F. avenaceum evolution, while no geographic lineage structure indicates widespread distribution of genotypes that resulted in nullifying the effects of geographic isolation on the evolution of this species. Moreover, significant incongruence between all individual tree topologies and little clonality is consistent with frequent recombination within F. avenaceum.

Molecular epidemiology of extraintestinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospital care settings

To assess the molecular epidemiology and prevalence of antibiotic resistance in Escherichia coli causing urinary tract infections of elderly patients from community and hospital settings. Also, to determine whether the possession of antibiotic resistance and virulence-associated genes can be linked to patient location or the clonal group of the organisms in question. E. coli were isolated from the urine samples of elderly patients from the Nottingham area, and subjected to antibiotic susceptibility testing, virulence gene detection by PCR and multilocus sequence typing. No correlation was observed between community- or hospital-derived strains with regard to antibiotic resistance levels or virulence gene profiles. E. coli ST131 (where ST stands for sequence type) was the predominant ST found in both hospital and community samples, and demonstrated high levels of antibiotic resistance to the test panel, but did not possess a significantly larger array of virulence genes or a specific gene profile compared with other STs. The level of antibiotic resistance or virulence gene possession in uropathogenic E. coli is not directly associated with the healthcare setting of the patient, but there is a variation in antibiotic resistance and virulence gene possession depending on clonal group. ST131 is highly virulent and demonstrates high levels of antibiotic resistance, but its virulence does not appear to be attributable to the possession of a specific virulence-associated gene set or the possession of any virulence-associated gene in significantly higher levels than in any other ST.

First report of Fusarium oxysporum causing potato dry rot in Solanum tuberosum in Colombia

Fusarium oxysporum has a worldwide distribution and is responsible for vascular wilt or root rot in many plants. Strains are classified into formae speciales based on their high degree of host specificity, for which multilocus sequence…

Clonal Differences between Non-Typhoidal Salmonella (NTS) Recovered from Children and Animals Living in Close Contact in The Gambia

BackgroundNon-Typhoidal Salmonella (NTS) is an important cause of invasive bacterial disease and associated with mortality in Africa. However, little is known about the environmental reservoirs and predominant modes of transmission. Our study aimed to study the role of domestic animals in the transmission of NTS to humans in rural area of The Gambia.MethodologyHuman NTS isolates were obtained through an active population-based case-control surveillance study designated to determine the aetiology and epidemiology of enteric infections covering 27,567 Gambian children less than five years of age in the surveillance area. Fourteen children infected with NTS were traced back to their family compounds and anal swabs collected from 210 domestic animals present in their households. Identified NTSs were serotyped and genotyped by multi-locus sequencing typing.Principal FindingsNTS was identified from 21/210 animal sources in the households of the 14 infected children. Chickens carried NTS more frequently than sheep and goats; 66.6%, 28.6% and 4.8% respectively. The most common NTS serovars were S. Colindale in humans (21.42%) and S. Poona in animals (14.28%). MLST on the 35 NTS revealed four new alleles and 24 sequence types (ST) of which 18 (75%) STs were novel. There was no overlap in serovars or genotypes of NTS recovered from humans or animal sources in the same household.ConclusionOur results do not support the hypothesis that humans and animals in close contact in the same household carry genotypically similar Salmonella serovars. These findings form an important baseline for future studies of transmission of NTS in humans and animals in Africa.

Trends of invasive serotype 6C pneumococci in Spain: emergence of a new lineage

To analyse the epidemiology of isolates of serotype 6C among invasive pneumococci isolated from children and adults in Spain between 1997 and 2009, and to characterize serotype 6C clones and macrolide and quinolone resistance mechanisms. Antimicrobial susceptibility was determined following CLSI guidelines. Phenotypic characterization of macrolide-resistant isolates was performed by the double disc diffusion method. Genes associated with resistance to erythromycin and tetracycline were sought by PCR, while quinolone resistance was analysed by restriction fragment length polymorphism of the quinolone resistance-determining region. Isolates were typed by multilocus sequence typing. Seven hundred and eighty-nine of 866 serotype 6A pneumococci collected from 1997 to 2009 were available. Of these, 213 (27.0%) were serotype 6C; 16/163 (9.8%) in the 1997-2001 (pre-PCV7) period, 37/322 (11.5%) in the 2002-05 (early-PCV7) period and 160/381 (42.0%) in the 2006-09 (late-PCV7) period. The overall proportions of serotype 6C increased from 0.1% (pre-PCV7) to 1% (late-PCV7) for paediatric isolates and from 0.3% to 1.7% among adult isolates. A major serotype 6C lineage (ST224/ST1150/ST4821), accounting for 66.7% of the isolates, was identified across the whole period. In the late-PCV7 period the antimicrobial non-susceptibility of serotype 6C increased in association with the emergence of the ST386/ST4310/ST4825 lineage, which carried a Tn6002 transposon [erm(B) and tet(M) genes]. Serotype 6C pneumococci were identified in Spain during the period 1997-2009. The increase in serotype 6C in the late-PCV7 period was associated with the spread of the ST224/ST1150/ST4821 lineage and the emergence of the ST386/ST4310/ST4825 lineage.

Molecular analysis of NDM-1-producing enterobacterial isolates from Geneva, Switzerland

To analyse the mechanisms responsible for decreased susceptibility or resistance to carbapenems in several enterobacterial isolates recovered in 2009-10 in Geneva University Hospitals, Switzerland. PCR and sequencing were used to identify β-lactamases, 16S RNA methylases and plasmid-mediated quinolone resistance genes. The transferable properties of the plasmids were analysed, as well as their plasmid type. The strains were typed by multilocus sequence typing. Three patients were found to be positive for NDM-1-producing enterobacterial isolates (one with Escherichia coli and Klebsiella pneumoniae, one with K. pneumoniae only and one with Proteus mirabilis), where NDM-1 stands for New Delhi metallo-β-lactamase-1. The bla(NDM-1) carbapenemase gene was detected in all isolates in addition to genes encoding narrow-spectrum β-lactamases (TEM-1, SHV-11, OXA-1, OXA-9 and OXA-10), extended-spectrum β-lactamases (CTX-M-15, CMY-16 and CMY-30), ArmA and quinolone resistance determinants (Qnr). The bla(NDM-1) gene was located on conjugative IncA/C- or IncF-type plasmids. Upstream of the bla(NDM-1) gene, part of ISAba125, previously identified in NDM-1-negative Acinetobacter baumannii, was found. Downstream of the bla(NDM-1) gene, variable sequences were found. This work constitutes the first identification of NDM-1 producers in Switzerland. Interestingly, patients from whom these NDM-1-producing isolates were recovered had a link with the Indian subcontinent or the Balkans.

Multi locus sequence type comparison of invasive and commensal Haemophilus influenzae isolates from Delhi

Haemophilus influenzae is a major public health concern in the developing world. The most virulent strain is H. influenzae Type b (Hib). Hib also constitutes a major portion of nasopharyngeal commensal flora in otherwise healthy individuals. Through dendogram based on composite gene sequences of seven multi locus sequence type genes, it was observed that invasive and commensal isolates made two completely separate clusters which are indicative of independent evolution of these two groups of H. influenzae in the Indian subcontinent.

Predominance of ST320 among Streptococcus pneumoniae serotype 19A isolates from 10 Asian countries

After 7-valent pneumococcal conjugate vaccine (PCV7) introduction, non-vaccine serotypes such as 19A are increasing among Streptococcus pneumoniae. However, only limited data on 19A S. pneumoniae are available in Asian countries. Out of 1637 S. pneumoniae clinical pneumonia isolates collected during 2008 and 2009 from 10 Asian countries (Korea, Malaysia, Taiwan, Thailand, Saudi Arabia, Hong Kong, India, Japan, the Philippines and Vietnam), 91 serotype 19A S. pneumoniae isolates were identified. Capsular swelling reaction identified serotype 19A isolates. Antimicrobial susceptibility testing was performed on the serotype 19A isolates using the broth microdilution method, and the genotypes of the isolates were assessed using multilocus sequence typing. Thirty different sequence types (STs) were identified. The most prevalent clone was ST320 (46 isolates, 51.1%). ST320 was found in Hong Kong, India, Korea, Malaysia, Saudi Arabia and Taiwan. ST320 isolates were mostly multidrug resistant (MDR) and showed significantly higher resistance rates than other STs for cefuroxime, clindamycin, and trimethoprim/sulfamethoxazole. Although diverse clones were identified among 19A S. pneumoniae isolates, MDR ST320 was the predominant clone in Asian countries. Its predominance, even in countries with no or low coverage of PCV7, may indicate that its emergence and dissemination was due to more than just vaccine selection pressure in Asian countries. A longitudinal investigation of the change of serotypes and genotypes since the introduction of PCV7 is required to understand the emergence and dissemination mechanisms of a certain clone of 19A S. pneumoniae isolates.

Changing trends in vancomycin-resistant enterococci in French hospitals, 2001-08

Unprecedented outbreaks of vancomycin-resistant enterococci (VRE) have occurred in French hospitals since 2004. The aim of this study was to provide a picture of the spread and control of VRE in France and to characterize the isolates. Notification of VRE cases to Institut de Veille Sanitaire has been mandatory since 2001. Isolates of VRE were sent to the National Reference Centre for species and vancomycin-resistance gene identification. Isolates were tested for antimicrobial susceptibility and typed by PFGE and multilocus sequence typing. Five hundred and four VRE notifications from 195 hospitals were recorded, corresponding to 2475 cases of infection (n=243) or colonization (n=2232) and 74 episodes of clustered cases. Outbreaks were controlled by implementation of infection control measures, although the number of new hospitals reporting isolation of VRE was increasing. The majority of 902 VRE isolated from 2006 to 2008 were Enterococcus faecium (94.8%) with the vanA or vanB gene. No isolate was resistant to linezolid, tigecycline or fusidic acid. PFGE analysis showed 161 different patterns. Generally a few predominant clones and several minor clones spread in a single hospital. In a subset of 46 representatives of PFGE clones, 13 different sequence types were characterized, all belonging to clonal complex CC17, while the esp and hyl genes were inconsistently detected. The national mandatory notification of unusual nosocomial events allowed rapid identification of VRE outbreaks and early implementation of control measures that have proved effective. However, VRE continue to emerge in a growing number of hospitals.

Staphylococcus aureuscarriage among GPs in the Netherlands

The extent to which GPs serve as a reservoir for antibiotic-resistant Staphylococcus aureus is unknown and not well studied. To determine the prevalence of nasal S. aureus carriage among GPs in the Netherlands, as well as the antimicrobial resistance and the genotypes of isolated S. aureus. Observational, point-prevalence, and cross-sectional study. GPs attending the annual conference of the Dutch College of General Practitioners in 2006. Nasal swabs were randomly taken from 395 GPs and analysed for the presence of S. aureus. Antimicrobial susceptibility was determined by a microbroth dilution method and the genotypes by spa typing, which was associated with multilocus sequence typing. Of the GPs, 129/395 (33%; 95% confidence interval [CI] = 28 to 37%) were carriers of S. aureus. No meticillin-resistant S. aureus (MRSA) was found. Resistance was observed to penicillin (71%; 95% CI = 63 to 79%), fusidic acid (7%; 95% CI = 3 to 13%), and clarithromycin (6%; 95% CI = 3 to 12%). In 72% of the isolates, an MRSA-related genotype of S. aureus was found. The low antibiotic resistance found among S. aureus of GPs suggests that GPs are not a reservoir of antibiotic-resistant S. aureus strains. The relatively high resistance to fusidic acid, which has not previously been described in the Netherlands and is mostly because of antibiotic use, suggests that patients infect GPs and not the other way round. GPs may be at risk for nasal carriage of S. aureus with an MRSA-related genotype.

Characterisation of Australian MRSA strains ST75- and ST883-MRSA-IV and analysis of their accessory gene regulator locus.

BackgroundCommunity-acquired methicillin-resistant Staphylococcus aureus have become a major problem in Australia. These strains have now been isolated throughout Australia including remote Indigenous communities that have had minimal exposure to healthcare facilities. Some of these strains, belonging to sequence types ST75 and ST883, have previously been reported to harbour highly divergent alleles of the housekeeping genes used in multilocus sequence typing.Methodology/Principal FindingsST75-MRSA-IV and ST883-MRSA-IV isolates were characterised in detail. Morphological features as well as 16S sequences were identical to other S. aureus strains. Although a partial rnpB gene sequence was not identical to previously known S. aureus sequences, it was found to be more closely related to S. aureus than to other staphylococci. Isolates also were screened using diagnostic DNA microarrays. These isolates yielded hybridisation results atypical for S. aureus. Primer directed amplification assays failed to detect species markers (femA, katA, sbi, spa). However, arbitrarily primed amplification indicated the presence of unknown alleles of these genes. Isolates could not be assigned to capsule types 1, 5 or 8. The allelic group of the accessory gene regulator (agr) locus was not determinable. Sequencing of a region of agrB, agrC and agrD (approximately 2,100 bp) revealed a divergent sequence. However, this sequence is more related to S. aureus agr alleles I and IV than to agr sequences from other Staphylococcus species. The predicted auto-inducing peptide (AIP) sequence of ST75 was identical to that of agr group I, while the predicted AIP sequence of ST883 was identical to agr group IV.Conclusions/SignificanceThe genetic properties of ST75/ST883-MRSA may be due to a series of evolutionary events in ancient insulated S. aureus strains including a convergent evolution leading to agr group I- or IV-like AIP sequences and a recent acquisition of SCCmec IV elements.

Multiple cryptic species with divergent substrate affinities in the Serpula himantioides species complex

Serpula himantioides is a widespread saprotrophic morphospecies mainly colonising coniferous wood in nature, but it appears frequently in buildings as well. From an earlier study, it is known that at least three divergent lineages occur within the S. himantioides species complex. In this study, a broader sample of S. himantioides isolates has been analysed by multi-locus sequencing, including new isolates from Asia, North and South America. Altogether five phylogenetical species (PS1–5) were detected, all recognised across independent gene phylogenies. A new southern South American phylogenetic species (PS1) was found, representing an early diverging lineage within the S. himantioides species complex. The two closely related PS2 and PS3 lineages included isolates from North America only, and PS4 was also dominated by North American isolates. Most of the investigated isolates (76%) clustered into PS5, a lineage that has been found on most continents, including North America. Overall, little phylogeographical structure was found in PS5, indicating frequent and recent long-distance dispersal events within this widespread lineage. Our analyses indicate that South and North America are the centres of divergence for the S. himantioides species complex. Some of the lineages seem adapted to various substrates, but PS5 is able to decay a wide array of angiosperms and gymnosperms, which may have facilitated the spread of this lineage throughout the world.

Test of a Novel Streptococcus pneumoniae Serotype 6C Type Specific Polyclonal Antiserum (Factor Antiserum 6d) and Characterisation of Serotype 6C Isolates in Denmark

BackgroundIn 2007, Park et al. identified a novel serotype among Streptococcus pneumoniae serogroup 6 which they named serotype 6C. The aim of this study was to evaluate with the Neufeld test a novel S. pneumoniae serotype 6C type specific polyclonal antiserum. In addition, serotype 6C isolates found in Denmark in 2007 and 2008 as well as eight old original serotype 6A isolates were characterised.MethodsIn this study, 181 clinical Streptococcus pneumoniae isolates from Denmark 2007 and 2008 were examined; 96 isolates had previously been typed as serotype 6A and 85 as serotype 6B. In addition, eight older isolates from 1952 to 1987, earlier serotyped as 6A, were examined. Serotype 6C isolates were identified by PCR and serotyping with the Neufeld test using the novel type specific polyclonal antiserum, factor antiserum 6 d, in addition to factor antisera 6b, 6b* (absorbed free for cross-reactions to serotype 6C) and 6c. All antisera are commercially available and antiserum 6b obtained from the supplier after 1 January 2009 is antiserum 6b*. All serotype 6C isolates were further characterised using multi-locus sequence typing.ResultsWhen retesting all 96 original serotype 6A isolates by PCR and the Neufeld test, 29.6% (24 of 81) of the invasive isolates in Denmark from 2007 and 2008 were recognised as serotype 6C. In addition, three of eight old isolates originally serotyped as 6A were identified to be serotype 6C. The oldest serotype 6C isolate was from 1962. The serotype 6C isolates belonged to eleven different sequence types (ST) and nine clonal complexes (CC), ST1692 (CC395), ST386 (CC386) and ST481 (CC460) were the predominant types.ConclusionsWe tested a novel polyclonal antiserum 6 d, as well as modified antiserum 6b*, provided a scheme for the serotyping of S. pneumoniae serogroup 6 using the Neufeld test and compared the serotyping method with PCR based methods. The two types of methods provided the same results. In future, it will, therefore, be possible to test also serotype 6C in accordance to the standard method for serotyping of S. pneumoniae recommended by WHO.Among all invasive isolates from Denmark 2007 and 2008, serotype 6C constituted 29.6% of the original serotype 6A isolates. The serotype 6C isolates were found to be diverse belonging to a number of different STs and CCs of which most have been observed in other countries previously. Serotype 6C is regarded as an "old" serotype being present among S. pneumoniae isolates in Denmark for at least 48 years. The genetic diversity of serotype 6C isolates and their genetic relationship to other serotypes suggested that serotype 6C strains may have arisen from several different independent recombination events involving different parental strains such as serotypes 6A, 6B, 23F and 4.

Genotyping isolates of the entomopathogenic fungus Beauveria bassiana sensu lato by multi-locus polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis

Multi-locus denaturing gradient gel electrophoresis (DGGE) analysis was developed to investigate the genotypes of Beauveria bassiana sensu lato. Sensitive tests indicated all isolates with one or more nucleotide differences at EF-1 and Bloc could be distinguished by DGGE except for one pair of strains that differed at four nucleotide positions. Ten, twelve and five genotypes were identified at the EF-1, Bloc and ITS locus, respectively, among seventeen isolates, which together differentiated 13 genotypes. These results demonstrated that multi-locus DGGE is a potentially useful molecular marker for genotyping, identifying and tracking the fates of experimentally released strains of B. bassiana sensu lato. Moreover, by multi-locus DGGE for scanning B. bassiana sensu lato isolates with different multilocus sequences, genetic diversity of B. bassiana sensu lato was effectively investigated with substantially reduced time and cost in subsequent DNA sequencing.

Fusarium culmorum is a single phylogenetic species based on multilocus sequence analysis

Fusarium culmorum is a major pathogen of wheat and barley causing head blight and crown rot in cooler temperate climates of Australia, Europe, West Asia and North Africa. To better understand its evolutionary history we partially sequenced single copy nuclear genes encoding translation elongation factor 1-α (TEF), reductase (RED) and phosphate permease (PHO) in 100 F. culmorum isolates with 11 isolates of Fusarium crookwellense, Fusarium graminearum and Fusarium pseudograminearum. Phylogenetic analysis of multilocus sequence (MLS) data using Bayesian inference and maximum parsimony analysis showed that F. culmorum from wheat is a single phylogenetic species with no significant linkage disequilibrium and little or no lineage development along geographic origin. Both MLS and TEF and RED gene sequence analysis separated the four Fusarium species used and delineated three to four groups within the F. culmorum clade. But the PHO gene could not completely resolve isolates into their respective species. Fixation index and gene flow suggest significant genetic exchange between the isolates from distant geographic regions. A lack of strong lineage structure despite the geographic separation of the three collections indicates a frequently recombining species and/or widespread distribution of genotypes due to international trade, tourism and long-range dispersal of macroconidia. Moreover, the two mating type genes were present in equal proportion among the F. culmorum collection used in this study, leaving open the possibility of sexual reproduction.

Trichoderma asperellum sensu lato consists of two cryptic species

Analysis of a worldwide collection of strains of Trichoderma asperellum sensu lato using multilocus genealogies of four genomic regions (tef1, rpb2, act, ITS1, 2 and 5.8s rRNA), sequence polymorphism-derived (SPD) markers, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) of the proteome and classical mycological techniques revealed two morphologically cryptic sister species within T. asperellum, T. asperellum, T. asperelloides sp. nov. and a third closely related but morphologically distinct species. T. yunnanense. Trichoderma asperellum and T. asperelloides have wide sympatric distribution on multiple continents; T. yunnanense is represented by a single strain from China. Several strains reported in the literature or represented in GenBank as T. asperellum are re-identified as T. asperelloides. Four molecular SPD typing patterns (I–IV) were found over a large geographic range. Patterns I–III were produced only by T. asperellum and pattern IV by T. asperelloides and T. yunnanense. Pattern I was found in North America, South America, Africa and Europe and Asia (Saudi Arabia). Pattern III was found in Africa, North America, South America and Asia, not in Europe. Pattern II was found only in Cameroon (central Africa) and Peru. Pattern IV was found in all continents. All SPD II pattern strains formed a strongly supported subclade within the T. asperellum clade in the phylogenetic tree based on rpb2 and MLS (combined multilocus sequence). The diversity of DNA sequences, SPD markers and polypeptides in T. asperellum suggests that further speciation is under way within T. asperellum. MALDI-TOF MS distinguished T. yunnanense from related taxa by UPGMA clustering, but separation between T. asperellum and T. asperelloides was less clear.