Candidemia by Species of the Candida parapsilosis Complex in Children’s Hospital: Prevalence, Biofilm Production and Antifungal Susceptibility

Opportunistic infections are an increasingly common problem in hospitals, and the yeast Candida parapsilosis has emerged as an important nosocomial pathogen. The aims of this study were to determine and compare (i) the prevalence rate among C. parapsilosis complex organisms isolated from blood in a public children’s hospital in São Paulo state, (ii) the ability of the complex C. parapsilosis species identified to produce biofilm and (iii) the antifungal susceptibility profiles. Forty-nine (49) specimens of isolated blood yeast were analyzed, previously identified as C. parapsilosis by conventional methods. After the molecular analysis, the isolates were characterized as C. parapsilosis sensu stricto (83.7 %), C. orthopsilosis (10.2 %) and C. metapsilosis (6.1 %). All species were able to form biofilm. The species with the highest biofilm production was C. parapsilosis sensu stricto, followed by C. orthopsilosis and further by C. metapsilosis. All of the strains have demonstrated similar susceptibility to fluconazole, caspofungin, voriconazole, cetoconazole and 5-flucytosine. Only one strain of C. parapsilosis was resistant to amphotericin B. Regarding itraconazole, 66.6 and 43.9 % isolates of C. metapsilosis and C. parapsilosis, respectively, have demonstrated to be susceptible dose-dependent, with one isolate of the latter species resistant to the drug. Candida parapsilosis sensu stricto has demonstrated to be the less susceptible, mainly to amphotericin B, caspofungin and “azoles” such as fluconazole. Therefore, C. metapsilosis and C. orthopsilosis are still involved in a restricted number of infections, but these data have become essential for there are very few studies of these species in Latin America.     

Simple,Low-Cost Detection of Candida parapsilosis Complex Isolates and Molecular Fingerprinting of Candida orthopsilosis Strains in Kuwait by ITS Region Sequencing and Amplified Fragment Length Polymorphism Analysis

Candida parapsilosis has now emerged as the second or third most important cause of healthcare-associated Candida infections. Molecular studies have shown that phenotypically identified C. parapsilosis isolates represent a complex of three species, namely, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Lodderomyces elongisporus is another species phenotypically closely related to the C. parapsilosis-complex. The aim of this study was to develop a simple, low cost multiplex (m) PCR assay for species-specific identification of C. parapsilosis complex isolates and to study genetic relatedness of C. orthopsilosis isolates in Kuwait. Species-specific amplicons from C. parapsilosis (171 bp), C. orthopsilosis (109 bp), C. metapsilosis (217 bp) and L. elongisporus (258 bp) were obtained in mPCR. Clinical isolates identified as C. parapsilosis (n = 380) by Vitek2 in Kuwait and an international collection of 27 C. parapsilosis complex and L. elongisporus isolates previously characterized by rDNA sequencing were analyzed to evaluate mPCR. Species-specific PCR and DNA sequencing of internal transcribed spacer (ITS) region of rDNA were performed to validate the results of mPCR. Fingerprinting of 19 clinical C. orthopsilosis isolates (including 4 isolates from a previous study) was performed by amplified fragment length polymorphism (AFLP) analysis. Phenotypically identified C. parapsilosis isolates (n = 380) were identified as C. parapsilosis sensu stricto (n = 361), C. orthopsilosis (n = 15), C. metapsilosis (n = 1) and L. elongisporus (n = 3) by mPCR. The mPCR also accurately detected all epidemiologically unrelated C. parapsilosis complex and L. elongisporus isolates. The 19 C. orthopsilosis isolates obtained from 16 patients were divided into 3 haplotypes based on ITS region sequence data. Seven distinct genotypes were identified among the 19 C. orthopsilosis isolates by AFLP including a dominant genotype (AFLP1) comprising 11 isolates recovered from 10 patients. A rapid, low-cost mPCR assay for detection and differentiation of C. parapsilosis, C. orthopsilosis, C. metapsilosis and L. elongisporus has been developed.     

Comparison of Commercial Methods and the CLSI Broth Microdilution to Determine the Antifungal Susceptibility of Candida parapsilosis Complex Bloodstream Isolates from Three Health Institutions in Rio de Janeiro,Brazil

Two commercial methods, the Etest and Vitek 2, were compared with the Clinical and Laboratory Standards Institute broth microdilution method to determine the susceptibility of Candida parapsilosis complex to amphotericin B, caspofungin, fluconazole, voriconazole, and itraconazole. One-hundred bloodstream isolates of C. parapsilosis complex from three hospitals in Rio de Janeiro city, Brazil, between 1998 and 2006 were analyzed. C. parapsilosis sensu stricto (61 %) was the predominant species, followed by C. orthopsilosis (37 %) and C. metapsilosis (2 %). Most isolates were susceptible to the tested drugs. However, one C. parapsilosis sensu stricto isolate was considered resistant for amphotericin B. The essential agreement was 100 % between the methods, except for itraconazole (96.3 %). The categorical agreement varied for fluconazole and itraconazole by Etest and for amphotericin B and fluconazole by Vitek 2. This study reinforces the suitability of the commercial methods in routine clinical microbiology laboratories for antifungal susceptibility testing.     

Differential Sensitivity of the Species of Candida parapsilosis Sensu Lato Complex Against Statins

Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are human fungal pathogens with clinical importance. The recently reclassified three closely related species have significant variation in virulence, clinical prevalence and susceptibility characteristics to different antifungal compounds. The aim of this study was to investigate the in vitro activity of atorvastatin and fluvastatin against C. metapsilosis, C. orthopsilosis and C. parapsilosis. Susceptibility tests showed that C. parapsilosis was the most sensitive while C. orthopsilosis was the least susceptible species to both drugs. On the basis of the differential sensitivity, we developed a simple, reliable and highly cost-effective plate assay to distinguish these closely related species. Applying this method, 54 isolates belonging to the C. parapsilosis sensu lato complex deposited in Szeged Microbial Collection could be sorted into the three species with 100 % probability.     

Killing kinetics of anidulafungin,caspofungin and micafungin against Candida parapsilosis species complex: Evaluation of the fungicidal activity

Background

Candida parapsilosis, Candida metapsilosis and Candida orthopsilosis are emerging as relevant causes of candidemia. Moreover, they show differences in their antifungal susceptibility and virulence. The echinocandins are different in terms of in vitro antifungal activity against Candida. Time-kill (TK) curves represent an excellent approach to evaluate the fungicidal activity of antifungal drugs.

Candida orthopsilosis fungemias in a Spanish tertiary care hospital: Incidence,epidemiology and antifungal susceptibility

BackgroundFew studies exist on prevalence of fungemia by Candida orthopsilosis, with variable results.AimsTo study the incidence, epidemiology and antifungal susceptibility of C. orthopsilosis strains isolated from fungemias over two years at a tertiary hospital.MethodsCandidemia episodes between June 2007 and June 2009 in a university hospital (Puerta del Mar, Cádiz, Spain) were studied. The strains initially identified as Candida parapsilosis were genotypically screened for C. parapsilosis sensu stricto, C. orthopsilosis and Candida metapsilosis, and their antifungal susceptibility was evaluated.ResultsIn this period 52 cases of candidemia were documented. Of the 19 strains originally identified as C. parapsilosis, 13 were confirmed as C. parapsilosis sensu stricto and 6 as C. orthopsilosis. Of the 52 isolates, the most frequent species were Candida albicans (30.8%), C. parapsilosis sensu stricto (25%), C. orthopsilosis, Candida tropicalis and Candida glabrata in equal numbers (11.5%). C. orthopsilosis isolates were susceptible to amphotericin B, caspofungin, voriconazole and fluconazole, with no significant differences in MIC values with C. parapsilosis sensu stricto. The source of isolates of C. orthopsilosis were neonates (50%) and surgery (50%), and 100% were receiving parenteral nutrition; however C. parapsilosis sensu stricto was recovered primarily from patients over 50 years (69.2%) and 46.1% were receiving parenteral nutrition.ConclusionsThese findings show that C. orthopsilosis should be considered as human pathogenic yeast and therefore its accurate identification is important. Despite our small sample size our study suggests that a displacement of some epidemiological characteristics previously attributed to C. parapsilosis to C. orthopsilosis may be possible.     

Prevalence and distribution profiles of Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis responsible for superficial candidiasis in a Chinese university hospital

The Candida parapsilosis complex consists of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Recently, many studies described the prevalence of this species complex mainly in invasive candidiasis. Additionally, data showed that these three species are different in virulence and in vitro drug susceptibility. However, to our knowledge, the prevalence and distribution of the species complex in superficial candidiasis is not very clear to date. In this study, 2,128 Candida isolates from specimens of superficial candidiasis were collected over a 1-year period. Combination of routine and molecular tools, a total of 214 samples were identified to be positive for the C. parapsilosis complex (10.1%), of which 198 (92.5%) were monofungal and 16 (7.5%) were polyfungal. Among the 198 monofungal isolates, 191 (96.5%) were identified as C. parapsilosis sensu stricto, 5 (2.5%) as C. metapsilosis, and 2 (1.0%) as C. orthopsilosis species based on the molecular method. All C. parapsilosis complex isolates from the 16 polyfungal populations were found to be C. parapsilosis sensu stricto. Further analysis showed that the distribution profiles of the C. parapsilosis complex in adult patients were different from that in pediatric patients, and the prevalence rate of it varied greatly by sites of isolation. This study provides insight into the epidemiology of the species complex in superficial candidiasis.     

In Vitro Evaluation of Phospholipase,Proteinase, and Esterase Activities of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida metapsilosis</Emphasis>

The aim of this study is to characterize extracellular phospholipase, proteinase, and esterase activities of Candida parapsilosis and C. metapsilosis isolated from clinical sources. Using PCR-restriction fragment length polymorphism (PCR–RFLP) of the secondary alcohol dehydrogenase (SADH) gene fragment, we identified 20 as C. parapsilosis and 11 as C. metapsilosis from 31 isolates of C. parapsilosis species complex. No C. orthopsilosis was identified. A significantly high isolation frequency of C. metapsilosis (35.5%) was observed. Subsequent evaluation of enzymatic profile showed that 90.5% of C. parapsilosis and 91.7% of C. metapsilosis isolates were phospholipase producers. No difference in phospholipase activity was observed between two species. In terms of proteinase, 81.0% of C. parapsilosis and 83.3% of C. metapsilosis isolates were positive. A higher level of proteinase activity was detected in C. parapsilosis. A remarkably high proportion of both C. parapsilosis and C. metapsilosis isolates exhibited strong phospholipase and proteinase activities, suggesting that the production of these two enzymes might be common for them. On the other hand, both species similarly displayed rare esterase activity, with only one C. parapsilosis and two C. metapsilosis isolates being positive. Our data may further add to the confusion concerning the hydrolytic enzymatic activities of the C. parapsilosis complex, and a wider collection of isolates and standardized methods may help to address the issue.     

Species distribution and antifungal susceptibility among clinical isolates of Candida parapsilosis complex from India

Background

Candida parapsilosis is recognized as a species complex: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are three distinct but closely related species.

Killer behavior within the Candida parapsilosis complex

A group of 29 isolates of Candida parapsilosis sensu stricto, 29 of Candida orthopsilosis, and 4 of Candida metapsilosis were assayed for the presence of killer activity using Saccharomyces cerevisiae ATCC 26609 as a sensitive strain. All C. metapsilosis isolates showed killer activity at 25 °C while strains of C. parapsilosis sensu stricto or C. orthopsilosis did not exhibit this activity. Sensitivity to killer toxins was evaluated using a set of previously reported killer strains of clinical origin. Only 11 isolates of the C. parapsilosis complex were inhibited by at least one killer isolate without resulting in any clear pattern, except for C. parapsilosis sensu stricto ATCC 22019, which was inhibited by every killer strain with the exception of C. parapsilosis and Candida utilis. The lack of sensitivity to killer activity among isolates of the genus Candida suggests that their toxins belong to the same killer type. Differentiation of species within the C. parapsilosis complex using the killer system may be feasible if a more taxonomically diverse panel of killer strains is employed.     

Characterization of Virulence Properties in the C. parapsilosis Sensu Lato Species

The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto, C . orthopsilosis and C . metapsilosis . Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12 C . metapsilosis and 18 C . orthopsilosis isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the C . metapsilosis strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto, C . orthopsilosis and C . metapsilosis were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to C . orthopsilosis and C . metapsilosis isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and C . metapsilosis strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of Galleria mellonella larvae. The mortality rate of G . mellonella larvae infected with C . metapsilosis isolates was significantly lower than those infected with C. parapsilosis sensu stricto or C . orthopsilosis strains. Taken together, our findings demonstrate that C . metapsilosis is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions.     

Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007–2014

The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it reinforces the relevance of using commercially available microbiological methods to assess antifungal susceptibility. These data improve our knowledge of the national distribution of species of the psilosis group, as there are very few studies of these species in Italy.     

Genome analysis of five recently described species of the CUG-Ser clade uncovers Candida theae as a new hybrid lineage with pathogenic potential in the Candida parapsilosis species complex

Candida parapsilosis species complex comprises three important pathogenic species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. The majority of C. orthopsilosis and all C. metapsilosis isolates sequenced thus far are hybrids, and most of the parental lineages remain unidentified. This led to the hypothesis that hybrids with pathogenic potential were formed by the hybridization of non-pathogenic lineages that thrive in the environment. In a search for the missing hybrid parentals, and aiming to get a better understanding of the evolution of the species complex, we sequenced, assembled and analysed the genome of five close relatives isolated from the environment: Candida jiufengensis, Candida pseudojiufengensis, Candida oxycetoniae, Candida margitis and Candida theae. We found that the linear conformation of mitochondrial genomes in Candida species emerged multiple times independently. Furthermore, our analyses discarded the possible involvement of these species in the mentioned hybridizations, but identified C. theae as an additional hybrid in the species complex. Importantly, C. theae was recently associated with a case of infection, and we also uncovered the hybrid nature of this clinical isolate. Altogether, our results reinforce the hypothesis that hybridization is widespread among Candida species, and potentially contributes to the emergence of lineages with opportunistic pathogenic behaviour.     

Complete DNA sequences of the mitochondrial genomes of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis: insight into the evolution of linear DNA genomes from mitochondrial telomere mutants

We determined complete mitochondrial DNA sequences of the two yeast species, Candida orthopsilosis and Candida metapsilosis, and compared them with the linear mitochondrial genome of their close relative, C.parapsilosis. Mitochondria of all the three species harbor compact genomes encoding the same set of genes arranged in the identical order. Differences in the length of these genomes result mainly from the presence/absence of introns. Multiple alterations were identified also in the sequences of the ribosomal and transfer RNAs, and proteins. However, the most striking feature of C.orthopsilosis and C.metapsilosis is the existence of strains differing in the molecular form of the mitochondrial genome (circular-mapping versus linear). Their analysis opens a unique window for understanding the role of mitochondrial telomeres in the stability and evolution of molecular architecture of the genome. Our results indicate that the circular-mapping mitochondrial genome derived from the linear form by intramolecular end-to-end fusions. Moreover, we suggest that the linear mitochondrial genome evolved from a circular-mapping form present in a common ancestor of the three species and, at the same time, the emergence of mitochondrial telomeres enabled the formation of linear monomeric DNA forms. In addition, comparison of isogenic C.metapsilosis strains differing in the form of the organellar genome suggests a possibility that, under some circumstances, the linearity and/or the presence of telomeres provide a competitive advantage over a circular-mapping mitochondrial genome.     

Ten-Year Study of Species Distribution and Antifungal Susceptibilities of Candida Bloodstream Isolates at a Brazilian Tertiary Hospital

To describe the incidence and susceptibility profile of Candida bloodstream infections in a tertiary-care hospital, we performed a retrospective observational study from 1998 to 2007. Comorbidities and risk factors were compiled from all cases. In vitro susceptibility testing to fluconazole, itraconazole, voriconazole, and amphotericin B was performed for 100 isolates, and caspofungin was tested for C. parapsilosis complex. In a ten-year evaluation of candidemias, 44?% were caused by C. albicans, and species of the C. parapsilosis complex were the second most frequent agents (37?%). Other species presented lower incidences (C. tropicalis, 13?%, C. glabrata, 5?%, and C. krusei, 1?%). Neither C. dubliniensis nor C. metapsilosis were observed in this study. C. orthopsilosis (3?%) and C. parapsilosis stricto sensu (34?%) were also found. Species distribution was independent of catheterization, mechanical ventilation, or previous use of antifungals or corticoids. Parenteral nutrition administration was strongly related to C. glabrata infection, and the highest mortality (80?%) was observed in patients infected by this species. All C. albicans isolates showed high susceptibility to all tested drugs. However, two C. parapsilosis stricto sensu isolates presented high minimum inhibitory concentration (MIC) (4?mg/L each) to fluconazole, and one exhibited voriconazole MIC of 0.25?mg/L, highlighting the cross-resistance to these azoles. All isolates of C. tropicalis and C. glabrata showed no resistance to any drug tested. No difference was noted between C. parapsilosis and C. orthopsilosis susceptibilities to caspofungin. Our results suggest that resistance to amphotericin B, fluconazole, voriconazole, itraconazole, and caspofungin in Brazilian Candida bloodstream isolates is still uncommon.     

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of yeasts is contingent on robust reference spectra

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods.

Methods

MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster.

Principal Findings

Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70–1.90 provided correct species assignment despite being identified to “genus-level”. MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results.

Conclusions

MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility.     

Viridans Group Streptococci Clinical Isolates: MALDI-TOF Mass Spectrometry versus Gene Sequence-Based Identification

Viridans Group Streptococci (VGS) species-level identification is fundamental for patients management. Matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF MS) has been used for VGS identification but discrimination within the Mitis group resulted difficult. In this study, VGS identifications with two MALDI-TOF instruments, the Biotyper (Bruker) and the VITEK MS (bioMérieux) have been compared to those derived from tuf, soda and rpoB genes sequencing. VGS isolates were clustered and a dendrogram constructed using the Biotyper 3.0 software (Bruker). RpoB gene sequencing resulted the most sensitive and specific molecular method for S. pneumonia identification and was used as reference method. The sensitivity and the specificity of the VITEK MS in S. pneumonia identification were 100%, while the Biotyper resulted less specific (92.4%). In non pneumococcal VGS strains, the group-level correlation between rpoB and the Biotyper was 100%, while the species-level correlation was 61% after database upgrading (than 37% before upgrading). The group-level correlation between rpoB and the VITEK MS was 100%, while the species-level correlation was 36% and increases at 69% if isolates identified as S. mitis/S. oralis are included. The less accurate performance of the VITEK MS in VGS identification within the Mitis group was due to the inability to discriminate between S. mitis and S. oralis. Conversely, the Biotyper, after the release of the upgraded database, was able to discriminate between the two species. In the dendrogram, VGS strains from the same group were grouped into the same cluster and had a good correspondence with the gene-based clustering reported by other authors, thus confirming the validity of the upgraded version of the database. Data from this study demonstrated that MALDI-TOF technique can represent a rapid and cost saving method for VGS identification even within the Mitis group but improvements of spectra database are still recommended.     

The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis

Candida metapsilosis is a rarely-isolated, opportunistic pathogen that belongs to a clade of pathogenic yeasts known as the C. parapsilosis sensu lato species complex. To gain insight into the recent evolution of C. metapsilosis and the genetic basis of its virulence, we sequenced the genome of 11 clinical isolates from various locations, which we compared to each other and to the available genomes of the two remaining members of the complex: C. orthopsilosis and C. parapsilosis. Unexpectedly, we found compelling genomic evidence that C. metapsilosis is a highly heterozygous hybrid species, with all sequenced clinical strains resulting from the same past hybridization event involving two parental lineages that were approximately 4.5% divergent in sequence. This result indicates that the parental species are non-pathogenic, but that hybridization between them formed a new opportunistic pathogen, C. metapsilosis, that has achieved a worldwide distribution. We show that these hybrids are diploid and we identified strains carrying loci for both alternative mating types, which supports mating as the initial mechanism for hybrid formation. We trace the aftermath of this hybridization at the genomic level, and reconstruct the evolutionary relationships among the different strains. Recombination and introgression -resulting in loss of heterozygosis- between the two subgenomes have been rampant, and includes the partial overwriting of the MTLa mating locus in all strains. Collectively, our results shed light on the recent genomic evolution within the C. parapsilosis sensu lato complex, and argue for a re-definition of species within this clade, with at least five distinct homozygous lineages, some of which having the ability to form hybrids.     

Evaluation of MALDI-TOF mass spectroscopy methods for determination of Escherichia coli pathotypes

It is rapidly becoming apparent that many E. coli pathotypes cause a considerable burden of human disease. Surveillance of these organisms is difficult because there are few or no simple, rapid methods for detecting and differentiating the different pathotypes. MALDI-TOF mass spectroscopy has recently been rapidly and enthusiastically adopted by many clinical laboratories as a diagnostic method because of its high throughput, relatively low cost, and adaptability to the laboratory workflow. To determine whether the method could be adapted for E. coli pathotype differentiation the Bruker Biotyper methodology and a second methodology adapted from the scientific literature were tested on isolates representing eight distinct pathotypes and two other groups of E. coli. A total of 136 isolates was used for this study. Results confirmed that the Bruker Biotyper methodology that included extraction of proteins from bacterial cells was capable of identifying E. coli isolates from all pathotypes to the species level and, furthermore, that the Bruker extraction and MALDI-TOF MS with the evaluation criteria developed in this work was effective for differentiating most pathotypes.