Stability and Qualification of a Legacy Fungal Collection.

Background: Microbial culture collections are valuable repositories for qualified and diverse microorganisms, playing a pivotal role in research, education, innovation, as well as in our response to current and emerging public health and societal challenges. However, such precious holdings, when not integrated in professional biobank infrastructures, may be vulnerable to major risks such as staff retirement, changes in the institutional strategy, or natural disasters. The process of preserving and rescuing "historical" collections can be long and treacherous with a loss of a part of the collection. At the Biological Resource Center of Institut Pasteur, we undertook the challenge of rescuing the dormant legacy fungal collection. Materials and Methods: A total of 64 freeze-dried strains, including yeasts and filamentous fungi, were characterized by using a polyphasic approach combining morphological features and molecular data. We assessed the viability, purity, and authenticity of selected strains isolated from multiple sources and stored for more than 20 years. Results: Our preliminary results show long-term stability of the selected strains and successful qualification in terms of purity and authentication. Moreover, based on the most recent taxonomic revisions, we updated and revised the nomenclature, where applicable. Conclusion: Our findings demonstrated the potential value of reviving historical microbial collections for biobanking and research activities and reassure us about the collection's future reopening.

The speciation and hybridization history of the genus Salmonella.

Bacteria and archaea make up most of natural diversity, but the mechanisms that underlie the origin and maintenance of prokaryotic species are poorly understood. We investigated the speciation history of the genus Salmonella, an ecologically diverse bacterial lineage, within which S. enterica subsp. enterica is responsible for important human food-borne infections. We performed a survey of diversity across a large reference collection using multilocus sequence typing, followed by genome sequencing of distinct lineages. We identified 11 distinct phylogroups, 3 of which were previously undescribed. Strains assigned to S. enterica subsp. salamae are polyphyletic, with two distinct lineages that we designate Salamae A and B. Strains of the subspecies houtenae are subdivided into two groups, Houtenae A and B, and are both related to Selander's group VII. A phylogroup we designate VIII was previously unknown. A simple binary fission model of speciation cannot explain observed patterns of sequence diversity. In the recent past, there have been large-scale hybridization events involving an unsampled ancestral lineage and three distantly related lineages of the genus that have given rise to Houtenae A, Houtenae B and VII. We found no evidence for ongoing hybridization in the other eight lineages, but detected subtler signals of ancient recombination events. We are unable to fully resolve the speciation history of the genus, which might have involved additional speciation-by-hybridization or multi-way speciation events. Our results imply that traditional models of speciation by binary fission and divergence are not sufficient to account for Salmonella evolution.

Carriage of a Single Strain of Nontoxigenic Corynebacterium diphtheriae bv. Belfanti (Corynebacterium belfantii) in Four Patients with Cystic Fibrosis.

Cystic fibrosis (CF) patients are commonly colonized by bacterial pathogens, which can induce persistent lung inflammation and may contribute to clinical deterioration. Colonization of CF patients and cross-transmission by Corynebacterium diphtheriae have not been reported so far. The aim of this article was to investigate the possibility of a cross-transmission of C. diphtheriae biovar Belfanti between four patients of a CF center. C. diphtheriae biovar Belfanti (now formally called C. belfantii) isolates were collected from four patients in a single CF care center over a period of 6 years and analyzed by microbiological methods and whole-genome sequencing. Epidemiological links among patients were investigated. Ten isolates were collected from 4 patients. Whole-genome sequencing of one isolate from each patient showed that a single strain was shared among them. In addition, one patient was found to have the same strain in two consecutive samplings performed 9 months apart. The strain was nontoxigenic and was susceptible to most antimicrobial agents. Ciprofloxacin resistance was observed in one patient. The idea of transmission of the strain among patients was supported by the occurrence of same-day visits to the CF center. This study demonstrated colonization of CF patients by C. diphtheriae biovar Belfanti (C. belfantii), and the data suggest persistence and transmission of a unique strain during at least 6 years in a single CF patient care center.

Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination.

Severe liver abscess infections caused by hypervirulent clonal-group CG23 Klebsiella pneumoniae have been increasingly reported since the mid-1980s. Strains typically possess several virulence factors including an integrative, conjugative element ICEKp encoding the siderophore yersiniabactin and genotoxin colibactin. Here we investigate CG23's evolutionary history, showing several deep-branching sublineages associated with distinct ICEKp acquisitions. Over 80% of liver abscess isolates belong to sublineage CG23-I, which emerged in ~1928 following acquisition of ICEKp10 (encoding yersiniabactin and colibactin), and then disseminated globally within the human population. CG23-I's distinguishing feature is the colibactin synthesis locus, which reportedly promotes gut colonisation and metastatic infection in murine models. These data show circulation of CG23 K. pneumoniae decades before the liver abscess epidemic was first recognised, and provide a framework for future epidemiological and experimental studies of hypervirulent K. pneumoniae. To support such studies we present an open access, completely sequenced CG23-I human liver abscess isolate, SGH10.

Rhinoscleroma pathogenesis: The type K3 capsule of Klebsiella rhinoscleromatis is a virulence factor not involved in Mikulicz cells formation.

Rhinoscleroma is a human specific chronic granulomatous infection of the nose and upper airways caused by the Gram-negative bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. Although considered a rare disease, it is endemic in low-income countries where hygienic conditions are poor. A hallmark of this pathology is the appearance of atypical foamy monocytes called Mikulicz cells. However, the pathogenesis of rhinoscleroma remains poorly investigated. Capsule polysaccharide (CPS) is a prominent virulence factor in bacteria. All K. rhinoscleromatis strains are of K3 serotype, suggesting that CPS can be an important driver of rhinoscleroma disease. In this study, we describe the creation of the first mutant of K. rhinoscleromatis, inactivated in its capsule export machinery. Using a murine model recapitulating the formation of Mikulicz cells in lungs, we observed that a K. rhinoscleromatis CPS mutant (KR cps-) is strongly attenuated and that mice infected with a high dose of KR cps- are still able to induce Mikulicz cells formation, unlike a K. pneumoniae capsule mutant, and to partially recapitulate the characteristic strong production of IL-10. Altogether, the results of this study show that CPS is a virulence factor of K. rhinoscleromatis not involved in the specific appearance of Mikulicz cells.

Genome-wide mosaicism within Mycobacterium abscessus: evolutionary and epidemiological implications.

BACKGROUND: In mycobacteria, conjugation differs from the canonical Hfr model, but is still poorly understood. Here, we quantified this evolutionary processe in a natural mycobacterial population, taking advantage of a large clinical strain collection of the emerging pathogen Mycobacterium abscessus (MAB). RESULTS: Multilocus sequence typing confirmed the existence of three M. abscessus subspecies, and unravelled extensive allelic exchange between them. Furthermore, an asymmetrical gene flow occurring between these main lineages was detected, resulting in highly admixed strains. Intriguingly, these mosaic strains were significantly associated with cystic fibrosis patients with lung infections or chronic colonization. Genome sequencing of those hybrid strains confirmed that half of their genomic content was remodelled in large genomic blocks, leading to original tri-modal 'patchwork' architecture. One of these hybrid strains acquired a locus conferring inducible macrolide resistance, and a large genomic insertion from a slowly growing pathogenic mycobacteria, suggesting an adaptive gene transfer. This atypical genomic architecture of the highly recombinogenic strains is consistent with the distributive conjugal transfer (DCT) observed in M. smegmatis. Intriguingly, no known DCT function was found in M. abscessus chromosome, however, a p-RAW-like genetic element was detected in one of the highly admixed strains. CONCLUSION: Taken together, our results strongly suggest that MAB evolution is sporadically punctuated by dramatic genome wide remodelling events. These findings might have far reaching epidemiological consequences for emerging mycobacterial pathogens survey in the context of increasing numbers of rapidly growing mycobacteria and M. tuberculosis co-infections.

Resequencing microarray technology for genotyping human papillomavirus in cervical smears.

There are more than 40 human papillomaviruses (HPVs) belonging to the alpha genus that cause sexually transmitted infections; these infections are among the most frequent and can lead to condylomas and anogenital intra-epithelial neoplasia. At least 18 of these viruses are causative agents of anogenital carcinomas. We evaluated the performance of a resequencing microarray for the detection and genotyping of alpha HPV of clinical significance using cloned HPV DNA. To reduce the number of HPV genotypes tiled on microarray, we used reconstructed ancestral sequences (RASs) as they are more closely related to the various genotypes than the current genotypes are among themselves. The performance of this approach was tested by genotyping with a set of 40 cervical smears already genotyped using the commercial PapilloCheck kit. The results of the two tests were concordant for 70% (28/40) of the samples and compatible for 30% (12/40). Our findings indicate that RASs were able to detect and identify one or several HPV in clinical samples. Associating RASs with homonym sequences improved the genotyping of HPV present in cases of multiple infection. In conclusion, we demonstrate the diagnostic potential of resequencing technology for genotyping of HPV, and illustrate its value both for epidemiological studies and for monitoring the distribution of HPV in the post-vaccination era.

Carbapenem-resistant Klebsiella pneumoniae exhibit variability in capsular polysaccharide and capsule associated virulence traits.

BACKGROUND: Novel therapies are urgently needed to treat carbapenem-resistant Klebsiella pneumoniae (CR-Kp)-mediated infection, which constitute a major health threat in the United States. In order to assess if it is feasible to develop anticapsular antibodies as a potential novel therapy, it is crucial to first systematically characterize capsular polysaccharide (CPS) and virulence traits in these strains. METHODS: Forty CR-Kp were genotyped by pulsed field gel electrophoresis, multilocus sequence typing (MLST), and molecular capsule typing (C-patterns and wzi sequencing). Their biofilm formation, serum resistance, macrophage-mediated killing, and virulence in Galleria mellonella were compared. MAb (1C9) was generated by co-immunization with 2 CPSs, and cross-reactivity was investigated. RESULTS: MLST assigned 80% of CR-Kp isolates to the ST258-clone. Molecular capsule typing identified new C-patterns, including C200/wzi-154, which was widely represented and associated with blaKPC-3-bearing strains. Heterogeneity was detected in biofilm formation and macrophage-mediated killing. Differences in serum resistance correlated with virulence in G. mellonella. ST258 strains carrying blaKPC-3 were less virulent than those with blaKPC-2. MAb 1C9 cross-reacted with 58% of CR-Kp CPSs. CONCLUSIONS: CR-Kp ST258 strains exhibit variability of virulence-associated traits. Differences were associated with the type of KPC gene and CPS. Identification of cross-reacting anti-CPS mAbs encourages their development as adjunctive therapy.

Multilocus sequence typing scheme for the Mycobacterium abscessus complex.

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world.

A novel murine model of rhinoscleroma identifies Mikulicz cells, the disease signature, as IL-10 dependent derivatives of inflammatory monocytes.

Rhinoscleroma is a human specific chronic disease characterized by the formation of granuloma in the airways, caused by the bacterium Klebsiella pneumoniae subspecies rhinoscleromatis, a species very closely related to K. pneumoniae subspecies pneumoniae. It is characterized by the appearance of specific foamy macrophages called Mikulicz cells. However, very little is known about the pathophysiological processes underlying rhinoscleroma. Herein, we characterized a murine model recapitulating the formation of Mikulicz cells in lungs and identified them as atypical inflammatory monocytes specifically recruited from the bone marrow upon K. rhinoscleromatis infection in a CCR2-independent manner. While K. pneumoniae and K. rhinoscleromatis infections induced a classical inflammatory reaction, K. rhinoscleromatis infection was characterized by a strong production of IL-10 concomitant to the appearance of Mikulicz cells. Strikingly, in the absence of IL-10, very few Mikulicz cells were observed, confirming a crucial role of IL-10 in the establishment of a proper environment leading to the maturation of these atypical monocytes. This is the first characterization of the environment leading to Mikulicz cells maturation and their identification as inflammatory monocytes.

Optimized Multilocus variable-number tandem-repeat analysis assay and its complementarity with pulsed-field gel electrophoresis and multilocus sequence typing for Listeria monocytogenes clone identification and surveillance.

Populations of the food-borne pathogen Listeria monocytogenes are genetically structured into a small number of major clonal groups, some of which have been implicated in multiple outbreaks. The goal of this study was to develop and evaluate an optimized multilocus variable number of tandem repeat (VNTR) analysis (MLVA) subtyping scheme for strain discrimination and clonal group identification. We evaluated 18 VNTR loci and combined the 11 best ones into two multiplexed PCR assays (MLVA-11). A collection of 255 isolates representing the diversity of clonal groups within phylogenetic lineages I and II, including representatives of epidemic clones, were analyzed by MLVA-11, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). MLVA-11 had less discriminatory power than PFGE, except for some clones, and was unable to distinguish some epidemiologically unrelated isolates. Yet it distinguished all major MLST clones and therefore constitutes a rapid method to identify epidemiologically relevant clonal groups. Given its high reproducibility and high throughput, MLVA represents a very attractive first-line screening method to alleviate the PFGE workload in outbreak investigations and listeriosis surveillance.

Multilocus sequence typing as a replacement for serotyping in Salmonella enterica.

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.

Expansion of European vacA and cagA alleles to East-Asian Helicobacter pylori strains in Cambodia.

Helicobacter pylori infection is associated with gastric cancer (GC). The highest incidence rates have been described in Asia, but regional variations exist that do not match the distribution of infection prevalence rates. The aim of the study was to examine the possible contribution of H. pylori virulence factors to geographic differences in the incidence of GC across East and Southeast Asia. We studied 66 isolates from Cambodian patients that had previously been assigned to two genetic populations based on sequences of seven housekeeping genes, namely hpEurope (n = 34, 51.5%) and hpEastAsia, subpopulation hspEAsia (n = 32, 48.5%). These strains were characterized with respect to vacA polymorphism and cagA status by PCR, and the CagA C-terminal region was sequenced. We also sequenced the complete cagA gene from 10 hpEurope and 10 hspEAsia strains chosen at random. The cagA gene was present in 92.4% of the 66 isolates and was mainly of Western type (n = 36, 59.0%). hspEAsia strains carrying East-Asian CagA and the m1-type vacA allele (15.2%) were less frequent among the 66 Cambodian isolates than reported in East Asian countries, a finding that might partly explain the intermediate incidence of GC in Cambodia, and by extension, in Southeast Asia (except for Vietnam). The observed high prevalence of s1a alleles (34.4%) and Western CagA (28.1%) among hspEAsia strains indicates frequent introgression of European vacA and cagA alleles into East Asian H. pylori strains. This expansion might have severe consequences for individual disease outcome.

Evolutionary history of Helicobacter pylori sequences reflect past human migrations in Southeast Asia.

The human population history in Southeast Asia was shaped by numerous migrations and population expansions. Their reconstruction based on archaeological, linguistic or human genetic data is often hampered by the limited number of informative polymorphisms in classical human genetic markers, such as the hypervariable regions of the mitochondrial DNA. Here, we analyse housekeeping gene sequences of the human stomach bacterium Helicobacter pylori from various countries in Southeast Asia and we provide evidence that H. pylori accompanied at least three ancient human migrations into this area: i) a migration from India introducing hpEurope bacteria into Thailand, Cambodia and Malaysia; ii) a migration of the ancestors of Austro-Asiatic speaking people into Vietnam and Cambodia carrying hspEAsia bacteria; and iii) a migration of the ancestors of the Thai people from Southern China into Thailand carrying H. pylori of population hpAsia2. Moreover, the H. pylori sequences reflect iv) the migrations of Chinese to Thailand and Malaysia within the last 200 years spreading hspEasia strains, and v) migrations of Indians to Malaysia within the last 200 years distributing both hpAsia2 and hpEurope bacteria. The distribution of the bacterial populations seems to strongly influence the incidence of gastric cancer as countries with predominantly hspEAsia isolates exhibit a high incidence of gastric cancer while the incidence is low in countries with a high proportion of hpAsia2 or hpEurope strains. In the future, the host range expansion of hpEurope strains among Asian populations, combined with human motility, may have a significant impact on gastric cancer incidence in Asia.

PCR-based identification of Klebsiella pneumoniae subsp. rhinoscleromatis, the agent of rhinoscleroma.

Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma.

Species distribution and ribotype diversity of Burkholderia cepacia complex isolates from French patients with cystic fibrosis.

A total of 153 Burkholderia cepacia strains obtained from 153 French patients with cystic fibrosis were identified as Burkholderia multivorans (51.6%) or Burkholderia cenocepacia (45.1%). Eighty-two genotypes were identified using PvuII and EcoRI ribotyping. B. multivorans genotype A (found in 32 French patients) and two other genotypes were also identified among isolates from Austrian, German, Italian, and Canadian patients.