Cindy Fevre

Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization.

Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedländers pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23K1 and clone CC82K1, were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82K1 indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23K1 remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of important virulent clones associated with specific infections and provide an evolutionary framework for research into the links between clones, virulence and other genomic features in K. pneumoniae.

Six Groups of the OXY β-Lactamase Evolved over Millions of Years in Klebsiella oxytoca

ABSTRACT The diversity and evolution of the class A OXY β-lactamase from Klebsiella oxytoca were investigated and compared to housekeeping gene diversity. The entire blaOXY coding region was sequenced in 18 clinical isolates representative of the four K. oxytoca β-lactamase gene groups blaOXY-1 to blaOXY-4 and of two new groups identified here, blaOXY-5 (with four isolates with pI 7.2 and one with pI 7.7) and blaOXY-6 (with four isolates with pI 7.75 and three with pI 8.1). Genes blaOXY-5 and blaOXY-6 showed 99.8% within-group nucleotide similarity but differed from each other by 4.2% and from blaOXY-1, their closest relative, by 2.5% and 2.9%, respectively. Antimicrobial susceptibility to β-lactams was similar among OXY groups. Nucleotide sequence diversity of the 16S rRNA (1,454 bp), rpoB (940 bp), gyrA (383 bp), and gapDH (573 bp) genes was in agreement with the β-lactamase gene phylogeny. Strains with blaOXY-1, blaOXY-2, blaOXY-3, blaOXY-4, and blaOXY-6 genes formed five phylogenetic groups, named KoI, KoII, KoIII, KoIV, and KoVI, respectively. Isolates harboring blaOXY-5 appeared to represent an emerging lineage within KoI. We estimated that the blaOXY gene has been evolving within K. oxytoca for approximately 100 million years, using as calibration the 140-million-year estimation of the Escherichia coli-Salmonella enterica split. These results show that the blaOXY gene has diversified along K. oxytoca phylogenetic lines over long periods of time without concomitant evolution of the antimicrobial resistance phenotype.

Variants of the Klebsiella pneumoniae OKP Chromosomal Beta-Lactamase Are Divided into Two Main Groups, OKP-A and OKP-B

ABSTRACT Two blaOKP subgroups were found, diverging by 4.2%. Subgroups blaOKP-A (10 enzyme variants, pIs from 7.1 to 8.3) and blaOKP-B (11 variants, pI 7.1) showed similar antibiotic susceptibilities. Sequencing of rpoB, gyrA, and mdh demonstrated a concordant subdivision of Klebsiella pneumoniae phylogenetic group KpII into two subgroups, KpII-A and KpII-B.

Comparison of PFGE and multilocus sequence typing for analysis of Klebsiella pneumoniae isolates.

Klebsiella pneumoniae represents an important nosocomial pathogen causing urinary, respiratory and blood infections (Brisse et al., 2006; Podschun & Ullmann, 1998). Hospital outbreaks due to K. pneumoniae are frequent and especially feared when caused by multidrug-resistant strains, such as extended-spectrum blactamase producers (Paterson & Bonomo, 2005). DNA-based strain typing methods are used to distinguish K. pneumoniae clinical isolates in order to understand transmission patterns and to help management of hospital infections. Molecular serotyping, based on PCR– RFLP of the cps operon responsible for capsular polysaccharide expression, has a higher discriminatory ability than traditional K typing (Brisse et al., 2004), and ribotyping is also applicable to K. pneumoniae (Brisse & Verhoef, 2001). Nevertheless, the most commonly used method is PFGE analysis of macrorestriction fragments (Arlet et al., 1994). The main advantage of PFGE lies in its high discriminatory power (Hansen et al., 2002), but PFGE is technically demanding and requires a high level of coordination (e.g. http://www.cdc.gov/ pulsenet) to achieve inter-laboratory reproducibility. In contrast, multilocus sequence typing (MLST) provides unambiguous data that are suitable for global epidemiology and evolutionary studies (Maiden et al., 1998). A MLST method was previously developed for K. pneumoniae, and analysis of nosocomial isolates showed that MLST can discriminate among epidemiologically unrelated isolates (Diancourt et al., 2005). However, the discriminatory power of MLST was not compared to that of PFGE. In our previous study (Diancourt et al., 2005), 28 isolates belonged to 11 groups that were not distinguished by MLST nor by ribotyping. Among these 11 groups, 5 comprised isolates from distinct countries or separated by large sampling times. For these apparently unrelated cases the isolates could be suspected as being genotypically undistinguishable due to an insufficient discriminatory power of MLST and ribotyping, rather than due to an undocumented epidemiological link. We report here on the comparison of the discriminatory power of PFGE with previously reported methods.

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.

Listeria monocytogenes-associated biliary tract infections: a study of 12 consecutive cases and review.

AbstractAt present, little is known regarding Listeria monocytogenes-associated biliary tract infection, a rare form of listeriosis.In this article, we will study 12 culture-proven cases reported to the French National Reference Center for Listeria from 1996 to 2013 and review the 8 previously published cases.Twenty cases were studied: 17 cholecystitis, 2 cholangitis, and 1 biliary cyst infection. Half were men with a median age of 69 years (32–85). Comorbidities were present in 80%, including cirrhosis, rheumatoid arthritis, and diabetes. Five patients received immunosuppressive therapy, including corticosteroids and anti-tumor necrosis factor biotherapies. Half were afebrile. Blood cultures were positive in 60% (3/5). Gallbladder histological lesions were analyzed in 3 patients and evidenced acute, chronic, or necrotic exacerbation of chronic infection. Genoserogroup of the 12 available strains were IVb (n = 6), IIb (n = 5), and IIa (n = 1). Their survival in the bile was not enhanced when compared with isolates from other listeriosis cases. Adverse outcome was reported in 33% (5/15): 3 deaths, 1 recurrence; 75% of the patients with adverse outcome received inadequate antimicrobial therapy (P = 0.033).Biliary tract listeriosis is a severe infection associated with high mortality in patients not treated with appropriate therapy. This study provides medical relevance to in vitro and animal studies that had shown Listeria monocytogenes ability to survive in bile and induce overt biliary infections.

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.

Intestinal Organoids as a Novel Tool to Study Microbes-Epithelium Interactions.

The gut, particularly the colon, is the host of approximately 1000 bacterial species, the so-called gut microbiota. The relationship between the gut microbiota and the host is symbiotic and mutualistic, influencing many aspects of the biology of the host. This homeostatic balance can be disrupted by enteric pathogens, such as Shigella flexneri or Listeria monocytogenes, which are able to invade the epithelial layer and consequently subvert physiological functions. To study the host-microbe interactions in vitro, the crypt culture model, known as intestinal organoids, is a powerful tool. Intestinal organoids provide a model in which to examine the response of the epithelium, particularly the response of intestinal stem cells, to the presence of bacteria. Furthermore, the organoid model enables the study of pathogens during the early steps of enteric pathogen invasion.Here, we describe methods that we have established to study the cellular microbiology of symbiosis between the gut microbiota and host intestinal surface and secondly the disruption of host homeostasis due to an enteric pathogen.

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.

Peyer’s patch myeloid cells infection by Listeria signals through gp38+ stromal cells and locks intestinal villus invasion

The foodborne pathogen Listeria monocytogenes (Lm) crosses the intestinal villus epithelium via goblet cells (GCs) upon the interaction of Lm surface protein InlA with its receptor E-cadherin. Here, we show that Lm infection accelerates intestinal villus epithelium renewal while decreasing the number of GCs expressing luminally accessible E-cadherin, thereby locking Lm portal of entry. This novel innate immune response to an enteropathogen is triggered by the infection of Peyer’s patch CX3CR1+ cells and the ensuing production of IL-23. It requires STAT3 phosphorylation in epithelial cells in response to IL-22 and IL-11 expressed by lamina propria gp38+ stromal cells. Lm-induced IFN-&ggr; signaling and STAT1 phosphorylation in epithelial cells is also critical for Lm-associated intestinal epithelium response. GC depletion also leads to a decrease in colon mucus barrier thickness, thereby increasing host susceptibility to colitis. This study unveils a novel innate immune response to an enteropathogen, which implicates gp38+ stromal cells and locks intestinal villus invasion, but favors colitis.