Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola.

BACKGROUND: Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species. RESULT: This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates. CONCLUSIONS: This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

Lower IgG somatic hypermutation rates during acute dengue virus infection is compatible with a germinal center-independent B cell response.

BACKGROUND: The study of human B cell response to dengue virus (DENV) infection is critical to understand serotype-specific protection and the cross-reactive sub-neutralizing response. Whereas the first is beneficial and thus represents the ultimate goal of vaccination, the latter has been implicated in the development of severe disease, which occurs in a small, albeit significant, fraction of secondary DENV infections. Both primary and secondary infections are associated with the production of poly-reactive and cross-reactive IgG antibodies. METHODS: To gain insight into the effect of DENV infection on the B cell repertoire, we used VH region high-throughput cDNA sequencing of the peripheral blood IgG B cell compartment of 19 individuals during the acute phase of infection. For 11 individuals, a second sample obtained 6 months later was analyzed for comparison. Probabilities of sequencing antibody secreting cells or memory B cells were estimated using second-order Monte Carlo simulation. RESULTS: We found that in acute disease there is an increase in IgG B cell diversity and changes in the relative use of segments IGHV1-2, IGHV1-18, and IGHV1-69. Somewhat unexpectedly, an overall low proportion of somatic hypermutated antibody genes was observed during the acute phase plasmablasts, particularly in secondary infections and those cases with more severe disease. CONCLUSIONS: Our data are consistent with an innate-like antiviral recognition system mediated by B cells using defined germ-line coded B cell receptors, which could provide a rapid germinal center-independent antibody response during the early phase of infection. A model describing concurrent T-dependent and T-independent B cell responses in the context of DENV infection is proposed, which incorporates the selection of B cells using hypomutated IGHV segments and their potential role in poly/cross-reactivity. Its formal demonstration could lead to a definition of its potential implication in antibody-dependent enhancement, and may contribute to rational vaccine development efforts.

Draft Genome Sequences of Klebsiella variicola Plant Isolates.

Three endophytic Klebsiella variicola isolates-T29A, 3, and 6A2, obtained from sugar cane stem, maize shoots, and banana leaves, respectively-were used for whole-genome sequencing. Here, we report the draft genome sequences of circular chromosomes and plasmids. The genomes contain plant colonization and cellulases genes. This study will help toward understanding the genomic basis of K. variicola interaction with plant hosts.

Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain.

BACKGROUND: Despite the potential to produce antibodies that can neutralize different virus (heterotypic neutralization), there is no knowledge of why vaccination against influenza induces protection predominantly against the utilized viral strains (homotypic response). Identification of structural patterns of the B cell repertoire associated to heterotypic neutralization may contribute to identify relevant epitopes for a universal vaccine against influenza. METHODS: Blood samples were collected from volunteers immunized with 2008/2009 trivalent inactivated vaccine (TIV), pandemic H1N1 (pdmH1N1) monovalent inactivated vaccine (MIV) and the 2014/2015 TIV. Neutralization was assessed by hemagglutination and microneutralization test. IgG V(H) amplicons derived from peripheral blood RNA from pre-immune and 7 days post vaccination were subjected to 454-Roche sequencing. Full reconstruction of the sampled repertoires was done with ImmunediveRsity. RESULTS: The TIV induced a predominantly homotypic neutralizing serologic response, while the 09 MIV induced a heterotypic neutralizing seroconversion in 17% of the individuals. Both the 08/09 and the 14/15 TIV were associated with a reduction in clonotypic diversity, whereas 09 MIV was the opposite. Moreover, TIV and MIV induced distinctive patterns of IGHV segment use that are consistent with B cell selection by conserved antigenic determinants shared by the pre-pandemic and the pandemic strains. However, low somatic hypermutation rates in IgG after 09 MIV immunization, but not after 08/09 and 14/15 TIV immunization were observed. Furthermore, no evidence of the original antigenic sin was found in the same individuals after vaccination with the three vaccines. CONCLUSIONS: Immunization with a new influenza virus strain (2009 pdmH1N1) induced unique effects in the peripheral B cell repertoire clonal structure, a stereotyped response involving distinctive IGHV segment use and low somatic hypermutation levels. These parameters were contrastingly different to those observed in response to pre-pandemic and post-pandemic vaccination, and may be the result of clonal selection of common antigenic determinants, as well as germinal center-independent responses that wane as the pandemic strain becomes seasonal. Our findings may contribute in the understanding of the structural and cellular basis required to develop a universal influenza vaccine.