Genotypic and phenotypic modifications of Neisseria meningitidis after an accidental human passage.
Bottom Line: By comparing the number of SNP in all three isolates and knowing the number of passages between Z5463 and Z5463PI, we concluded that around 25 bacterial divisions occurred in the human body.Different pilin variants were found after the in vivo passage, which expressed different properties of adhesion.Furthermore the deletion of one gene involved in LOS biosynthesis (lgtB) results in Z5463BC expressing a different LOS than the L9 immunotype of Z2491.
Affiliation: INSERM U1002, Paris, France.
A scientist in our laboratory was accidentally infected while working with Z5463, a Neisseria meningitidis serogroup A strain. She developed severe symptoms (fever, meningism, purpuric lesions) that fortunately evolved with antibiotic treatment to complete recovery. Pulse-field gel electrophoresis confirmed that the isolate obtained from the blood culture (Z5463BC) was identical to Z5463, more precisely to a fourth subculture of this strain used the week before the contamination (Z5463PI). In order to get some insights into genomic modifications that can occur in vivo, we sequenced these three isolates. All the strains contained a mutated mutS allele and therefore displayed an hypermutator phenotype, consistent with the high number of mutations (SNP, Single Nucleotide Polymorphism) detected in the three strains. By comparing the number of SNP in all three isolates and knowing the number of passages between Z5463 and Z5463PI, we concluded that around 25 bacterial divisions occurred in the human body. As expected, the in vivo passage is responsible for several modifications of phase variable genes. This genomic study has been completed by transcriptomic and phenotypic studies, showing that the blood strain used a different haemoglobin-linked iron receptor (HpuA/B) than the parental strains (HmbR). Different pilin variants were found after the in vivo passage, which expressed different properties of adhesion. Furthermore the deletion of one gene involved in LOS biosynthesis (lgtB) results in Z5463BC expressing a different LOS than the L9 immunotype of Z2491. The in vivo passage, despite the small numbers of divisions, permits the selection of numerous genomic modifications that may account for the high capacity of the strain to disseminate.
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Mentions: The week before becoming ill, the patient had been working with a derivative of Z5463. Therefore, we first aimed at assessing whether the strain isolated from the blood culture was a derivative of Z5463. A PFGE analysis was performed (Fig. 1A) using material obtained directly from the frozen stocks of Z5463, Z5463PI, the isolate manipulated the week before the accident, and Z5463BC, the isolate obtained directly from the blood culture of the patient. Z5463PI had initially been selected from a colony of Z5463 for its ability to produce high amounts of the MDA phage proteins on colony immunoblots using an antibody against a peptide of NMA1796, predicted to be the main capsid protein of the MDA phage. Four passages took place between the frozen stock of Z5463 and that of Z5463PI. All three strains have an identical PFGE profile when digested by BglII (Fig. 1A), NheI or SpeI (data not shown). Furthermore, considering that Z5463PI had been selected on the basis of the level of production of phage proteins, a Southern-blot using an internal probe of the MDA phage was performed on the BglII digested PFGE gel (Fig. 1B). This experiment identified an additional occurence of the MDA prophage in Z5463PI and Z5463BC chromosomes. The locations of the MDA prophage in the chromosomes were mapped by LM-PCR as described in the experimental procedure section. Both strains had two copies of the MDA phage in the same places (Fig. 1C). The first copy corresponded to the initial insertion of the phage in dRS3 repeats localized between NMA1791 and NMA1801 (gpm). The second phage copy was localised in dRS3 repeats localized between NMA1110 and NMA1111. The precise location of this phage was found to be exactly the same in both strains: in a dinucleotide CT at position 1058887 of the Z2491 chromosome, in the middle of the dRS3 sequence. The flanking regions of the phage occurrences sharing no homology, we concluded for an active mechanism involved in the MDA phage duplication. Altogether, these data demonstrate that the infecting strain was Z5463PI, which has two copies of the MDA prophage inserted in target dRS3 sequences.