Maria Leticia Zarantonelli

Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis

ABSTRACT Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA ). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

The duality of virulence and transmissibility in Neisseria meningitidis

Neisseria meningitidis is a commensal bacterium of the human nasopharynx that occasionally provokes invasive disease. Carriage strains of N. meningitidis are heterogeneous, more frequent in nature and are transmitted among carriers. Disease is not a part of this transmission cycle and is caused by virulent strains. N. meningitidis is highly variable and variants that are modified in their virulence and/or transmissibility are continually generated. These events probably occur frequently, thus explaining not only the heterogeneous nature of meningococcal populations in carriers but probably also the evolutionary success of this human-restricted bacterium.

A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice

We developed a model of sequential influenza A virus (IAV)-Neisseria meningitidis serogroup C (Nm) infection in BALB/c mice. Mice infected intranasally with a sublethal IAV dose (260 pfu) were superinfected intranasally with Nm. Fatal meningococcal pneumonia and bacteremia were observed in IAV-infected mice superinfected with Nm on day 7, but not in those superinfected on day 10. The susceptibility of mice to Nm superinfection was correlated with the peak interferon-gamma production in the lungs and decrease in IAV load. After Nm challenge, both IAV-infected and uninfected control mice produced the inflammatory cytokines interleukin (IL)-1 and IL-6. However, IL-10 was detected in susceptible mice superinfected on day 7 after IAV infection, but not in resistant mice. This model of dual IAV-Nm infection was also used to evaluate the role of bacterial virulence factors in the synthesis of the capsule. A capsule-defective mutant was cleared from the lungs, whereas a mutant inactivated for the crgA gene, negatively regulating expression of the pili and capsule, upon contact with host cells, retained invasiveness. Therefore, this model of meningococcal disease in adult mice reproduces the pathogenesis of human meningococcemia with fatal sepsis, and is useful for analyzing known or new genes identified in genomic studies.

Influenza A Virus Neuraminidase Enhances Meningococcal Adhesion to Epithelial Cells through Interaction with Sialic Acid-Containing Meningococcal Capsules

ABSTRACT The underlying mechanisms of the epidemiological association between influenza virus infections and Neisseria meningitidis invasive infections are not fully understood. Here we report that adhesion of N. meningitidis to human Hec-1-B epithelial cells is enhanced by influenza A virus (IAV) infection. A potential role of the viral neuraminidase (NA) in facilitating meningococcal adhesion to influenza virus-infected epithelial cells was examined. Expression of a recombinant IAV NA in Hec-1-B human epithelial cells increased the adhesion of strains of N. meningitidis belonging to the sialic acid-containing capsular serogroups B, C, and W135 but not to the mannosamine phosphate-containing capsular serogroup A. Adhesion enhancement was not observed with an inactive NA mutant or in the presence of an NA inhibitor (zanamivir). Furthermore, purified IAV NA was shown to cleave sialic acid-containing capsular polysaccharides of N. meningitidis. On the whole, our findings suggest that a direct interaction between the NA of IAV and the capsule of N. meningitidis enhances bacterial adhesion to cultured epithelial cells, most likely through cleavage of capsular sialic acid-containing polysaccharides. A better understanding of the association between IAV and invasive meningococcal infections should help to set up improved control strategies against these seasonal dual viral-bacterial infections.

Rifampin-resistant Neisseria meningitidis.

To the Editor: Immediate management of meningococcal disease requires antimicrobial drug treatment of patients with β-lactams and chemoprophylaxis of contact persons with rifampin. High-level resistance to rifampin (MIC >32 mg/L) in Neisseria meningitidis is provoked by mutations (most frequently at the residue His 552) in the rpoB gene encoding the b subunit of RNA polymerase (1,2). Resistance may lead to chemoprophylaxis failure and must be rapidly detected (3). Concerns have been raised about the clonal spread of resistant isolates (1); however, rifampin-resistant isolates are rarely reported. We tested 6 N. meningitidis isolates corresponding to 3 pairs of linked cases of meningococcal disease. In each pair, the index case was due to a rifampin-susceptible isolate and was followed by the secondary case due to a resistant isolate in a contact person. Phenotyping and genotyping of the isolates showed that each pair belonged to a different major serogroup (A, B, and C) and to a different genetic lineage (ST-7, ST-32, and ST-2794) (Figure). We next amplified a fragment in rpoB between codons 421 and 701 by using oligonucleotide rpoBF1 (5´gttttcccagtcacgacgttgtaCTGTCCGAAGCCCAACAAAACTCTTGG3´) and rpoBR1 (5´ttgtgagcggataacaatttcTTCCAAGAATGGAATCAGGGATGCTGC3´). The 2 oligonucleotides harbor adaptors (in lower case) corresponding to universal forward and reverse oligonucleotides that can be used for sequencing after amplification. We also analyzed 2 cerebrospinal fluid (CSF) samples corresponding to 2 linked culture-negative cases of meningococcal disease in which the second case was believed to have been caused by rifampin-resistant N. meningitidis. These 2 cases were diagnosed by polymerase chain reaction (PCR) detection of meningococcal DNA, as previously described (4). Figure Blood bacterial counts in 6-week-old female BALB/c mice (Janvier, France), challenged intraperitoneally with standardized inocula of 107 colony forming units (CFU) of rifampin-susceptible (RifS) isolates and their corresponding rifampin-resistant (RifR) ... The 3 rifampin-susceptible isolates harbored a wild-type rpoB sequence (His 552), as did the first CSF sample. All 3 rifampin-resistant isolates harbored a His→Tyr mutation, while analysis of the second CSF sample showed a His→Asn mutation (Figure). Both mutations have been observed in N. meningitidis (3). No other difference in the sequence was seen among all isolates on the amplified fragment. This approach can rapidly detect rpoB mutations and can be applied to culture-negative clinical samples. The virulence of the isolates was evaluated through their ability to provoke bacteremia in mice after 6-week-old female BALB/c mice (Janvier, France) were injected intraperitoneally. Bacteremia is a good indicator of bacterial virulence as it reflects bacterial survival upon invasion of the bloodstream. The experimental design was approved by the Institut Pasteur Review Board. The rifampin-resistant clinical isolate LNP22330 showed substantially reduced bacteremia when compared to the corresponding susceptible isolate LNP21362 (Figure). Such a reduction was not significant for the other 2 pairs (LNP18278/LNP18378 and LNP18368/LNP18491), but these strains were all less virulent than LNP21362, with ≈1 log10 lower blood bacterial loads. The 3 pairs of isolates belonged to different genetic lineages according to the multilocus sequence typing typing. Indeed, we have recently proved that virulence of meningococcal isolates in the mouse model depends on the genetic lineage of the tested isolate (5). To better study the impact of rpoB mutation on meningococcal virulence we constructed an isogenic mutant strain, NM05-08, by transforming the susceptible isolate LNP21362 with a PCR-amplified fragment from a resistant isolate (LNP22330), as previously described (6). The PCR fragment corresponded to the product of amplification between the oligonucleotides ropB1UP (5´ggccgtctgaaCTGTCCGAAGCCCAACAAAACTCTTGG3´) and rpoBR1. The oligonucleotide RpoB1UP is the same as the upstream rpoBF1 but with a DNA uptake sequence (in lower case) that was added at the 5´ end to permit DNA transformation (7). The transformant strain NM05-08 was resistant to rifampin (MIC >32 mg/L), and the sequence of the rpoB gene confirmed the His→Tyr mutation. When compared to the parental isolate (LNP21362), strain NM05-08 showed reduced virulence. Indeed, bacterial loads were similar to those observed for the resistant isolate LNP22330 (Figure). These results strongly suggest a direct negative impact of rpoB mutations on meningococcal virulence. Mutations in the rpoB gene have been reported to confer pleiotropic phenotypes (8). The data reported here show that rifampin-resistant isolates were not clonal but belonged to different genetic lineages. The results of virulence assays in mice suggest that mutations in rpoB in resistant isolates may have a major biological cost for N. meningitidis, which can be defined as lower bacterial fitness in terms of survival in the bloodstream. This biological cost could explain the lack of clonal expansion of meningococcal isolates that acquired resistance to rifampin.

Differential Role of Lipooligosaccharide of Neisseria meningitidis in Virulence and Inflammatory Response during Respiratory Infection in Mice

ABSTRACT Meningococcal lipooligosaccharide (LOS) induces a strong proinflammatory response in humans during meningococcal infection. We analyzed the role of LOS in the inflammatory response and virulence during the early infectious process in a mouse model of meningococcal respiratory challenge. An lpxA mutant strain (serogroup B) devoid of LOS (strain Z0204) could not persist in the lungs and did not invade the blood. The persistence in the lungs and invasion of the bloodstream by a rfaD mutant expressing truncated LOS with only lipid A and 3-deoxy-d-manno-2-octulosonic acid molecules (strain Z0401) was intermediate between those of the wild-type and Z0204 strains. Both LOS mutants induced acute pneumonia with the presence of infiltrating polymorphonuclear leukocytes in lungs. Although tumor necrosis factor alpha production was reduced in mice infected with the mutant of devoid LOS, both LOS mutants induced production of other proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and the murine IL-8 homolog KC. Together, these results suggest that meningococcal LOS plays a role during the early infectious and invasive process, and they further confirm that other, nonlipopolysaccharide components of Neisseria meningitidis may significantly contribute to the inflammatory reaction of the host.

Interlaboratory Comparison of PCR-Based Methods for Detection of Penicillin G Susceptibility in Neisseria meningitidis

ABSTRACT We carried out a study for the nonculture detection of susceptibility of Neisseria meningitis to penicillin G in three laboratories of the European Monitoring Group on Meningococci (EMGM). Thirteen clinical samples (cerebrospinal fluids) and corresponding bacterial isolates from 13 cases of invasive meningococcal infection were distributed to the three laboratories. The MICs of penicillin G were determined for the isolates. Each laboratory used an “in-house” PCR-based method to determine alterations to the penA gene, which is associated with a reduced susceptibility to penicillin G. Nucleotide sequences from the 3′ end of the penA gene were also determined. We observed a good correlation between genotyping of penA and the phenotypic determination (MIC) of susceptibility to penicillin G. The results obtained by the three methods for penA in the samples correlated very well with those obtained in bacterial isolates and with sequence data. The kappa coefficient that was used to estimate the level of agreement between genotypic results varied between 0.65 and 1, indicating a good agreement. This suggests that genotyping can predict susceptibility of N. meningitidis to penicillin G. These data strongly suggest that genotyping of penA should be used to determine meningococcal susceptibility to penicillin G in culture-negative cases. Although the nucleotide sequence of penA may be the gold standard in genotyping of penA, the less expensive PCR-based approach reported in this study may be quicker when a large number of isolates and clinical samples need to be tested.

Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml±0.1), CD80 (LOGEC50 = 4.88 µg/ml±0.15) and CD86 (LOGEC50 = 5.36 µg/ml±0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7±5.1% cells versus 12±2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed that PBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.