Kingella kingae and Osteoarticular Infections.

OBJECTIVES: In this study, we aimed to contrast the bacteriologic epidemiology of osteoarticular infections (OAIs) between 2 patient groups in successive 10-year periods, before and after the extensive use of nucleic acid amplification assays in the diagnostic process. METHODS: Epidemiologic data and bacteriologic etiologies of all children presenting with OAIs on admission to our institution over 20 years (1997-2016) were assessed retrospectively. The population was divided into 2 cohorts, using the standardized use of polymerase chain reaction as the cutoff point (2007). The conventional cohort included children with OAIs mainly investigated by using classic cultures, whereas the molecular cohort referred to patients also investigated by using molecular assays. RESULTS: Kingella kingae was the most frequently isolated pathogen, responsible for 51% of OAIs, whereas other classic pathogens were responsible for 39.7% of cases in the molecular cohort. A statistically significant increase in the mean incidence of OAIs was observed, as was a decrease in the mean age at diagnosis after 2007. After 2007, the pathogen remained unidentified in 21.6% of OAIs in our pediatric population. CONCLUSIONS: Extensive use of nucleic acid amplification assays improved the detection of fastidious pathogens and has increased the observed incidence of OAI, especially in children aged between 6 and 48 months. We propose the incorporation of polymerase chain reaction assays into modern diagnostic algorithms for OAIs to better identify the bacteriologic etiology of OAIs.

Genomics of the new species Kingella negevensis: diagnostic issues and identification of a locus encoding a RTX toxin.

Kingella kingae, producing the cytotoxic RTX protein, is a causative agent of serious infections in humans such as bacteremia, endocarditis and osteoarticular infection, especially in young children. Recently, Kingella negevensis, a related species, has been isolated from the oral cavity of healthy children. In this study, we report the isolation of K. negevensis strain eburonensis, initially misidentified as K. kingae with MALDI-TOF MS, from a vaginal specimen of a patient suffering of vaginosis. The genome sequencing and analysis of this strain together with comparative genomics of the Kingella genus revealed that K. negevensis possesses a full homolog of the rtx operon of K. kingae involved in the synthesis of the RTX toxin. We report that a K. kingae specific diagnostic PCR, based on the rtxA gene, was positive when tested on K. negevensis strain eburonensis DNA. This cross-amplification, and risk of misidentification, was confirmed by in silico analysis of the target gene sequence. To overcome this major diagnostic issue we developed a duplex real-time PCR to detect and distinguish K. kingae and K. negevensis. In addition to this, the identification of K. negevensis raises a clinical issue in term of pathogenic potential given the production of a RTX hemolysin.

CEACAM1 negatively regulates IL-1ß production in LPS activated neutrophils by recruiting SHP-1 to a SYK-TLR4-CEACAM1 complex.

LPS-activated neutrophils secrete IL-1ß by activation of TLR-4. Based on studies in macrophages, it is likely that ROS and lysosomal destabilization regulated by Syk activation may also be involved. Since neutrophils have abundant expression of the ITIM-containing co-receptor CEACAM1 and Gram-negative bacteria such as Neisseria utilize CEACAM1 as a receptor that inhibits inflammation, we hypothesized that the overall production of IL-1ß in LPS treated neutrophils may be negatively regulated by CEACAM1. We found that LPS treated neutrophils induced phosphorylation of Syk resulting in the formation of a complex including TLR4, p-Syk, and p-CEACAM1, which in turn, recruited the inhibitory phosphatase SHP-1. LPS treatment leads to ROS production, lysosomal damage, caspase-1 activation and IL-1ß secretion in neutrophils. The absence of this regulation in Ceacam1⁻/⁻ neutrophils led to hyper production of IL-1ß in response to LPS. The hyper production of IL-1ß was abrogated by in vivo reconstitution of wild type but not ITIM-mutated CEACAM1 bone marrow stem cells. Blocking Syk activation by kinase inhibitors or RNAi reduced Syk phosphorylation, lysosomal destabilization, ROS production, and caspase-1 activation in Ceacam1⁻/⁻ neutrophils. We conclude that LPS treatment of neutrophils triggers formation of a complex of TLR4 with pSyk and pCEACAM1, which upon recruitment of SHP-1 to the ITIMs of pCEACAM1, inhibits IL-1ß production by the inflammasome. Thus, CEACAM1 fine-tunes IL-1ß production in LPS treated neutrophils, explaining why the additional utilization of CEACAM1 as a pathogen receptor would further inhibit inflammation.

Neisseria meningitidis escape from the bactericidal activity of a monoclonal antibody is mediated by phase variation of lgtG and enhanced by a mutator phenotype.

Bacteria adapt to environmental changes through high-frequency switches in expression of specific phenotypes. Localized hypermutation mediated by simple sequence repeats is an important mechanism of such phase variation (PV) in Neisseria meningitidis. Loss or gain of nucleotides in a poly(C) tract located in the reading frame results in switches in expression of lgtG and determines whether a glucose or a phosphoethanolamine (PEtn) is added at a specific position in the inner core lipopolysaccharide (LPS). Monoclonal antibody (MAb) B5 is bactericidal for N. meningitidis strain 8047 when PEtn is present in the inner core LPS and lgtG is switched "off." Escape from the bactericidal activity of this antibody was examined by subjecting strain 8047 to multiple cycles of growth in the presence of MAb B5 and human serum. Escape variants with alterations in the lgtG repeat tract rapidly accumulated in bacterial populations during selection with this antibody. Strain 8047 was outcompeted in this assay by the 8047 Delta mutS strain due to the elevated PV rate of this mismatch repair mutant and hence the greater proportion of preexisting phase variants of lgtG in the inoculum. This mutS mutant was also more virulent than strain 8047 during escape from passive protection by MAb B5 in an in vivo infant rat model of bacteremia. These results provide an example of how PV rates can modulate the occurrence and severity of infection and have important implications for understanding the evolution of bacterial fitness in species subject to environmental variations that occur during persistence within and transmission between hosts.

Mutator clones of Neisseria meningitidis in epidemic serogroup A disease.

Serogroup A Neisseria meningitidis has repeatedly caused widespread epidemics of meningitis and septicemia throughout the 20th century. Recently, in a limited collection of strains, epidemic serogroup A isolates were found to have elevated mutation rates that was caused by defects in mismatch repair pathways. To ascertain the role of these mutators in the epidemic spread of this serogroup, the prevalence of hypermutability in a collection of 95 serogroup A N. meningitidis invasive isolates was determined. Overall mutability in Neisseriae can be described by measuring both missense mutation rates as well as phase variation frequencies of "contingency loci." Fifty-seven percent of serogroup A isolates possessed elevated mutability, which could be divided into two classes: intermediate and high level. Eleven of 20 high-level mutators, with phase variation rates >100-fold higher than wild-type isolates, were defective in mismatch repair. Ten of the 34 intermediate mutators possessing >10-fold increases in phase variation rates could be partially complemented by a wild-type mutL allele. A high prevalence of mutators in epidemic isolates indicates that hypermutability may play a major role in the transmission of this pathogen. The added diversity derived from increased phase variation rates may allow fixation of mutator alleles more frequently during epidemic spread.

CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae.

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.

Analysis of Moraxella catarrhalis by DNA typing: evidence for a distinct subpopulation associated with virulence traits.

Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains. Both methods were found to be highly concordant, generating a dendrogram with 2 main branches. The division of the M. catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences. Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene. In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies. The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains. These findings suggest the existence of subpopulations of M. catarrhalis that differ in virulence, and they may have implications for vaccine development.

Polymorphism of the complement C8A and -B genes in two families with C8 beta deficiency and neisserial infections.

Serum samples from members of two Italian families with complement C8 beta deficiency were studied by SDS-PAGE under nonreducing conditions and by IEF. The proband of family I had suffered from two episodes of purulent meningitis and two of her uncles had suffered from only one episode, while the proband of family II had suffered from three different episodes. In contrast to previous findings, where C8 beta deficiency was cosegregating with C8A (alpha-gamma) allotype A, the proband of family II had the C8A allotype B. In addition, in one of her sons a novel variant of the C8 beta chain was detected. Studies at the DNA level in family I, using a recently described PCR system, demonstrate the presence of a C-T exchange, which creates a stop codon in exon 9 of the C8B gene, as cause for the C8 beta deficiency. The mutated allele does not carry the TaqI polymorphic restriction site located in intron 11 of the C8B gene. These findings provide evidence for multiple recombination events within the genetic region of the defective C8B gene.