We herein report the isolation and characterization of 21 Gram-stain-negative strains cultivated from the oropharynx of healthy children in Israel and Switzerland. Initially described as small colony variants of Kingella kingae, phenotypic analysis, biochemical analysis, phylogenetic analysis based on sequencing of the partial 16S rRNA gene and five housekeeping genes (abcZ, adk, G6PD, groEL and recA), and whole genome sequencing and comparison between members of the genera Kingella and Neisseria provided evidence for assigning them to the genus Kingella. Cellular fatty acids included important amounts of C12 : 0, C14 : 0, C16 : 0 and C16 : 1n7. Digital DNA-DNA hybridization between the isolates Sch538T and K. kingae ATCC 23330T revealed relatedness of 19.9 %. Comparative analysis of 16S rRNA gene sequences available in GenBank allowed matches to strains isolated in the USA, suggesting a wider geographical distribution. A novel species named Kingella negevensis sp. nov. is proposed, as most strains have been isolated in the Negev, a desert region of southern Israel. The type strain is Sch538T (=CCUG 69806T=CSUR P957).

is an important invasive pathogen in early childhood. The organism elaborates an RTX toxin presumably restricted to this species. Consequently, real-time quantitative PCR (qPCR) assays targeting the RTX locus have been developed in recent years and are gaining increasing use for the molecular diagnosis of infections. However, the present study shows that, a species newly identified in young children, harbors an identical RTX locus, raising the question of whether can be misidentified as by clinical microbiology laboratories. comparison of sp. RTX and genes and studies provided evidence that targeting the and genes could not differentiate between strains of and, whereas targeting the gene could. This prompted the design of a highly specific and sensitive qPCR assay targeting (). Ninety-nine culture-negative osteoarticular specimens from 99 children younger than 4 years of age were tested with a conventional 16S rRNA gene-based broad-range PCR assay and -specific, -specific (), and qPCR assays. Forty-two specimens were positive, including 41 that were also positive and 1 (the remaining one) that was positive. Thus, this study discloses an invasive infection caused by in humans and demonstrates that targeting the RTX locus cannot be used for the formal diagnosis of infections. These findings stress the need for further studies on the epidemiology of asymptomatic carriage and invasive infections caused by in humans.

is a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting the gene and the RTX locus of However, recent studies showed that real-time PCR (RT-PCR) assays targeting the sp. RTX locus that are currently available for the diagnosis of infection lack specificity because they could not distinguish between and the recently described species. Furthermore, analysis of the gene from a large collection of 45 strains showed that primers and probes from-based RT-PCR assays display a few mismatches with variations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative to- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, a -specific RT-PCR assay targeting the malate dehydrogenase () gene was developed for predicting no mismatch between primers and probe and 18 variants of the gene from 20 distinct sequence types of This novel -specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnose infections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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K. kingae outbreaks displayed severe K. kingae diseases that were poorly confirmed with culture methods. We argue for the use of genomic technologies to investigate further K. kingae outbreaks.

We present an evolutionary hypothesis assuming that signals marking nucleotide synthesis (DNA replication and RNA transcription) evolved from multi- to unidimensional structures, and were carried over from transcription to translation. This evolutionary scenario presumes that signals combining secondary and primary nucleotide structures are evolutionary transitions. Mitochondrial replication initiation fits this scenario. Some observations reported in the literature corroborate that several signals for nucleotide synthesis function in translation, and vice versa. (a) Polymerase-induced frameshift mutations occur preferentially at translational termination signals (nucleotide deletion is interpreted as termination of nucleotide polymerization, paralleling the role of stop codons in translation). (b) Stem-loop hairpin presence/absence modulates codon-amino acid assignments, showing that translational signals sometimes combine primary and secondary nucleotide structures (here codon and stem-loop). (c) Homopolymer nucleotide triplets (AAA, CCC, GGG, TTT) cause transcriptional and ribosomal frameshifts. Here we find in recently described human mitochondrial RNAs that systematically lack mono-, dinucleotides after each trinucleotide (delRNAs) that delRNA triplets include 2x more homopolymers than mitogenome regions not covered by delRNA. Further analyses of delRNAs show that the natural circular code X (a little-known group of 20 translational signals enabling ribosomal frame retrieval consisting of 20 codons {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} universally overrepresented in coding versus other frames of gene sequences), regulates frameshift in transcription and translation. This dual transcription and translation role confirms for X the hypothesis that translational signals were carried over from transcriptional signals.

Primary epiphyseal subacute osteomyelitis (PESAO) caused by Mycobacterium species in young children is poorly recognized. We aimed to define the spectrum of this uncommon condition and to propose a novel diagnostic approach. We performed a systematic review of the literature on the PubMed website by selecting all reports of isolated infantile PESAO caused by Mycobacterium species since 1975. We identified 350 citations, of which 174 were assessed for eligibility based on title and abstract. The full text of 81 eligible citations was screened, and relevant data of 15 children under 4 years of age with mycobacterial PESAO were extracted. These data were pooled with those from our Institution. Data from 16 children were reviewed. The median age was 16 ± 7 months and the male:female ratio 1.7. The knee was the most common infection site (94%). The diagnosis of mycobacterial disease was delayed in all cases (range, 2 weeks to 6 months), and initially presumed by histology in 15 children (94%). Microbiologically proven diagnosis was confirmed by bone cultures in 8 of the 15 children (53%), and by specific PCR in 2 of the 3 culture-negative bone specimens (67%). Three children experienced long-term orthopedic complications despite surgical drainage and prolonged antimycobacterial regimens. All recently reported cases came from high-burden tuberculosis areas. Mycobacterium species contribute to the burden of infantile PESAO in endemic tuberculosis areas and may cause growth disturbances. We argue in favor of the early recognition of mycobacterial disease by specific molecular assays in children with infantile PESAO living in high-burden areas.

We report the investigation methods for the diagnosis of an epidemic and culture-negative Kingella kingae endocarditis complicating a severe outbreak of hand, foot and mouth disease in a childcare center. The diagnosis was confirmed by polymerase chain reaction testing performed from cardiac tissue. Our findings argue for the systematic investigation of K. kingae outbreaks by using molecular tools in such context.