Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo, Which Can Be Used as a Target for Bactericidal Antibody

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.Nontypeable Haemophilus influenzae is an important pathogen in middle ear infections in children, sinusitis in adults, and acute bronchitis in individuals with chronic obstructive lung disease. The organism is very well adapted to the human host environment, and this has hindered successful development of an effective vaccine. In this article, we describe a mechanism by which the bacteria decorates its surface lipooligosaccharide with a sugar unique to Gram-negative bacteria, ketodeoxyoctanoate (KDO). This sugar decoration is present during active infection and we have shown that an antibody directed against this sugar can result in killing of the organism. These data demonstrate that the lipooligosaccharide ketodeoxyoctanoate epitope may be a novel NTHi-specific candidate vaccine antigen

Characterizing the bioburden of ESBL-producing organisms in a neonatal unit using chromogenic culture media: a feasible and efficient environmental sampling method

Introduction Infections due to extended spectrum beta-lactamase producing organisms (ESBL) have emerged as the leading cause of sepsis among hospitalized neonates in Botswana and much of sub-Saharan Africa and south Asia. Yet, ESBL reservoirs and transmission dynamics within the neonatal intensive care unit (NICU) environment are not well-understood. This study aimed to assess the efficiency and feasibility of a chromogenic-culture-media-based environmental sampling approach to characterize the ESBL bioburden within a NICU. Methods A series of four point-prevalence surveys were conducted at a 36-bed NICU at a public tertiary referral hospital in Botswana from January-June 2021. Samples were collected on 4 occasions under semi-sterile technique using 1) flocked swabs & templates (flat surfaces); 2) sterile syringe & tubing (water aspiration); and 3) structured swabbing techniques (hands & equipment). Swabs were transported in physiological saline-containing tubes, vortexed, and 10 µL was inoculated onto chromogenic-agar that was selective and differential for ESBL (CHROMagar™ ESBL, Paris, France), and streaking plates to isolate individual colonies. Bacterial colonies were quantified and phenotypically characterized using biochemical identification tests. Results In total, 567 samples were collected, 248 (44%) of which grew ESBL. Dense and consistent ESBL contamination was detected in and around sinks and certain high-touch surfaces, while transient contamination was demonstrated on medical equipment, caregivers/healthcare worker hands, insects, and feeding stations (including formula powder). Results were available within 24–72 h of collection. To collect, plate, and analyse 50 samples, we estimated a total expenditure of $269.40 USD for materials and 13.5 cumulative work hours among all personnel. Conclusions Using basic environmental sampling and laboratory techniques aided by chromogenic culture media, we identified ESBL reservoirs (sinks) and plausible transmission vehicles (medical equipment, infant formula, hands of caregivers/healthcare workers, & insects) in this NICU environment. This strategy was a simple and cost-efficient method to assess ESBL bioburden and may be feasible for use in other settings to support ongoing infection control assessments and outbreak investigations.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofReviewedFacultyOthe

Quantification of recA gene expression as an indicator of repair potential in marine bacterioplankton communities of Antarctica

Marine bacteria in surface waters must cope daily with the damaging effects of exposure to solar radiation (containing both UV-A and UV-B wavelengths), which produces lesions in their DNA. As the stratospheric ozone layer is depleted, these coping mechanisms are likely to play an even more important role in the viability of marine bacterial communities. The recA gene is ubiquitous among eubacteria and is highly conserved both in nucleotide and amino acid sequence. Besides its role in generalized recombination, the gene's translational product, RecA, is the regulator of 'dark repair' activity (DNA-repair mechanisms that do not require visible light as a cofactor). We have taken advantage of this function and used recA gene expression as a barometer of the DNAdamage repair capacity of bacterial assemblages in the Southern Ocean. Studies were conducted in the Gerlache Strait, Antarctica, in the austral springs of 1995 and 1996. Analysis of both recA mRNA and RecA protein extracted from natural communities indicated that the level of expression of this gene varied in a diel fashion, suggesting an increased repair capacity in these organisms. These included an early morning rise in RecA levels followed by a plateau or even a reduction in RecA concentration during the remainder of the day. A much greater increase in RecA was consistently observed after sunset, followed by a constant decrease during the night. Microcosm experiments with a RecA+ Gerlache Strait g-proteobacteria isolate, RM11001, demonstrated a similar diel pattern of expression. These studies demonstrate the usefulness of RecA as a biological indicator of DNA repair capacity in natural bacterial assemblages. They indicate that 'dark repair' of DNA damage is an important coping mechanism for bacteria in the marine environment of Antarctica.Peer reviewedMicrobiology and Molecular Genetic