Sequential markerless genetic manipulations of species from the Neisseria genus.

The development of simple and highly efficient strategies for genetic modifications is essential for postgenetic studies aimed at characterizing gene functions for various applications. We sought to develop a reliable system for Neisseria species that allows for both unmarked and accumulation of multiple genetic modifications in a single strain. In this work, we developed and validated three-gene cassettes named RPLK and RPCC, comprising of an antibiotic resistance marker for positive selection, the phenotypic selection marker lacZ or mCherry, and the counterselection gene rpsL. These cassettes can be transformed with high efficiency across the Neisseria genus while significantly reducing the number of false positives compared with similar approaches. We exemplified the versatility and application of these systems by obtaining unmarked luminescent strains (knock-in) or mutants (knock-out) in different pathogenic and commensal species across the Neisseria genus in addition to the cumulative deletion of six loci in a single strain of Neisseria elongata.

PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production in Citrobacter rodentium.

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-type Citrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline. C. rodentium uses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, the C. rodentium ΔpmrAB strain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription of pmrC (also known as eptA) and cptA OMV production in strains lacking either pmrC (eptA) or cptA was similarly increased in comparison to that of the wild type. Importantly, plasmid complementation of C. rodentium strains with either pmrC (eptA) or cptA resulted in a drastic inhibition of OMV production. Finally, we showed that ß-lactamase and CroP, two enzymes found in the C. rodentium periplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by which C. rodentium and possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genes pmrC (eptA) and cptAIMPORTANCE Although OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogen Citrobacter rodentium In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron in C. rodentium These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria.

Population-Based Estimate of Melioidosis, Kenya.

Melioidosis is thought to be endemic, although underdiagnosed, in Africa. We identified 5 autochthonous cases of Burkholderia pseudomallei infection in a case series in Kenya. Incidence of B. pseudomallei bacteremia in Kenya's Kilifi County is low, at 1.5 cases per million person-years, but this result might be an underestimate.

Standardization of Laboratory Methods for the PERCH Study.

The Pneumonia Etiology Research for Child Health study was conducted across 7 diverse research sites and relied on standardized clinical and laboratory methods for the accurate and meaningful interpretation of pneumonia etiology data. Blood, respiratory specimens, and urine were collected from children aged 1-59 months hospitalized with severe or very severe pneumonia and community controls of the same age without severe pneumonia and were tested with an extensive array of laboratory diagnostic tests. A standardized testing algorithm and standard operating procedures were applied across all study sites. Site laboratories received uniform training, equipment, and reagents for core testing methods. Standardization was further assured by routine teleconferences, in-person meetings, site monitoring visits, and internal and external quality assurance testing. Targeted confirmatory testing and testing by specialized assays were done at a central reference laboratory.

Detection of Pneumococcal DNA in Blood by Polymerase Chain Reaction for Diagnosing Pneumococcal Pneumonia in Young Children From Low- and Middle-Income Countries.

BACKGROUND.: We investigated the performance of polymerase chain reaction (PCR) on blood in the diagnosis of pneumococcal pneumonia among children from 7 low- and middle-income countries. METHODS.: We tested blood by PCR for the pneumococcal autolysin gene in children aged 1-59 months in the Pneumonia Etiology Research for Child Health (PERCH) study. Children had World Health Organization-defined severe or very severe pneumonia or were age-frequency-matched community controls. Additionally, we tested blood from general pediatric admissions in Kilifi, Kenya, a PERCH site. The proportion PCR-positive was compared among cases with microbiologically confirmed pneumococcal pneumonia (MCPP), cases without a confirmed bacterial infection (nonconfirmed), cases confirmed for nonpneumococcal bacteria, and controls. RESULTS.: In PERCH, 7.3% (n = 291/3995) of cases and 5.5% (n = 273/4987) of controls were blood pneumococcal PCR-positive (P < .001), compared with 64.3% (n = 36/56) of MCPP cases and 6.3% (n = 243/3832) of nonconfirmed cases (P < .001). Blood pneumococcal PCR positivity was higher in children from the 5 African countries (5.5%-11.5% among cases and 5.3%-10.2% among controls) than from the 2 Asian countries (1.3% and 1.0% among cases and 0.8% and 0.8% among controls). Among Kilifi general pediatric admissions, 3.9% (n = 274/6968) were PCR-positive, including 61.7% (n = 37/60) of those with positive blood cultures for pneumococcus. DISCUSSION.: The utility of pneumococcal PCR on blood for diagnosing childhood pneumococcal pneumonia in the 7 low- and middle-income countries studied is limited by poor specificity and by poor sensitivity among MCPP cases.

Evaluation of Pneumococcal Load in Blood by Polymerase Chain Reaction for the Diagnosis of Pneumococcal Pneumonia in Young Children in the PERCH Study.

BACKGROUND.: Detection of pneumococcus by lytA polymerase chain reaction (PCR) in blood had poor diagnostic accuracy for diagnosing pneumococcal pneumonia in children in 9 African and Asian sites. We assessed the value of blood lytA quantification in diagnosing pneumococcal pneumonia. METHODS.: The Pneumonia Etiology Research for Child Health (PERCH) case-control study tested whole blood by PCR for pneumococcus in children aged 1-59 months hospitalized with signs of pneumonia and in age-frequency matched community controls. The distribution of load among PCR-positive participants was compared between microbiologically confirmed pneumococcal pneumonia (MCPP) cases, cases confirmed for nonpneumococcal pathogens, nonconfirmed cases, and controls. Receiver operating characteristic analyses determined the "optimal threshold" that distinguished MCPP cases from controls. RESULTS.: Load was available for 290 of 291 cases with pneumococcal PCR detected in blood and 273 of 273 controls. Load was higher in MCPP cases than controls (median, 4.0 × 103 vs 0.19 × 103 copies/mL), but overlapped substantially (range, 0.16-989.9 × 103 copies/mL and 0.01-551.9 × 103 copies/mL, respectively). The proportion with high load (≥2.2 log10 copies/mL) was 62.5% among MCPP cases, 4.3% among nonconfirmed cases, 9.3% among cases confirmed for a nonpneumococcal pathogen, and 3.1% among controls. Pneumococcal load in blood was not associated with respiratory tract illness in controls (P = .32). High blood pneumococcal load was associated with alveolar consolidation on chest radiograph in nonconfirmed cases, and with high (>6.9 log10 copies/mL) nasopharyngeal/oropharyngeal load and C-reactive protein ≥40 mg/L (both P < .01) in nonconfirmed cases but not controls. CONCLUSIONS.: Quantitative pneumococcal PCR in blood has limited diagnostic utility for identifying pneumococcal pneumonia in individual children, but may be informative in epidemiological studies.

Molecular epidemiology of Klebsiella pneumoniae invasive infections over a decade at Kilifi County Hospital in Kenya.

Multidrug resistant (MDR) Klebsiella pneumoniae is a common cause of nosocomial infections worldwide. Recent years have seen an explosion of resistance to extended-spectrum ß-lactamases (ESBLs) and emergence of carbapenem resistance. Here, we examine 198 invasive K. pneumoniae isolates collected from over a decade in Kilifi County Hospital (KCH) in Kenya. We observe a significant increase in MDR K. pneumoniae isolates, particularly to third generation cephalosporins conferred by ESBLs. Using whole-genome sequences, we describe the population structure and the distribution of antimicrobial resistance genes within it. More than half of the isolates examined in this study were ESBL-positive, encoding CTX-M-15, SHV-2, SHV-12 and SHV-27, and 79% were MDR conferring resistance to at least three antimicrobial classes. Although no isolates in our dataset were found to be resistant to carbapenems we did find a plasmid with the genetic architecture of a known New Delhi metallo-ß-lactamase-1 (NDM)-carrying plasmid in 25 isolates. In the absence of carbapenem use in KCH and because of the instability of the NDM-1 gene in the plasmid, the NDM-1 gene has been lost in these isolates. Our data suggests that isolates that encode NDM-1 could be present in the population; should carbapenems be introduced as treatment in public hospitals in Kenya, resistance is likely to ensue rapidly.

Invasive Group A Streptococcus Infection among Children, Rural Kenya.

To determine the extent of group A Streptococcus (GAS) infections in sub-Saharan Africa and the serotypes that cause disease, we analyzed surveillance data for 64,741 hospital admissions in Kilifi, Kenya, during 1998-2011. We evaluated incidence, clinical presentations, and emm types that cause invasive GAS infection. We detected 370 cases; of the 369 for which we had data, most were skin and soft tissue infections (70%), severe pneumonia (23%), and primary bacteremia (14%). Overall case-fatality risk was 12%. Incidence of invasive GAS infection was 0.6 cases/1,000 live births among neonates, 101/100,000 person-years among children <1 year of age, and 35/100,000 among children <5 years of age. Genome sequencing identified 88 emm types. GAS causes serious disease in children in rural Kenya, especially neonates, and the causative organisms have considerable genotypic diversity. Benefit from the most advanced GAS type-specific vaccines may be limited, and efforts must be directed to protect against disease in regions of high incidence.

Evolution of longitudinal division in multicellular bacteria of the Neisseriaceae family.

Rod-shaped bacteria typically elongate and divide by transverse fission. However, several bacterial species can form rod-shaped cells that divide longitudinally. Here, we study the evolution of cell shape and division mode within the family Neisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the genera Alysiella, Simonsiella and Conchiformibius, which can be found in the oral cavity of mammals, are multicellular and divide longitudinally. We use comparative genomics and ultrastructural microscopy to infer that longitudinal division within Neisseriaceae evolved from a rod-shaped ancestor. In multicellular longitudinally-dividing species, neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan. In these bacteria, peptidoglycan insertion does not appear concentric, i.e. from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, we identify genes and alleles associated with multicellularity and longitudinal division, including the acquisition of amidase-encoding gene amiC2, and amino acid changes in proteins including MreB and FtsA. Introduction of amiC2 and allelic substitution of mreB in a rod-shaped species that divides by transverse fission results in shorter cells with longer septa. Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria, and suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability.

The Etiology of Pneumonia in HIV-uninfected Children in Kilifi, Kenya: Findings From the Pneumonia Etiology Research for Child Health (PERCH) Study.

BACKGROUND: In the 1980s, Streptococcus pneumoniae and Haemophilus influenzae were identified as the principal causes of severe pneumonia in children. We investigated the etiology of severe childhood pneumonia in Kenya after introduction of conjugate vaccines against H. influenzae type b, in 2001, and S. pneumoniae, in 2011. METHODS: We conducted a case-control study between August 2011 and November 2013 among residents of the Kilifi Health and Demographic Surveillance System 28 days to 59 months of age. Cases were hospitalized at Kilifi County Hospital with severe or very severe pneumonia according to the 2005 World Health Organization definition. Controls were randomly selected from the community and frequency matched to cases on age and season. We tested nasal and oropharyngeal samples, sputum, pleural fluid, and blood specimens and used the Pneumonia Etiology Research for Child Health Integrated Analysis, combining latent class analysis and Bayesian methods, to attribute etiology. RESULTS: We enrolled 630 and 863 HIV-uninfected cases and controls, respectively. Among the cases, 282 (44%) had abnormal chest radiographs (CXR positive), 33 (5%) died in hospital, and 177 (28%) had diagnoses other than pneumonia at discharge. Among CXR-positive pneumonia cases, viruses and bacteria accounted for 77% (95% CrI: 67%-85%) and 16% (95% CrI: 10%-26%) of pneumonia attribution, respectively. Respiratory syncytial virus, S. pneumoniae and H. influenza, accounted for 37% (95% CrI: 31%-44%), 5% (95% CrI: 3%-9%), and 6% (95% CrI: 2%-11%), respectively. CONCLUSIONS: Respiratory syncytial virus was the main cause of CXR-positive pneumonia. The small contribution of H. influenzae type b and pneumococcus to pneumonia may reflect the impact of vaccine introductions in this population.

The Mycobacterial Cell Envelope: A Relict From the Past or the Result of Recent Evolution?

Mycobacteria are well known for their taxonomic diversity, their impact on global health, and for their atypical cell wall and envelope. In addition to a cytoplasmic membrane and a peptidoglycan layer, the cell envelope of members of the order Corynebacteriales, which include Mycobacterium tuberculosis, also have an arabinogalactan layer connecting the peptidoglycan to an outer membrane, the so-called "mycomembrane." This unusual cell envelope composition of mycobacteria is of prime importance for several physiological processes such as protection from external stresses and for virulence. Although there have been recent breakthroughs in the elucidation of the composition and organization of this cell envelope, its evolutionary origin remains a mystery. In this perspectives article, the characteristics of the cell envelope of mycobacteria with respect to other actinobacteria will be dissected through a molecular evolution framework in order to provide a panoramic view of the evolutionary pathways that appear to be at the origin of this unique cell envelope. In combination with a robust molecular phylogeny, we have assembled a gene matrix based on the presence or absence of key determinants of cell envelope biogenesis in the Actinobacteria phylum. We present several evolutionary scenarios regarding the origin of the mycomembrane. In light of the data presented here, we also propose a novel alternative hypothesis whereby the stepwise acquisition of core enzymatic functions may have allowed the sequential remodeling of the external cell membrane during the evolution of Actinobacteria and has led to the unique mycomembrane of slow-growing mycobacteria as we know it today.

Population effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and non-typeable Haemophilus influenzae in Kilifi, Kenya: findings from cross-sectional carriage studies.

BACKGROUND: The effect of 7-valent pneumococcal conjugate vaccine (PCV) in developed countries was enhanced by indirect protection of unvaccinated individuals, mediated by reduced nasopharyngeal carriage of vaccine-serotype pneumococci. The potential indirect protection of 10-valent PCV (PCV10) in a developing country setting is unknown. We sought to estimate the effectiveness of introduction of PCV10 in Kenya against carriage of vaccine serotypes and its effect on other bacteria. METHODS: PCV10 was introduced into the infant vaccination programme in Kenya in January, 2011, accompanied by a catch-up campaign in Kilifi County for children aged younger than 5 years. We did annual cross-sectional carriage studies among an age-stratified, random population sample in the 2 years before and 2 years after PCV10 introduction. A nasopharyngeal rayon swab specimen was collected from each participant and was processed in accordance with WHO recommendations. Prevalence ratios of carriage before and after introduction of PCV10 were calculated by log-binomial regression. FINDINGS: About 500 individuals were enrolled each year (total n=2031). Among children younger than 5 years, the baseline (2009-10) carriage prevalence was 34% for vaccine-serotype Streptococcus pneumoniae, 41% for non-vaccine-serotype Streptococcus pneumoniae, and 54% for non-typeable Haemophilus influenzae. After PCV10 introduction (2011-12), these percentages were 13%, 57%, and 40%, respectively. Adjusted prevalence ratios were 0·36 (95% CI 0·26-0·51), 1·37 (1·13-1·65), and 0·62 (0·52-0·75), respectively. Among individuals aged 5 years or older, the adjusted prevalence ratios for vaccine-serotype and non-vaccine-serotype S pneumoniae carriage were 0·34 (95% CI 0·18-0·62) and 1·13 (0·92-1·38), respectively. There was no change in prevalence ratio for Staphylococcus aureus (adjusted prevalence ratio for those <5 years old 1·02, 95% CI 0·52-1·99, and for those ≥5 years old 0·90, 0·60-1·35). INTERPRETATION: After programmatic use of PCV10 in Kilifi, carriage of vaccine serotypes was reduced by two-thirds both in children younger than 5 years and in older individuals. These findings suggest that PCV10 introduction in Africa will have substantial indirect effects on invasive pneumococcal disease. FUNDING: GAVI Alliance and Wellcome Trust.