Neonatal invasive disease caused by Streptococcus agalactiae in Europe: the DEVANI multi-center study.

PURPOSE: Group B streptococcus (GBS) remains a leading cause of invasive disease, mainly sepsis and meningitis, in infants < 3 months of age and of mortality among neonates. This study, a major component of the European DEVANI project (Design of a Vaccine Against Neonatal Infections) describes clinical and important microbiological characteristics of neonatal GBS diseases. It quantifies the rate of antenatal screening and intrapartum antibiotic prophylaxis among cases and identifies risk factors associated with an adverse outcome. METHODS: Clinical and microbiological data from 153 invasive neonatal cases (82 early-onset [EOD], 71 late-onset disease [LOD] cases) were collected in eight European countries from mid-2008 to end-2010. RESULTS: Respiratory distress was the most frequent clinical sign at onset of EOD, while meningitis is found in > 30% of LOD. The study revealed that 59% of mothers of EOD cases had not received antenatal screening, whilst GBS was detected in 48.5% of screened cases. Meningitis was associated with an adverse outcome in LOD cases, while prematurity and the presence of cardiocirculatory symptoms were associated with an adverse outcome in EOD cases. Capsular-polysaccharide type III was the most frequent in both EOD and LOD cases with regional differences in the clonal complex distribution. CONCLUSIONS: Standardizing recommendations related to neonatal GBS disease and increasing compliance might improve clinical care and the prevention of GBS EOD. But even full adherence to antenatal screening would miss a relevant number of EOD cases, thus, the most promising prophylactic approach against GBS EOD and LOD would be a vaccine for maternal immunization.

Induction of Susceptibility to Disseminated Infection with IgA1 Protease-Producing Encapsulated Pathogens Streptococcus pneumoniae, Haemophilus influenzae Type b, and Neisseria meningitidis.

Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the principal causes of bacterial meningitis. It is unexplained why only occasional individuals develop invasive infection, while the vast majority remain healthy and develop immunity when encountering these pathogens. A capsular polysaccharide and an IgA1 protease are common to these pathogens. We tested the hypothesis that patients are primed to susceptibility to invasive infection by other bacteria that express the same capsular polysaccharide but no IgA1 protease. Thereby, the subsequently colonizing pathogen may protect its surface with IgA1 protease-generated Fab fragments of IgA1 devoid of Fc-mediated effector functions. Military recruits who remained healthy when acquiring meningococci showed a significant response of inhibitory antibodies against the IgA1 protease of the colonizing clone concurrent with serum antibodies against its capsular polysaccharide. At hospitalization, 70.8% of meningitis patients carried fecal bacteria cross-reactive with the capsule of the actual pathogen, in contrast to 6% of controls (P < 0.0001). These were Escherichia coli K100, K1, and K92 in patients with infection caused by H. influenzae type b and N. meningitidis groups B and C, respectively. This concurred with a significant IgA1 response to the capsule but not to the IgA1 protease of the pathogen. The demonstrated multitude of relationships between capsular types and distinct IgA1 proteases in pneumococci suggests an alternative route of immunological priming associated with recombining bacteria. The findings support the model and offer an explanation for the rare occurrence of invasive diseases in spite of the comprehensive occurrence of the pathogens. IMPORTANCE Why some individuals develop invasive infection, including meningitis, with Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b is unexplained. The vast majority of humans are colonized with the three pathogens but remain healthy and develop immunity. The findings of this study support the hypothesis that patients are primed for disease by time-shifted acquisition of two different bacteria, an immunogenic commensal followed by the pathogen, but both expressing the same capsular polysaccharide. The IgA1 protease common to the three pathogens cleaves the preexisting IgA1 antibodies induced by the commensal. This eliminates Fc-mediated protective mechanisms and releases capsule-binding monomeric Fab fragments that enhance bacterial adherence and block access of other isotypes of antibody molecules. This concept provides new insight into the pathogenesis of bacterial meningitis and potential new strategies for prevention.

Complement activation by human IgG antibodies to galactose-α-1,3-galactose.

Some human antibodies may paradoxically inhibit complement activation on bacteria and enhance pathogen survival in humans. This property was also claimed for IgG antibodies reacting with terminal galactose-α-1,3-galactose (Galα3Gal; IgG anti-αGal), a naturally occurring and abundant antibody in human plasma that targets numerous different pathogens. To reinvestigate these effects, we used IgG anti-αGal affinity isolated from a pool of normal human IgG and human hypogammaglobulinaemia serum as a complement source. Flow cytometry was performed to examine antibody binding and complement deposition on pig erythrocytes, Escherichia coli O86 and Streptococcus pneumoniae serotype 9V. Specific nanobodies were used to block the effect of single complement factors and to delineate the complement pathways involved. IgG anti-αGal was capable of activating the classical complement pathway on all the tested target cells. The degree of activation was exponentially related to the density of bound antibody on E. coli O86 and pig erythrocytes, but more linearly on S. pneumoniae 9V. The alternative pathway of complement amplified complement deposition. Deposited C3 fragments covered the activating IgG anti-αGal, obstructing its detection and highlighting this as a likely general caveat in studies of antibody density and complement deposition. The inherent capacity for complement activation by the purified carbohydrate reactive IgG anti-αGal was similar to that of normal human IgG. We propose that the previously reported complement inhibition by IgG anti-αGal relates to suboptimal assay configurations, in contrast to the complement activating property of the antibodies demonstrated in this paper.

Abundant human anti-Galα3Gal antibodies display broad pathogen reactivity.

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.

The distribution of clones of Streptococcus agalactiae (group B streptococci) among herdspersons and dairy cows demonstrates lack of host specificity for some lineages.

Streptococcus agalactiae (Group B streptococcus, GBS) is a commensal of the human intestinal tract and vagina and is also an opportunistic pathogen causing serious, potentially lethal, infections preferentially in newborns and in the elderly. In cattle, it is considered an udder-specific pathogen and a common cause of mastitis. Here we investigated the host specificity of GBS by examining their colonization at various anatomical sites in both cattle and humans, as well as the possible cross-species transmission in closed barn environments. We collected more than 800 swab samples from dairy cows and herdspersons at eight dairy farms in Denmark. GBS was isolated from 12% of the samples. The GBS strains (N = 105) were characterized by biochemical test, serology, and Pulsed-Field Gel Electrophoresis (PFGE). Based on the PFGE patterns, 25 strains were selected for whole genome sequencing followed by phylogenetic analyses. The genomes were compared to each other and to a collection of publicly available GBS genomes. The study revealed that GBS clones were shared by cows and herdspersons. In phylogenetic analyses, these shared clones clustered with GBS strains from persons with no relation to farming. Horizontal cross-species transmission of the contagion in both directions was found to be highly likely within the same environment; thus, some cases of bovine mastitis are probably antrophonotic.

The Immunomodulatory Drug Glatiramer Acetate is Also an Effective Antimicrobial Agent that Kills Gram-negative Bacteria.

Classic drug development strategies have failed to meet the urgent clinical needs in treating infections with Gram-negative bacteria. Repurposing drugs can lead to timely availability of new antibiotics, accelerated by existing safety profiles. Glatiramer acetate (GA) is a widely used and safe formulation for treatment of multiple sclerosis. It contains a large diversity of essentially isomeric polypeptides with the cationic and amphiphilic character of many antimicrobial peptides (AMP). Here, we report that GA is antibacterial, targeting Gram-negative organisms with higher activity towards Pseudomonas aeruginosa than the naturally-occurring AMP LL-37 in human plasma. As judged from flow cytometric assays, bacterial killing by GA occurred within minutes. Laboratory strains of Escherichia coli and P. aeruginosa were killed by a process of condensing intracellular contents. Efficient killing by GA was also demonstrated in Acinetobacter baumannii clinical isolates and approximately 50% of clinical isolates of P. aeruginosa from chronic airway infection in CF patients. By contrast, the Gram-positive Staphylococcus aureus cells appeared to be protected from GA by an increased formation of nm-scale particulates. Our data identify GA as an attractive drug repurposing candidate to treat infections with Gram-negative bacteria.

Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population.

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.

Temperature limitation may explain the containment of the trophozoites in the cornea during Acanthamoeba castellanii keratitis.

Acanthamoeba keratitis is a serious sight-threatening disease. The relatively low temperature of the cornea may explain why amoebic infections usually are localized in this tissue and rarely spread to other parts of the eye. In this study, the growth rate of the amoeba Acanthamoeba castellanii was examined at different temperatures. The aim was to establish the optimal growth temperature for A. castellanii and to examine the growth within the vicinity of the core body temperature. The growth rates of four clinical and two environmental strains of A. castellanii were estimated at different temperatures, and temperature limitations for the trophozoite stage was established. Movements influenced by temperature gradients were monitored for two clinical strains of A. castellanii. The highest growth rate for each of the six amoebic strains tested was found to be close to 32 °C. The growth of the trophozoites of all examined strains was greatly reduced or completely halted at temperatures above 36 °C and encysted at the elevated temperature. Thus, the optimal growth temperature for the four strains of A. castellanii is close to the surface temperature of the human cornea, while the higher body core-temperature induced encysting of the amoebae. This may explain why most amoebic eye infections are confined to the cornea.

Capsular gene typing of Streptococcus agalactiae compared to serotyping by latex agglutination.

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalactiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex agglutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant patterns in the capsular gene typing, long-range PCR targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A procedure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed.

International external quality assurance for laboratory identification and typing of Streptococcus agalactiae (Group B streptococci).

We report the results from the first international multicenter external quality assessment (EQA) studies for molecular and serological typing of group B streptococcus (GBS) strains as part of DEVANI (Design of a Vaccine against Neonatal Infections), a pan-European program. A questionnaire-based surveillance was undertaken among eight laboratories participating in DEVANI and six laboratories not participating in DEVANI from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis, and typing. GBS strains from three EQA distributions were characterized using molecular and serological methods based on GBS capsular polysaccharide typing. Participants were asked to test the first distribution using their current serotyping and genotyping methods. The Strep-B-Latex agglutination method was the most widely used method, with a typeability value of >90%. A multiplex PCR assay for GBS capsular gene typing was also used by 2 of 14 centers, which achieved a typeability value of 93%; this assay detected only 9 of 10 GBS capsular polysaccharide genes. From the second and third EQA studies, standardized protocols were prepared for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method and a novel multiplex PCR assay that detected all 10 GBS capsular types (Ia to IX). These standardized protocols are being used by many European laboratories, and as the use of these methods increases, it is imperative to continuously improve and assess laboratory performance and offer training to any laboratories that have technical difficulties.

Emergence and global dissemination of host-specific Streptococcus agalactiae clones.

To examine the global diversity of Streptococcus agalactiae (group B streptococci [GBS]) and to elucidate the evolutionary processes that determine its population genetics structure and the reported changes in host tropism and infection epidemiology, we examined a collection of 238 bovine and human isolates from nine countries on five continents. Phylogenetic analysis based on the sequences of 15 housekeeping genes combined with patterns of virulence-associated traits identified a genetically heterogeneous core population from which virulent lineages occasionally emerge as a result of recombination affecting major segments of the genome. Such lineages, like clonal complex 17 (CC17) and two distinct clusters of CC23, are exclusively adapted to either humans or cattle and successfully spread globally. The recent emergence and expansion of the human-associated and highly virulent sequence type 17 (ST17) could conceivably account, in part, for the increased prevalence of neonatal GBS infections after 1960. The composite structure of the S. agalactiae genome invalidates phylogenetic inferences exclusively based on multilocus sequence typing (MLST) data and thereby the previously reported conclusion that the human-associated CC17 emerged from the bovine-associated CC67.

[Ascariasis is a zoonosis in Denmark--secondary publication]. / Ascariasis er en zoonose i Danmark--sekundaerpublikation.

It has been claimed that Danish cases of human ascariasis have been acquired either during travel to other countries or through consumption of untreated food imported from areas where human ascariasis is endemic. An epidemiological survey in Viborg County indicated, however, that pigs are the primary source of the infection. Our population-genetic investigations have now confirmed this hypothesis. The main transmission route for human ascariasis in developed countries therefore seems to be from pigs to people; thus, it is essential that contact with pig manure be avoided, especially by young children.

Effect of capsulation of opportunistic pathogenic bacteria on binding of the pattern recognition molecules mannan-binding lectin, L-ficolin, and H-ficolin.

Mannan-binding lectin (MBL), L-ficolin, and H-ficolin are pattern recognition molecules of the innate immune system. We investigated their ability to bind to different serotypes and noncapsulated variants of two gram-positive bacterial species, Streptococcus pneumoniae and Staphylococcus aureus. MBL did not bind to capsulated S. aureus or capsulated S. pneumoniae but did bind to a noncapsulated S. aureus variant (Wood). L-ficolin bound to some capsulated S. aureus serotypes (serotypes 1, 8, 9, 11, and 12) and capsulated S. pneumoniae serotypes (11A, 11D, and 11F) but not to noncapsulated strains. H-ficolin did not bind to any of the S. pneumoniae and S. aureus serotypes included in this study but did bind to one strain of Aerococcus viridans. The concentrations of the three proteins in 97 plasma samples were estimated. The median concentrations were 0.8 mug per ml for MBL, 3.3 mug per ml for L-ficolin, and 18.4 mug per ml for H-ficolin.

Dynamics of Streptococcus agalactiae colonization in women during and after pregnancy and in their infants.

The population dynamics of Streptococcus agalactiae (group B streptococci [GBS]) colonization of the vagina and anorectal area was investigated in a cohort of 77 Danish women during and after their pregnancy by a new sensitive method. The mean carriage rate among individual observations was 36%, and the cumulative carriage rate over the entire observation period was 54%. Examination of more than 1500 GBS isolates by pulsed-field gel electrophoresis demonstrated that the GBS population was remarkably homogeneous and stable in each carrier. Virtually all carriers were colonized by a single GBS clone on all occasions spanning up to 2 years. Repeated detection of the same clone even in women who were recorded as intermittent carriers suggests that the actual carrier rate exceeds 50% but that fluctuations in the GBS proportions of the flora occasionally preclude their detection. Newborns and young infants usually carried the same GBS clone as their mothers. However, only twice were identical clones of GBS detected in different women in contrast to the observed clonal relationships of clinical isolates. These observations strongly suggest differences in the properties and epidemiology of virulent GBS clones compared to clones commonly carried by healthy individuals.

Method for quantitative detection and presumptive identification of group B streptococci on primary plating.

Maternal prenatal screening for group B streptococci (GBS) followed by offering of intrapartum chemoprophylaxis to carriers is one of the strategies used to reduce the incidence of neonatal early-onset GBS infections. Culturing of vaginal and anorectal swab specimens in selective broth is the screening procedure recommended by the Centers for Disease Control and Prevention. This technique is sensitive; it does not, however, allow either evaluation of the degree of colonization or detection of cocolonization with several GBS clones. We have examined the carriage rate and population dynamics of GBS in a group of Danish women during pregnancy and 1 year after delivery using a new detection method. In the present paper we describe a mixed blood agar medium (MB agar) that identifies GBS in the primary cultures by detection of a double hemolysis pattern consisting of characteristic, large zones of partial hemolysis ("CAMP zones") and of narrow zones of complete hemolysis. The MB agar was at least as sensitive as culturing in selective broth for detection of GBS in vaginal and anorectal swab specimens, and GBS strains could be identified directly on the primary plate due to the CAMP zones without the need for subculturing. The carriage rate of GBS in a group of Danish women was found to be more than 30%, a figure considerably higher than the rate that was reported previously.