Peripheral compartment innate immune response to Haemophilus influenzae and Streptococcus pneumoniae in chronic obstructive pulmonary disease patients.

Alterations in innate immunity that predispose to chronic obstructive pulmonary disease (COPD) exacerbations are poorly understood. We examined innate immunity gene expression in peripheral blood polymorphonuclear leukocytes (PMN) and monocytes stimulated by Haemophilus influenzae and Streptococcus pneumoniae. Thirty COPD patients (15 rapid and 15 non-rapid lung function decliners) and 15 smokers without COPD were studied. Protein expression of IL-8, IL-6, TNF-α and IFN-γ (especially monocytes) increased with bacterial challenge. In monocytes stimulated with S. pneumoniae, TNF-α protein expression was higher in COPD (non-rapid decliners) than in smokers. In co-cultures of monocytes and PMN, mRNA expression of TGF-β1 and MYD88 was up-regulated, and CD14, TLR2 and IFN-γ down-regulated with H. influenzae challenge. TNF-α mRNA expression was increased with H. influenzae challenge in COPD. Cytokine responses were similar between rapid and non-rapid decliners. TNF-α expression was up-regulated in non-rapid decliners in response to H. influenzae (monocytes) and S. pneumoniae (co-culture of monocytes and PMN). Exposure to bacterial pathogens causes characteristic innate immune responses in peripheral blood monocytes and PMN in COPD. Bacterial exposure significantly alters the expression of TNF-α in COPD patients, although not consistently. There did not appear to be major differences in innate immune responses between rapid and non-rapid decliners.

Genotyping streptococcus pneumoniae

Streptococcus pneumoniae is a potentially deadly human pathogen associated with high morbidity, mortality and global economic burden. The universally used bacterial genotyping methods are multilocus sequence typing and pulsed field gel electrophoresis. However, another highly discriminatory, rapid and less expensive genotyping technique,multilocus variable number of tandem repeat analysis (MLVA), has been developed. Unfortunately, no universal MLVA protocol exists, and some MLVA protocols do not amplify certain loci for all pneumococcal serotypes, leaving genotyping profiles incomplete. A number of other genotyping or characterization methods have been developed and will be discussed. This review examines the various protocols for genotyping S. pneumoniae and highlights the current direction technology and research is heading to understand this bacterium.

Modified MLVA for genotyping Queensland invasive Streptococcus pneumoniae

Background Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The ‘gold standard’ genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST. Methods Four different MLVA protocols, including a modified method, were applied to 317 isolates of serotyped invasive S. pneumoniae isolated from sterile body sites of Queensland children under 15 years from 2007–2012. MLST was applied to 202 isolates for comparison. Results The modified MLVA4 is significantly more discriminatory than the ‘gold standard’ MLST method. MLVA4 has similar discrimination compared to other MLVA techniques in this study). The failure to amplify particular loci in previous MLVA methods were minimised in MLVA4. Failure to amplify BOX-13 and Spneu19 were found to be serotype specific. Conclusion We have modified a highly discriminatory MLVA technique for genotyping Queensland invasive S. pneumoniae. MLVA4 has the ability to enhance our understanding of the pneumococcal epidemiology and the changing genetics of the pneumococcus in localised and short-term studies.

Investigating the population structure of Queensland invasive Streptococcus pneumoniae isolates in children: Using a modified multi-locus variable number of tandem repeat analysis and a novel minimum SNPs capsular typing method

This research investigated the use of DNA fingerprinting to characterise the bacteria Streptococcus pneumoniae or pneumococcus, and hence gain insight into the development of new vaccines or antibiotics. Different bacterial DNA fingerprinting methods were studied, and a novel method was developed and validated, which characterises different cell coatings that pneumococci produce. This method was used to study the epidemiology of pneumococci in Queensland before and after the introduction of the current pneumococcal vaccine. This study demonstrated that pneumococcal disease is highly prevalent in children under four years, that the bacteria can `switch' its cell coating to evade the vaccine, and that some DNA fingerprinting methods are more discriminatory than others. This has an impact on understanding which strains are more prone to cause invasive disease. Evidence of the excellent research findings have been published in high impact internationally refereed journals.

Infection and cellular defense dynamics in a novel 17beta-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (106–107 CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants.

Aims: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. Methods and Results: Streptococcus bovis strains were tested for production of bacteriocin-like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin-positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. Conclusions: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin+ trait is maintained in animals at the same location. The HC5-like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. Significance and Impact of the Study: The HC5-like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety-three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Ruppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S.agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S.agalactiae; genotyping of selected S.agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S.agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Streptococcus milleri -ryhmä : Tunnistus lajitasolle ja esiintyvyys tietyissä infektioissa

S. milleri -ryhmän bakteerit ovat osa ihmisen suun, nielun, suoliston ja genitaalialueen normaaliflooran bakteeristoa. Kommensaalien lisäksi ryhmään kuuluu myös merkittäviä patogeenejä, jotka esiintyvät varsin runsaina löydöksinä monenlaisissa märkivissä infektioissa. Ryhmään kuuluu kolme lajia: S. anginosus, S. constellatus ja S. intermedius. Lajit ovat varsin samankaltaisia ja raportoidaankin usein vain ryhmänimellä. Lajit ovat kuitenkin erotettavissa, sillä ne eroavat toisistaan tuottamiensa entsyymien suhteen ja esiintyvyydessään kehon eri osissa. Työn tarkoituksena oli tunnistaa erityyppisistä kliinisistä infektioista otetuista näytteistä eristettyjä S. milleri -ryhmään luokiteltuja kantoja lajitasolle ja selvittää niiden esiintymisyleisyyttä näissä infektioissa. Näytteenottopaikat jaettiin viiteen ryhmään: naisten urogenitaalialue (15 kantaa), miesten urogenitaalialue (8 kantaa), oraaliset (28 kantaa), umpisuoli (34 kantaa) ja "muut" (12 kantaa). Lajitunnistuksen lisäksi selvitettiin kantojen hemolyyttisyys ja mahdollinen Lancefield-seroryhmä (A, C, F, G). Lajien erottelu perustuu eroihin bakteerien kyvyissä hajottaa tiettyjä substraatteja (entsyymiprofilointi), hemolyyttisyys määritettiin verimaljalla ja seroryhmitys tehtiin kaupallisella vasta-aine-sakkautumistestillä (Streptex latex Z1- 50). Työssä testattiin käytössä olevia ja kehiteltiin uusia, lähinnä ennalta muodostuneiden entsyymien tunnistamiseen perustuvia erottelumenetelmiä. Vertailtavina oli kolme entsyymiprofiliontimenetelmää, joista yksi on fluorogeeninen (4-Metyyli-umbelliferyyli-subtraatit) ja kaksi kromogeenistä (Weetabs ja RoscoDiagnostic tablets). Kannoilta määritettiin seuraavat aktiivisuudet: ?-fukosidaasi, (?-glukosidaasi, glukosidaasi, ?-galaktosidaaasi, ?-N-asetyyli-galaktosaminidaasi, ?-N-asetyyli-glukosaminidaasi, sialidaasi ja hyaluronidaasi. Työhön sisältyy myös erilaisten kasvatusalustojen sekä pH:n vaikutusten arviointia bakteerienentsyymiaktiivisuuksiin ja testituloksiin. Lisäksi työssä testattiin kromatografisensoluseinärasvahappoanalyysin soveltuvuutta lajien erotteluun. Menetelmiä tarkasteltiin herkkyyden sekäkäytännön suorittamisen ja aiheutuvien kustannusten kannalta. Asetetut tavoitteet saavutettiin. Kaikki käytetyt menetelmät osoittautuivat toimiviksi. Entsyymitestien tuloksetkorreloivat keskenään ja kirjallisuuden kanssa hyvin. Kannat karakterisoitiin, tunnistettiin lajitasolle ja lajiensiintyvyyttä kehon eri osissa voitiin vertailla. Mikään entsyymitesti ei osoittautunut ylivoimaisesti parhaaksi tai huonoimmaksi, vaikkakin yksittäistensubstraattien kohdalla eri testien herkkyydet vaihtelivatkin huomattavasti. Rasvahappoanalyysi ei erotellutkantoja toivotulla tavalla, joten sen käytöstä luovuttiin työn melko varhaisessa vaiheessa. Tutkituista 97 S. milleri -kannasta tunnistettiin 58 S. anginosus-kantaa, 29 S. constellatus-kantaa ja 10 S.ntermedius-kantaa. Eri lajit noudattivat entsyymiprofiiliaan muutamaa poikkeusta lukuunottamatta hyvinkinsäännöllisesti. Lajien sisäinen variaatio hemolyysiominaisuuksissa oli merkittävää ja S. inilleri -ryhmän erilajien sekä hemolyysisltään ja seroryhmältään erilaisten kantojen esiintyvyydessä kehon eri osissa havaittiinselkeitä eroja. Avainsanat: entsyymiprofilointi, fluorogeeniset ja kromogeeniset substraatit, seroryhmä, hemolyysi, esiintyvyys

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Promotion of fibronectin independent invasion by C5a peptidase into epithelial cells in group A Streptococcus

Background & objectives: Group A Streptococcus, causative agent of several clinical manifestations codes for multiple protein invasins which help the bacterium to enter non-phagocytic cells. C5a peptidase (SCPA) is a surface protein conserved among different serotypes of M1 strain. The present study was taken up to study SCPA promoted fibronectin independent entry of GAS into epithelial cells. Methods: An isogenic 90226 emm1DeltaAB (M1(-)) mutant was constructed, with thermosensitive pGhost vector. This isogenic M1(-) mutant expressed SCPA on the surface as determined by Western blotting and immunofluorescence. Results: On preincubation with anti-SCPA serum, the isogenic M1(-) strain exhibited 54 per cent decreased invasion as compared to the bacteria incubated with control serum. Also, purified recombinant SCPA proteins blocked internalization of M1(-) streptococci into HEp-2 cells. The M1(-) strain invaded at the same efficiency in the presence or absence of fibronectin. Interpretation & conclusion: These results suggested that SCPA acted as a potential invasin of group A streptococcus and promoted invasion independent of fibronectin.

Interplay of Virulence Factors in Complement Resistance of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is a normal inhabitant of the human nasopharynx. Symptoms occur in only a small proportion of those who become carriers, but the ubiquity of the organism in the human population results in a large burden of disease. S. pneumoniae is the leading bacterial cause of pneumonia, sepsis, and meningitis worldwide, causing the death of a million children each year. Middle-ear infection is the most common clinical manifestation of mucosal pneumococcal infections. In invasive disease, S. pneumoniae gains access to the bloodstream and spreads to normally sterile parts of the body. The progression from asymptomatic colonization to disease depends on factors characteristic of specific pneumococcal strains as well as the status of host defenses. The polysaccharide capsule surrounding the bacterium is considered to be the most important factor affecting the virulence of pneumococci. It protects pneumococci from phagocytosis and also may determine its affinity to the respiratory epithelium. S. pneumoniae as a species comprises more than 90 different capsular serotypes, but not all of them are equally prevalent in human diseases. Invasive serotypes are rarely isolated from healthy carriers, but relatively often cause invasive disease. Serotypes that are carried asymptomatically for a long time behave like opportunistic pathogens, causing disease in patients who have impaired immune defenses. The complement system is a collection of blood and cell surface proteins that act as a major primary defense against invading microbes. Phagocytic cells with receptors for complement proteins can engulf and destroy pneumococcal cells opsonized with these proteins. S. pneumoniae has evolved a number of ways to subvert mechanisms of innate immunity, and this is likely to contribute to its pathogenicity. The capsular serotype, proteins essential for virulence, as well the genotype, may all influence the ability of pneumococcus to resist complement and its potential to cause disease. Immunization with conjugate vaccines produces opsonic antibodies, which enhance complement deposition and clearance of the bacteria. The pneumococcal vaccine included in the Finnish national immunization program in 2010 contains the most common serotypes causing invasive disease. Clinical data suggest that protection from middle-ear infection and possibly also from invasive disease depends largely on the capsular serotype, for reasons hitherto unknown. The general aim of this thesis is to assess the relative roles of the pneumococcal capsule and virulence proteins in complement evasion and subsequent opsonophagocytic killing. The main question is whether differences between serotypes to resist complement explain the different abilities of serotypes to cause disease. The importance of particular virulence factors to the complement resistance of a strain may vary depending on its genotype. Prior studies have evaluated the effect of the capsule and virulence proteins on complement resistance of S. pneumoniae by comparing only a few strains. In this thesis, the role of pneumococcal virulence factors in the complement resistance of the bacterium was studied in several genotypically different strains. The ability of pneumococci to inhibit deposition of the complement protein C3 on the bacterial surface was found to depend on the capsular serotype as well as on other features of the bacteria. The results suggest that pneumococcal histidine triad (Pht) proteins may play a role in complement inhibition, but their contribution depends on the bacterial genotype. The capsular serotype was found to influence complement resistance more than the bacterial genotype. A higher concentration of anticapsular antibodies was required for the opsonophagocytic killing of serotypes resistant to C3 deposition. The invasive serotypes were more resistant to C3 deposition than the opportunistic serotypes, suggesting that the former are better adapted to resist immune mechanisms controlling the development of invasive disease. The different susceptibilities of serotypes to complement deposition, opsonophagocytosis, and resultant antibody-mediated protection should be taken into account when guidelines for serological correlates for vaccine efficacy evaluations are made. The results of this thesis suggest that antibodies in higher quantity or quality are needed for efficient protection against the invasive serotypes.