Article

Experimental pneumococcal meningitis in mice: a model of intranasal infection.

Department of Pediatrics, University Hospital Vrije Universiteit, and Department of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands.
The Journal of Infectious Diseases (Impact Factor: 5.85). 05/2001; 183(7):1143-6. DOI: 10.1086/319271
Source: PubMed

ABSTRACT Effective laboratory animal models of bacterial meningitis are needed to unravel the pathophysiology of this disease. Previous models have failed to simulate human meningitis by using a directly intracerebral route of infection. Hyaluronidase is a virulence factor of Streptococcus pneumoniae. In this study, a novel model of murine meningitis is described. Intranasal administration of S. pneumoniae with hyaluronidase induced meningitis in 50% of inoculated mice, as defined by a positive cerebrospinal fluid (CSF) culture and an inflammatory infiltrate in the meninges. None of the mice inoculated without hyaluronidase developed meningitis. Hyaluronidase was found to facilitate pneumococcal invasion of the bloodstream after colonization of the upper respiratory tract. Meningitis was characterized by pleocytosis of CSF and the induction of proinflammatory cytokines and CXC chemokines in brain tissue. These results indicate that this murine model mimics important features of human disease and allow for the use of this model for studying issues related to the pathophysiology and the treatment of pneumococcal meningitis.

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    ABSTRACT: The evolutionary success of bacteria depends on genetic variability. This variability may be the result of beneficial mutations in the genome or from the uptake of genetic elements that increase viability under stress conditions. Bacteria are repeatedly exposed to agents such as antibiotics and host immune responses, which may induce in bacteria a variety of survival strategies, including mutagenic mechanisms. The development of antibiotic resistance is one of the serious consequences of these mechanisms. Bacteria increase genetic variability and survive DNA damage by using horizontal gene transfer, competence and DNA repair mechanisms such as the SOS response, homologous recombination and mismatch repair. Besides genetic rearrangements, bacteria have other mechanisms to persist in hostile environments. Biofilms are sessile, often polysaccharide-encased bacterial growth forms that are particularly tolerant to mechanical shearing, antibiotics and host immune responses. Bovine udder infection, or mastitis, is an inflammation of the mammary gland caused by bacteria and it is usually recognized by clinical signs including abnormalities in the milk and the udder. Mastitis is the most common disease in the dairy industry and account for a significant proportion of the antibiotic treatments in dairy cows. Streptococcus uberis is a Gram-positive pathogenic bacterium and a member of the pyogenic group of the genus Streptococcus. S. uberis mainly infects the udder from environmental sources and is one of the most common causative agents of bovine mastitis. However, the molecular biology of S. uberis has been relatively unknown. The members of the Streptococcaceae family have been considered to lack the classical SOS response. In the first part of this research, the stress tolerance and mutagenic mechanisms of S. uberis were characterized. In study I, a novel self-regulated SOS -response gene cassette was identified and the expression of this cassette was induced by UV -light as well as the antibiotic ciprofloxacin. S. uberis was shown to perform mutagenic DNA -repair after UV -exposure and the repair was mediated by error-prone polymerase UmuC coded by the SOS -response gene cassette. This mutagenesis was shown to promote the development of antibiotic resistance and, according to the database searches, the homologs of this SOS gene cassette are found in several other streptococcal species. In study II, exposure to the fluoroquinolone antibiotic ciprofloxacin was also shown to induce antibiotic resistance-promoting mutations in S. uberis, but the mutagenesis was not UmuC-mediated. Unlike the model organism of the SOS response, Escherichia coli, S. uberis has distinct mechanisms for UV- and ciprofloxacin-induced mutagenesis. In study III, the effects of a sublethal concentration of ciprofloxacin on S. uberis were studied by proteomics. As a result of ciprofloxacin stress, S. uberis differentially expressed 20 proteins. The proteins were identified by mass spectrometry as enzymes involved in oxidative stress tolerance, NADH generation and nucleotide biosynthesis. The results suggest that ciprofloxacin exposure causes oxidative damage in S. uberis. The changes in enzymes involved in nucleotide balance suggest that nucleotide biosynthesis is a mechanism to stimulate mutagenesis leading to the development of antibiotic resistance. In the final part of the work, the ability of S. uberis to form biofilms was characterized and the biofilm formation of clinical and subclinical S. uberis isolates was investigated. The strains differed in their ability to form biofilms, ranging from low-producing strains to strains producing thick, multi-layered biofilms. To determine whether biofilm production by S. uberis is an inducible event, the effect of proteins from the host was tested. Milk, the natural growth medium of mastitis bacteria, induced biofilm formation in most strains, even at low concentrations. Further analyses indicated that the milk components casein and especially α- and β-casein are the primary inducing agents of biofilm production. The proteolytic activity of S. uberis is involved in this induction process, possibly by releasing peptides from caseins. Research on stress inducible systems provides us with information on how bacteria evolve, develop antibiotic resistance and escape the host immune system. The effects of antibiotic usage on bacteria are one of the most important questions in modern medicine, and knowledge of these effects helps in evaluating the necessity of antibiotic therapy. The biofilm formation of S. uberis provides a possible explanation for the ability of S. uberis to cause persistant mastitis, independent of antibiotic treatment. Bakteerien menestyminen muuttuvassa ympäristössä on riippuvainen geneettisestä vaihtelusta. Tämä geneettinen vaihtelu voi olla seurausta hyödyllisten mutaatioiden syntymisestä tai bakteerien hankkimista DNA-elementeistä, jotka parantavat selviytymiskykyä stressitilanteissa. Bakteerit altistuvat jatkuvasti stressaaville ympäristötekijöille, kuten antibiooteille ja isäntäorganismin immuunijärjestelmälle, jotka saattavat aktivoida selviytymismekanismeja kuten mutaatiomekanismeja. Antibioottiresistenssin kehittyminen on yksi näiden mekanismien vakavista sivuvaikutuksista. Horisontaalinen geenisiirto, kompetenssi ja DNA-korjausmekanismit, kuten SOS-vaste, homologinen rekombinaatio ja mismatch-korjaus, ovat järjestelmiä, joiden avulla bakteerit lisäävät geneettistä muunteluaan ja selviävät DNA-vaurioista. Geneettisten uudelleenjärjestäytymisten lisäksi bakteereilla on myös muita keinoja, joiden avulla selvitä vaikeissa olosuhteissa. Biofilmit ovat alustaansa kiinnittyneitä bakteerikasvustoja, jotka ovat usein polysakkaridien ympäröimiä. Biofilmit kestävät erittäin hyvin mekaanista rasitusta, antibiootteja ja isäntäorganismin immuunipuolustuksen hyökkäyksiä. Naudan utaretulehdus eli mastiitti on bakteerien aiheuttama maitorauhasen tulehdus ja se diagnosoidaan useimmiten kliinisten oireiden, kuten utareen muutosten tai maidon epänormaalin koostumuksen, perusteella. Mastiitti on lypsykarjan yleisin sairaus ja yleisin antibioottihoitojen syy. Streptococcus uberis on Gram-positiivinen patogeeninen bakteeri, joka kuuluu Streptococcus -suvun pyogeeniseen ryhmään. S. uberis infektoi utareita enimmäkseen ympäristölähteistä ja on yksi yleisimmästä naudan mastiitin aiheuttajista. Tästä huolimatta S. uberis bakteerin molekyylibiologiasta tiedetään suhteellisen vähän. Aiemmin on uskottu, että Streptococcaceae heimon bakteereilta puuttuu tyypillinen DNA-vaurioissa aktivoituva SOS-stressivaste. Tämän tutkimuksen ensimmäisessä osassa karakterisoidaan S. uberis bakteerin stressinsietokykyä ja mutaatiomekanismeja. Osajulkaisussa I identifioitiin S. uberis bakteerista uusi SOS-vasteoperoni, joka aktivoituu UV-valon ja antibioottialtistuksen vaikutuksesta ja säätelee itse omaa synteesiään. UV-altistuksen seurauksena syntyneiden DNA-vaurioiden korjauksesta vastaa UmuC, joka on SOS-vasteoperonin koodaama mutaatioita tuottava polymeraasi. Tämän mutaatioita aiheuttavan prosessin havaittiin kehittävän antibioottiresistenssiä. Tietokantahakujen avulla kyseisen SOS-vasteoperonin kanssa homologisia geenialueita löydettiin myös useista muista streptokokkilajeista. Osajulkaisussa II osoitettiin myös fluorokinoloni-antibiootti siprofloksasiinille altistumisen aiheuttavan mutaatioita ja antibioottiresistenttiyttä S. uberis bakteerissa, mutta tämä mutageneesi ei ollut UmuC:n aiheuttamaa. Toisin kuin malliorganismi Escherichia coli bakteerilla, S. uberis bakteerilla on erilaiset mekanismit UV- ja siprofloksasiini-indusoidulle mutageneesille. Osajulkaisussa III tutkittiin subletaalien siprofloksasiini-konsentraatioiden vaikutusta S. uberis bakteeriin proteomiikan avulla. Siprofloksasiini-stressin vaikutuksesta 20 proteiinin ilmentyminen muuttui. Massaspektrometrian avulla nämä proteiinit tunnistettiin oksidatiivisen stressin sietokyvyssä, NADH:n synteesissä ja nukleotidien biosynteesissä toimiviksi entsyymeiksi. Tulosten perusteella siprofloksasiinialtistus aiheuttaa oksidatiivisia vaurioita S. uberis bakteerissa. Muutokset nukleotiditasapainosta vastaavien entsyymien määrässä puolestaan viittaavat siihen, että nukleotidien biosynteesi voi olla mutageneesiä stimuloiva mekanismi, joka johtaa antibioottiresistenssin kehittymiseen. Tutkimuksen viimeisessä osassa tutkittiin kliinisten ja subkliinisten S. uberis kantojen kykyä muodostaa biofilmiä. Kantojen muodostamat biofilmit vaihtelivat paksuudeltaan suuresti lähes olemattomasta biofilmistä paksuihin monikerroksisiin biofilmeihin. Selvittääksemme onko biofilmin muodostus indusoitavissa S. uberis bakteerissa, testasimme isäntälajista peräisin olevien proteiinien vaikutusta biofilmin muodostukseen. Maito, luonnollinen mastiittibakteerin kasvualusta, indusoi biofilmin muodostusta jopa pieninä pitoisuuksina useimmissa kannoissa. Jatkotutkimuksissa selvisi, että maidon komponentti kaseiini ja etenkin α- ja β-kaseiinit ovat ensisijaiset indusoijat biofilmin muodostuksessa. S. uberis bakteerin proteolyyttisellä aktiivisuudella havaittiin olevan merkitystä biofilmin muodostuksessa. Tämä mahdollisesti viittaa siihen, että bakteeri vapauttaa kaseiinista proteolyyttisesti peptidejä, jotka toimivat indusoijina. Stressi-indusoituvien systeemien tutkimus lisää tietoa siitä kuinka bakteerit kehittyvät, tulevat resistenteiksi antibiooteille ja suojautuvat isäntäorganismin immuunijärjestelmältä. Antibioottien käytön vaikutukset bakteerikantaan on yksi modernin lääketieteen tärkeimmistä kysymyksistä ja tieto näistä vaikutuksista auttaa arvioimaan antibioottiterapioiden tarpeellisuutta. Kyky muodostaa biofilmiä tarjoaa mahdollisen selityksen sille, miksi S. uberis aiheuttaa sitkeitä mastiitteja antibioottihoidoista huolimatta.
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