Streptococcus pneumoniae é uma bactéria encapsulada causadora de doenças infecciosas como pneumonia, bacteremia e meningite, infecções essas que estão entre as principais causas de morte entre crianças, idosos e imunodeprimidos, indivíduos que constituem o grupo de risco para tais infecções. A vacinação tem sido a mais eficaz forma de conter tais infecções. A vantagem das vacinas conjugadas em comparação às polissacarídicas é a capacidade de indução de uma resposta imune T-dependente o que garante proteção mesmo ao grupo de risco para infecções por S. pneumonia. A proposta do projeto foi estabelecer um protocolo para obtenção de um conjugado constituído pelo polissacarídeo capsular de S. pneumonia sorotipo 1 (PS1) e pela proteína de superfície pneumocócica A (PspA). A síntese do conjugado empregou uma metodologia inédita para o sorotipo 1. A avaliação da resposta imune humoral induzida pelo conjugado mostrou a indução de IgG anti-PS1 gerada pelas imunizações com o conjugado PS1-PspA. / Streptococcus pneumoniae is an encapsulated bacteria causing infectious diseases such as pneumonia, bacteremia and meningitis, these infections are among the leading causes of death among children, elderly and immunocompromised, who constituting individuals of risk group. The vaccination has been the more effective form to counter these infection. The advantage of conjugated vaccines compared to vaccines polysaccharide, is the ability to induce a T-dependent immune response which provides protection even at risk groups for infection by S. pneumoniae. The project proposal was establish a protocol for obtaining a conjugate consisting of the capsular polysaccharide of S. pneumoniae serotype 1 (PS1) and the pneumococcal surface protein A (PspA). The synthesis of conjugate employed a new methodology for serotype 1. The evaluation of humoral immune response induced by the conjugate showed anti-PS1 IgG induction generated by immunization with the PS1-PspA.

A proteína A de superfície de pneumococo (PspA) é indispensável para a virulência da bactéria e foi escolhida para a elaboração de uma nova vacina conjugada contra S. pneumoniae. Para tanto foi desenvolvido um processo industrial de produção e purificação do fragmento recombinante da PspA clado 3 em E. coli. Cultivos descontínuos alimentados foram estabelecidos com glicose ou glicerol em reator de 5L, obtendo-se 62g/L de células secas e 3g/L de PspA3. As células foram lisadas por homogeneizador contínuo de alta pressão com eficiência de 96,7%. A centrifugação foi definida como etapa de clarificação. A sequência cromatográfica troca aniônica seguida de afinidade por Ni+2 rendeu os melhores resultados de pureza (81%) e recuperação (70%). A cromatografia de troca catiônica foi selecionada como terceira etapa do processo, definindo assim um processo de produção e purificação escalonável que possibilitou a obtenção de PspA3 com alto grau de pureza (90%). / The pneumococcal surface protein A (PspA) is indispensable for virulence of S. pneumoniae and it was the first choice as carrier for a new conjugated vaccine against S.pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment PspA clade 3 (rfPspA3) in E. coli. Fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. Centrifugation was defined for the clarification step. The sequence with Q- followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%, respectively). Cation exchange was chosen for the last chromatography. In conclusion, an industrial production and purification process was developed and rfPspA3 was obtained with high purity (90%).

<p><em>Streptococcus pneumoniae</em> (pneumokok) je&nbsp; jedan&nbsp; od&nbsp; vodećih&nbsp; uzroka morbiditeta i mortaliteta &scaron;irom sveta, kada su u pitanju infektivne bolesti. Pretežno izaziva infekcije gornjih respiratornih puteva (sinuzitis, otitis) i konjunktivitis. Vodeći je uzročnih vanbolničkih pneumonija, bakterijskog meningitisa i sepse. Lekovi izbora u terapiji pneumokoknih bolesti su beta laktamski antibiotici i makrolidi. Iako se makrolidni antibiotici uveliko koriste u lečenju pneumokoknih infekcija &scaron;irom sveta, porast rezistencije na makrolide&nbsp; bi&nbsp; mogao&nbsp; da&nbsp; kompromituje&nbsp; njihovu&nbsp; upotrebu. Rezistencija pneumokoka na makrolide je posredovana putem dva glavna mehanizma: modifikacija ciljnog mesta delovanja leka&nbsp; i aktivni efluks leka. Metilaciju 23S ribozomalne ribonukleinske kiseline (rRNK) obavlja enzim metilaza, čiju sintezu kodira<em> ermB</em> gen. Kod ovog tipa rezistencije dolazi do ukr&scaron;tene rezistencije na makrolide (M), linkozamide (L) i streptogramine B (Sb). Ovakav vid rezistencije se ispoljava kao MLS_b - fenotip i karakteri&scaron;e ga visok nivo rezistencije. Može se javiti kao konstitutivni (cMLS) i inducibilni (iMLS). Drugi mehanizam rezistencije na makrolide je aktivni efluks leka, kodiran od strane <em>mefA</em>&nbsp; gena. Efluks antibiotik a determini&scaron;e rezistenciju samo na 14-člane i 15-člane makrolide, bez ukr&scaron;tene rezistencije. Ispoljava se kao M-fenotip, a karakteri&scaron;e ga niži stepen rezistencije. Cilj ove studije je bio&nbsp; da&nbsp; se&nbsp; odredi u čestalost&nbsp; makrolidne&nbsp; rezistencije <em>Streptococcus pneumoniae</em> među invazivnim i neinvazivnim izolatima kod dece i odraslih, da se odrediti u čestalost korezistencije i multiple rezistencije kod makrolid rezistentnih sojeva&nbsp; <em>Streptococcus pneumoniae</em>, da se fenotipski odredi tip rezistencije na makrolide i da se ispita genska osnova makrolidne rezistencije (detektovati prisustvo <em>ermB</em> i <em>mefA</em> gena). Analizirani su podaci o 326 sojeva <em>Streptococcus pneumoniae</em> rezistentnih na makrolide (MRSP) sakupljenih &scaron;irom&nbsp; Srbije&nbsp; u&nbsp; periodu&nbsp; od&nbsp; januara&nbsp; 2010.&nbsp; do&nbsp; decembra&nbsp; 2012.&nbsp; godine. Sakupljeni&nbsp; MRSP&nbsp; izolati&nbsp; su&nbsp; transportovani&nbsp; u&nbsp; Nacionalnu&nbsp; referentnu laboratoriju za streptokok radi daljih ispitivanja. Identifikacija je vr&scaron;ena na osnovu mikroskopskih, kulturelnih i biohemijskih osobina. Konzervacija je vr&scaron;ena u moždano-srčanom bujonu sa 10% sadržajem glicerola na -80&deg;C. Dvostruki&nbsp; disk&nbsp; difuzioni&nbsp; test,&nbsp; kombinovani&nbsp; difuzion odilucioni&nbsp; test&nbsp; i automatizovani VITEK 2 sistem su kori&scaron;ćeni za određivanje fenotipova rezistencije na makrolide. Geni koji kodiraju rezistenciju na makrolide su detektovani PCR metodom. Ukupna rezistencija sojeva <em>S.pneumoniae</em> na makrolide u Srbiji je iznosila 34%. Sojevi <em>S.pneumoniae</em> rezistentni na makrolide su če&scaron;će bili izolovani kod dece (36%) u odnosu na odrasle (29%) osobe, i če&scaron;će su izolovani iz neinvazivnih (35,5%) u odnosu na invazivne (27,4%) materijale. Dominantan fenotip rezistencije na makrolide je bio MLS_b fenotip (78,5%). Konstitutivan MLS fenotip je bio zastupljen kod 73,9%, a inducibilan MLS kod 4,6% MRSP izolata. Potvrđena je udruženost <em>mefA</em>&nbsp; gena i M fenotipa; <em>ermB</em> gena i iMLS fenotipa, kao i <em>ermB</em> gena i cMLS fenotipa. Prisustvo oba ermB i mefA gena rezistencije je potvrđeno kod 43,9 % izolata. Svi izolati sa koji su imali oba gena rezistencije su ispoljili&nbsp; MLS_b fenotip.&nbsp; Istovremena&nbsp; neosetljivost&nbsp; na&nbsp; penicilin&nbsp; je bila zastupljena kod 16% MRSP sojeva. Visok nivo rezistencije na penicilin je imalo svega 5,8% MRSP izolata. Među MRSP sojevima je bio prisutan visok nivo&nbsp; rezistencije&nbsp; na&nbsp; tetraciklin&nbsp; (81,3%)&nbsp; i&nbsp; trimetoprim-sulfametoksazol (74,3%). Multirezistenti sojevi, koji su bili rezistentni na tetracikline i trimetoprim-sulfametoksazol su predstavljali dve trećine (66,1%) MRSP izolata.&nbsp; Zastupljenost&nbsp; udružene&nbsp; rezistencije&nbsp; MRSP&nbsp; na&nbsp; tetraciklin i trimetoprim-sulfametoksazol je bila veća kod sojeva sa MLS fenotipom (73,1%)&nbsp; u&nbsp; odnosu&nbsp; na&nbsp; sojeve&nbsp; sa&nbsp; M&nbsp; fenotipom&nbsp; (36,7%). Zastupljenost istovremene rezistencije na makrolide i druge antibiotike među kojima su penicilin, amoksicilin, cefotaksim, tetraciklin, trimetoprim-sulfametoksazol, kao&nbsp; i&nbsp; multirezistentnih&nbsp; sojeva&nbsp; je&nbsp; bila&nbsp; veća&nbsp; kod pedijatrijskih&nbsp; izolata pneumokoka&nbsp; u&nbsp; odnosu&nbsp; na&nbsp; sojeve&nbsp; dobijene&nbsp; kod&nbsp; odraslih.&nbsp; U čestalost istovremene rezistencije na makrolide i druge antibiotike među kojima su tetraciklin i ofloksacin je bila vi&scaron;e prisutna među neinvazivnim u odnosu na invazivne MRSP izolate. Invazivni MRSP izolati iz likvora su pokazivali veću rezistenciju na beta laktamske antibiotike u odnosu neinvazivne sojeve. MRSP sojevi su pokazali veoma visok nivo osetljivosti na levofloksacin (99,6), telitromicin (98,4%), cefotaksim (93,5%), i mipenem (97,3%). MRSP sojevi su u potpunosti bili osetljivi na vankomicin, linezolid, moksifloksacin, sparfloksacin, rifampicin&nbsp; i&nbsp; pristinamicin.&nbsp; Među&nbsp; invazivnim&nbsp; sojevima <em>S.pneumoniae</em> rezistentnim na makrolide je nađeno 12 različitih serotipova. Polovina izolata je pripadala serotipovima 19F (25%) i 14 (23%), dok su sledeći po učestalosti bili 6A (10,4%) i 23F (8,3%). Istovremena rezistencija na makrolide, penicilin, tetracikline i trimetoprim-sulfametoksazol je nađena kod serotipova 19F, 14 i 23F, dok su serotpovi 12F i 31 bili neosetljivi samo na makrolide. Na&scaron;e istraživanje predstavlja prvu detaljnu analizu fenotipskih i&nbsp; genotipskih&nbsp; osobina&nbsp; sojeva&nbsp; pneumokoka&nbsp; rezistentnih&nbsp; na&nbsp; makrolidne antibiotike u Srbiji. Dobijeni rezultati ukazuju na&nbsp; potrebu za aktivnim nadzorom nad pneumokoknim infekcijama u Srbiji.</p> / <p><em>Streptococcus pneumoniae</em> (pneumococcus) is one of the leading morbidity and&nbsp; mortality&nbsp; causes&nbsp; all&nbsp; over&nbsp; the&nbsp; world&nbsp; with&nbsp; respect&nbsp; to&nbsp; infectious&nbsp; diseases. <em>Streptococcus&nbsp; pneumoniae</em> is&nbsp; a&nbsp; leading&nbsp; cause&nbsp; of upper&nbsp; respiratory&nbsp; tract infections&nbsp; (&nbsp; sinusitis,&nbsp; otitis)&nbsp; and&nbsp; conjunctivitis. It&nbsp; is&nbsp; also&nbsp; the&nbsp; most&nbsp; common cause&nbsp; of&nbsp; community-acquired&nbsp; pneumonia, bacterial&nbsp; meningitis&nbsp; and&nbsp; sepsis. Beta lactam and&nbsp; macrolide antibiotics remained a first choice for empirical treatment of pneumococcal infections. Although macrolides are widely used for&nbsp;&nbsp; treatment&nbsp;&nbsp; of&nbsp;&nbsp; pneumococcal&nbsp;&nbsp; infections, an&nbsp;&nbsp; increase&nbsp;&nbsp; in&nbsp;&nbsp; macrolide resistance&nbsp; might compromise&nbsp; their use. Pneumococcal&nbsp; macrolide resistance is&nbsp; mediated&nbsp; by&nbsp; two&nbsp; major&nbsp; mechanisms:&nbsp; target&nbsp; site&nbsp; modification&nbsp; and&nbsp; active drug&nbsp; efflux.&nbsp; Methylation&nbsp; of&nbsp; the&nbsp; 23S&nbsp; ribosomal&nbsp; ribonucleic&nbsp; acid&nbsp; (rRNA)&nbsp; is performed&nbsp;&nbsp; by&nbsp;&nbsp; the&nbsp;&nbsp; enzyme&nbsp;&nbsp; methylase,&nbsp;&nbsp; encoded&nbsp;&nbsp; by&nbsp;&nbsp; the<em> ermB </em>gene. Modification&nbsp; of&nbsp; ribosomal&nbsp; targets&nbsp; leads&nbsp; to&nbsp; cross-resistance to&nbsp; macrolides (M),&nbsp; lincosamides&nbsp; (L)&nbsp; and&nbsp; streptogramins&nbsp; B&nbsp; (Sb). It&nbsp; is&nbsp; expressed&nbsp; as&nbsp; the MLS_b &ndash;phenotype,&nbsp; which&nbsp; confers&nbsp; a&nbsp; high-level&nbsp; resistance. This&nbsp; phenotype&nbsp; can&nbsp;&nbsp; be&nbsp;&nbsp; either&nbsp;&nbsp; constitutively&nbsp;&nbsp; (cMLS)&nbsp;&nbsp; or&nbsp;&nbsp; inducibly&nbsp;&nbsp; (iMLS). expressed. Another macrolide resistance mechanism is the active drug efflux, encoded by&nbsp; the <em>mefA&nbsp;</em> gene.&nbsp; The&nbsp; drug&nbsp; efflux&nbsp; confers&nbsp; resistance&nbsp; to&nbsp; 14-&nbsp; and&nbsp; 15-membered&nbsp; macrolides&nbsp; only,&nbsp; with&nbsp; no&nbsp; cross-resistance.&nbsp; It&nbsp; is&nbsp; expressed&nbsp; as&nbsp; the M-phenotype,&nbsp; which&nbsp; confers&nbsp; low-level&nbsp; resistance.&nbsp; The&nbsp; objective of&nbsp; this study&nbsp;&nbsp; was&nbsp;&nbsp; :&nbsp;&nbsp; 1) to&nbsp;&nbsp; examine&nbsp;&nbsp; the&nbsp;&nbsp; prevalence of&nbsp;&nbsp; macrolide&nbsp;&nbsp; resistant <em>Streptococcus&nbsp;&nbsp; pneumoniae </em>(MRSP) among&nbsp;&nbsp; invasive&nbsp;&nbsp; and&nbsp;&nbsp; noninvasive isolates in children and adults, 2) to examine the prevalence of coresistance and multiple-resistance among MRSP strains, 3) to examine the prevalence of&nbsp; macrolide&nbsp; resistant&nbsp; phenotypes,&nbsp; and&nbsp; 4)&nbsp; to&nbsp; examine&nbsp; the&nbsp; prevalence&nbsp; of macrolide&nbsp; resistant&nbsp; genotypes&nbsp; (detect&nbsp; the&nbsp; presence of&nbsp; the <em>ermB</em>&nbsp;&nbsp; and <em>mefA</em> gene).&nbsp; A&nbsp; total&nbsp; of&nbsp;&nbsp; 326&nbsp; MRSP&nbsp; strains&nbsp; were&nbsp; analyzed,&nbsp; which&nbsp; were&nbsp; collecte dall&nbsp; over&nbsp; Serbia&nbsp; in&nbsp; the&nbsp; period&nbsp; from&nbsp; January,&nbsp; 2010&nbsp; - December,&nbsp; 2012.&nbsp; The collected&nbsp;&nbsp; MRSP&nbsp;&nbsp; isolates&nbsp;&nbsp; were&nbsp;&nbsp; referred&nbsp;&nbsp; to&nbsp;&nbsp; the&nbsp;&nbsp; National&nbsp; Reference Laboratory&nbsp; for&nbsp; streptococci&nbsp; and&nbsp; pneumococci for&nbsp; further&nbsp; investigation. Identification based on microscopic, culture and biochemical features of the isolates. Conservation was performed in the brain-heart infusion broth with a&nbsp; 10%&nbsp; glycerol&nbsp; content&nbsp; at&nbsp; -80&deg;C.&nbsp; Macrolide&nbsp; resistance&nbsp; phenotypes&nbsp; were determined by a double disc diffusion test, combine d diffusion-dilution test and&nbsp;&nbsp; automatized&nbsp;&nbsp; VITEK&nbsp; 2 system. Macrolide&nbsp;&nbsp; resistance&nbsp;&nbsp; genes&nbsp;&nbsp; were&nbsp; determined by PCR. Overall, macrolide nonsusceptibility rate in Serbia was 34%.&nbsp; MRSP&nbsp; isolates&nbsp; were&nbsp; more&nbsp; prevale nt&nbsp; among&nbsp; children&nbsp; (36%)&nbsp; than adults&nbsp; (29%),&nbsp; and&nbsp; were&nbsp; more&nbsp; prevalent&nbsp; among&nbsp;&nbsp; noninvasive&nbsp; (35.5%)&nbsp; than invasive&nbsp; (27.4%)&nbsp; samples.&nbsp; Predominant&nbsp; macrolide&nbsp; resistance&nbsp; phenotype was&nbsp; the&nbsp; MLS b&nbsp; phenotype&nbsp; (78.5%),&nbsp; from&nbsp; which&nbsp; 73.9 %&nbsp; belonged&nbsp; to&nbsp; cMLS and&nbsp; 4.6%&nbsp; to&nbsp; iMLS&nbsp; phenotype.&nbsp; All&nbsp; the&nbsp; strains&nbsp; assigne d&nbsp; to&nbsp; the&nbsp; MLS_b phenotype harbored<em> ermB</em> gene, while all the strains with M phenotype had the mefA gene.&nbsp; The&nbsp; presence&nbsp; of&nbsp; both ermB and mefA resistance&nbsp; genes&nbsp; was confirmed&nbsp; in&nbsp; 43.9&nbsp; %&nbsp; of&nbsp; isolates. All&nbsp; the&nbsp; isolates&nbsp; which&nbsp; harbored&nbsp; both resistance genes expressed the MLS_b phenotype. Among macrolide resistant strains,&nbsp; penicillin&nbsp; nonsusceptiblility&nbsp; was&nbsp; observed&nbsp;&nbsp; in&nbsp; 16% .&nbsp; A&nbsp; high&nbsp; level resistance was confirmed in 5. 8% of MRSP isolates. MRSP strains showed high&nbsp; resistance rates to tetracyclin&nbsp; (81.3%) and&nbsp; trimethoprim-sulfamethoxazole&nbsp; (74.3%).&nbsp; Multiresistant&nbsp; strains,&nbsp; resistant&nbsp; to&nbsp; tetracyclines and&nbsp; trimethoprim-sulfamethoxazole,&nbsp; made&nbsp; two&nbsp; thirds&nbsp; (66.1&nbsp; %)&nbsp; of&nbsp; MRSP isolates.&nbsp; Among&nbsp; MRSP,&nbsp; co-resistance&nbsp; to&nbsp; tetracycline&nbsp; and&nbsp; trimethoprim-sulfamethoxazole&nbsp; was&nbsp; more&nbsp; prevalent&nbsp; among&nbsp; MLS&nbsp; phenotypes&nbsp; (73.1%) than&nbsp; M&nbsp; phenotypes&nbsp; (36.7%).&nbsp; Co-resistance&nbsp; strains&nbsp; to&nbsp; macrolides&nbsp; and&nbsp; other antibiotics including&nbsp;&nbsp;&nbsp; penicillin,&nbsp;&nbsp; amoxicillin,&nbsp;&nbsp;&nbsp; cefotaxime,&nbsp; tetracyclin, trimethoprim-sulfamethoxazole and multiresistant&nbsp; strains&nbsp; were more prevalent among children than adult. Coresistance to macrolides and other antibiotics&nbsp; including&nbsp; tetracycline&nbsp; and&nbsp; ofloxacin&nbsp; was&nbsp; more&nbsp; prevalent&nbsp; among noninvasive&nbsp;&nbsp; than&nbsp;&nbsp; invasive&nbsp;&nbsp; strains.&nbsp;&nbsp; Invasive&nbsp;&nbsp; MRSP&nbsp;&nbsp; isolates&nbsp;&nbsp; from&nbsp;&nbsp; the cerebrospinal fluid showed a higher resistance rate to beta lactam antibiotics than&nbsp; noninvasive&nbsp; strains.&nbsp; MRSP&nbsp; strains&nbsp; had&nbsp; a&nbsp; high&nbsp; susceptibility&nbsp; rates&nbsp; to levofloxacin&nbsp;&nbsp; (99.6),&nbsp;&nbsp; telithromycin&nbsp;&nbsp; (98.4%),&nbsp;&nbsp; cefotak sime&nbsp;&nbsp; (93.5%)&nbsp;&nbsp; and imipenem&nbsp; (97.3%).&nbsp; MRSP&nbsp; strains&nbsp; were&nbsp; fully&nbsp; susceptible&nbsp; to&nbsp; vancomycin, linezolid, moxifloxacin, sparfloxacin, rifampicin a nd pristinamycin. Among macrolide&nbsp;&nbsp; resistant <em>S.pneumoniae</em> strains,&nbsp;&nbsp; 12 different&nbsp;&nbsp; serotypes&nbsp;&nbsp; were identified.&nbsp; One&nbsp; half&nbsp; of&nbsp; these&nbsp; isolates&nbsp; belonged&nbsp; to the&nbsp; 19F&nbsp; (27.1%)&nbsp; and&nbsp; 14 (22.&nbsp; 9%)&nbsp; serotype,&nbsp; followed&nbsp; in&nbsp; frequency&nbsp; by&nbsp; the&nbsp; 6A&nbsp; (10.41%)&nbsp; and&nbsp; 23F (8.3%)&nbsp; serotype .&nbsp; Multiresistant&nbsp; strains&nbsp; (macrolides,&nbsp; penicillin,&nbsp; tetracyclines and&nbsp; trimethoprim-sulfamethoxazole)&nbsp; belonged&nbsp; to&nbsp; serotypes 19F,&nbsp; 14&nbsp; and 23F, while the 12F and 31 serotype were resistant to macrolides only. This in vestigation&nbsp;&nbsp; represents&nbsp;&nbsp; the&nbsp;&nbsp; first&nbsp;&nbsp; detailed&nbsp;&nbsp; analysis of&nbsp;&nbsp; phenotypes&nbsp;&nbsp; and genotypes&nbsp; of&nbsp; macrolide&nbsp; resistant&nbsp; pneumococcal&nbsp; strains&nbsp; in&nbsp; Serbia.&nbsp; The obtained&nbsp; results suggest&nbsp; the need for an active surveillance&nbsp; of pneumococcal infections in Serbia.</p>

Background: S. pneumoniae is a major cause of meningitis, pneumonia and sepsis in children. In 2006 universal pneumococcal vaccination was recommended in Germany for all children up to their second birthday. We have compared the prevalence and outcome of IPD at a single hospital before and after the introduction of vaccination. Findings: 55 cases of IPD were identified over an 11 year period. Almost half of the patients were younger than 2 years of age. Most of the children were affected by pneumonia. The second highest incidence seen was for meningitis and sepsis. 17 patients exhibited additional complications. Significant pre-existing and predisposing disorders, such as IRAK 4 defect, ALPS or SLE were identified in 4 patients. Complete recovery was seen in 78% of affected children; 11% had a fatal outcome and 11% suffered from long term complications. Only 31% overall had been vaccinated. The most common serotype was 14. Serotypes not covered by any of the current vaccines were also found. Antibiotic treatment commenced with cephalosporins in over 90%. Conclusion: Frequency of IPD in our hospital did not decrease after initiation of the pneumococcal vaccination. This might be due to vaccinations not being administered satisfactorily as well as to poor education about the need of the vaccination. Pre-existing diseases must be monitored and treated accordingly and rare deficiencies taken into account when IPD takes a foudroyant course. In addition, antibiotic stewardship has been initiated at this hospital centre as a consequence of the high cephalosporin use detected in this study.

Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen responsible for >1.6 million annual deaths globally. Pneumococcal penicillin-resistance is conferred by acquisition of ‘altered’ penicillin-binding protein (pbp) genes. The first penicillin-nonsusceptible pneumococci were identified in the late 1960s. Global pneumococcal penicillin-nonsusceptibility rates rapidly increased in the 1980s/90s. Since 2000, protein-conjugate vaccines, targeting 7, 10 or 13 of the ≥94 different pneumococcal capsule types (serotypes), have been introduced in many countries. Following vaccine implementation there has been a decline in vaccine-type pneumococcal disease and an increase in non-vaccine-type disease. These epidemiological changes result from “serotype replacement” and/or “serotype switching”. The former describes the expansion of non-vaccine-type clones in the absence of vaccine-type pneumococci. The latter describes serotype change following recombination at the capsule polysaccharide synthesis (cps) locus. To fully understand how pneumococci respond to vaccine- and antibiotic-induced selective pressures, we must better understand the evolutionary history of this pathogen. This thesis describes the study of a global collection of 426 pneumococci, dated 1937 - 2007. Serotype, genotype and penicillin-susceptibility data were collected. Nucleotide sequences of three pbp genes (for 389 isolates) and whole-genome sequences (for 96 isolates) were also generated. The data demonstrated the long-term persistence of certain clones within pneumococcal populations, and that pbp and large-fragment (>30 kb) cps ± pbp recombination was occurring prior to both widespread antibiotic use and vaccine implementation. The data highlighted the promiscuous nature of the globally-distributed PMEN1 clone and its contribution to the spread of pneumococcal penicillin-resistance. PMEN1 also donated multiple, large regions (1.7 - 32.3 kb) of its genome to at least two un-related clones. Finally, six “Tn916-like” genetic elements, conferring resistance to non-penicillin antibiotics, were newly identified. These included two of the oldest ever described. These results provided a unique insight into the history of pneumococcal evolution and the importance of genetic recombination.

Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...

Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...

Streptococcus pneumoniae encodes single serine/threonine protein kinase StkP and its cognate protein phosphatase PhpP. This signalling couple phosphorylates/dephosphorylates many target proteins involved in various cellular processes. So far, only few ot them was characterized in detail. Global phosphoproteomic analysis in the ∆stkP mutant strain background resulted in the identification of protein Spr0175 as phosphorylated on threonine 7. The main aim of this work was to characterize this new substrate. The ∆spr0175 mutant strains were prepared in the wild type genetic background Rx and R6 and then monitored for their growth and cell morphology. Mutant strains exhibited morphological defects revealing potential involvement of Spr0175 in the process of cell division. In the wild type D39 the deletion was unsuccesful, which may entail possible essentiality of Spr0175 in D39 strain. The results obtained also confirmed that the Spr0175 is modified in in vitro and in vivo conditions at threonine 7. In vitro study also confirmed minor phosphorylation at T4 residue. By using co-immunoprecipitation assay we demonstrated that Spr0175 protein can form oligomeric structures. Another aim of this work was cellular localization of Spr0175. By using fluorescent microscopy we showed that GFP-Spr0175 fusion...

Streptococcus pneumoniae is a leading cause of pneumonia, bacteremia, meningitis, otitis media and sinusitis, and is responsible for significant morbidity and mortality worldwide. The burden of pneumococcal disease has been greatly impacted by the high prevalence of HIV, especially in developing countries. Macrolides are commonly used for the treatment of pneumococcal infections with the resulting effect of increasing resistance. Pneumococci develop resistance to macrolides predominantly by two mechanisms; target modification and drug efflux. Target modification occurs through the acquisition of an erm(B) gene (MLSB phenotype) or through ribosomal mutation, and drug efflux occurs through the acquisition of a mef(A) gene (M phenotype). Alternative protein synthesis-inhibiting antibiotics such as linezolid and telithromycin have been developed in response to the increasing level of antibiotic resistance. In this study, novel mechanisms of resistance to protein synthesis-inhibiting antibiotics, and the current prevalence and epidemiology of macrolide resistance in South Africa were investigated. Two clinical isolates of S. pneumoniae resistant to macrolides, linezolid and chloramphenicol were identified in the PROTEKT surveillance study and the ABCs program of the CDC. The isolates were found to each contain a 6 bp deletion, resulting in the deletion of two amino acids from a highly conserved region of ribosomal protein L4 (64PWRQ67 to 64P_Q67 and 67QKGT70 to 67Q_T70). The genes encoding the mutant ribosomal proteins transformed susceptible strain R6 to macrolide, linezolid and chloramphenicol resistance, proving that the ii deletions conferred the resistance on the isolates, and indicating that these antibiotics share a common binding site. Growth studies of the R6 transformants showed increased mass doubling times, suggesting that the L4 mutations were associated with a fitness cost, but the original strains showed evidence of fitness compensation. The L4 mutations in these isolates represent a novel mechanism of cross-resistance to macrolides, linezolid and chloramphenicol. A macrolide-resistant clinical isolate of S. pneumoniae with mutations in 23S rRNA showed a heterogeneous phenotype and genotype. A mutant gene encoding 23S rRNA from this isolate transformed susceptible strain R6 to resistance. Transformants displayed similar heterogeneity to the isolate. Culture of resistant strain R6 in the presence of antibiotic maintained resistance, however culture of the strain in the absence of antibiotic pressure resulted in a reversion to susceptibility. By DNA sequencing, gene conversion was shown to occur between the wild-type and mutant 23S rRNA alleles. Growth studies indicated that the resistant phenotype was associated with a fitness cost. Therefore, under antibiotic selective pressure alleles converted to the mutant form, and in the absence of selective pressure alleles reverted to wild-type, in order to regain fitness. Through gene conversion the pneumococcus has the ability to rapidly adapt to the environment, with implications for susceptibility testing and patient treatment. A rare clinical isolate of S. pneumoniae, highly resistant to telithromycin, was received from the Canadian Bacterial Surveillance Network and was investigated for the mechanism of resistance. The isolate was found to contain an erm(B) gene iii with a truncated control peptide, as well as a mutant ribosomal protein L4, containing a number of mutations. Transformation of susceptible strain PC13, containing a wild-type erm(B) gene, with the mutant erm(B) gene decreased the susceptibility of PC13 to telithromycin, but did not confer high-level resistance. Transformation of PC13 with the mutant L4 gene or a fragment of the L4 gene containing only the 69GTG71 to TPS mutation, conferred high-level resistance on PC13. In contrast, transformation of R6, which did not contain an erm(B) gene, with the L4 gene or L4 fragment only conferred reduced telithromycin susceptibility. High-level telithromycin resistance was therefore conferred by a combination of an erm(B) gene with a 69GTG71 to TPS mutation in a highly conserved region of ribosomal protein L4. The combination of mechanisms inhibited the binding of telithromycin to the ribosome, whereas neither mechanism individually was sufficient. A telithromycin-resistant clinical isolate of S. pneumoniae was received from the PROTEKT surveillance study and was investigated for the resistance mechanism. The isolate was found to contain a 136 bp deletion in the regulatory region of erm(B). This mutant gene was shown, by transformation studies, to confer resistance on susceptible strain PC13. Expression of erm(B) on the transcriptional level was quantified by real-time reverse transcription PCR. In the presence of erythromycin and telithromycin, erm(B) expression was significantly higher in the mutant PC13 strain than the wild-type strain. Growth studies showed that the mutant PC13 strain had a shorter lag phase than the wild-type strain in the presence of erythromycin. Telithromycin resistance was conferred by the mutant iv erm(B) gene that was expressed at a higher level than the wild-type gene, most likely resulting in higher ribosomal methylation levels sufficient to hinder telithromycin binding. Macrolide resistance in invasive pneumococcal disease in South Africa for the period 2000 to 2005 was investigated through a national laboratory-based surveillance system. Viable isolates (n=15982) collected during the six-year period were phenotypically characterised, by determination of MICs and serotyping. Two hundred and sixty random isolates from 2005 were genotypically screened for the presence of erm(B) and mef(A). Macrolide resistance increased significantly from 9% in 2000 to 14% in 2005. Resistant isolates were received from all provinces of South Africa, with Gauteng and the Western Cape having the highest incidence. Serotype 14 was the most common macrolide-resistant serotype and 96% of macrolide-resistant isolates in 2005 were serotypes included in the 7-valent pneumococcal conjugate vaccine and serotype 6A. Macrolide resistance was significantly higher in children <5 than in individuals 5 years and older. The majority of strains (75%) over the six-year period displayed the MLSB phenotype. Of the 260 strains genotypically screened, 57% were positive for erm(B), 27% were positive for mef(A), 15% contained both erm(B) and mef(A), and 1% were negative for both genes and were found to contain ribosomal mutations. Eighty percent of isolates containing both erm(B) and mef(A) were serotype 19F and were found to be clonal by PFGE and MLST. These multidrug-resistant isolates were related to the Taiwan19F-14 global clone. v Many protein synthesis-inhibiting antibiotics share overlapping binding sites on the large ribosomal subunit. Alterations in 23S rRNA and ribosomal proteins L4 and L22, within the binding pocket, confer resistance and often cross-resistance to many of these antibiotics. The ability of the pneumococcus to develop resistance and the global spread of resistant strains highlights the importance of monitoring resistance levels and understanding resistance mechanisms.

Acute respiratory tract infections are the most common cause o f illness and death in the pediatric population worldwide. It is estimated that 70 - 80% o f severe pneumonias in Africa are caused by S.pnewnoniae (the pneumococcus) followed by H. influenzae type b. Surveillance reveals that drug resistance is increasing worldwide, South Africa not being an exception. This has considerably complicated the management o f infections caused by both the pneumococcus and H. influenzae type b ( H ib ). It is widely accepted that colonization of the nasopharynx even briefly precedes middle ear infection and invasive pneumococcal disease. Early onset of colonization after birth has been associated with early onset o f middle ear infections. Furthermore, colonized children are able to transmit these organisms to other children. Carriage o f pneumococci commonly occurs in young children. The carriage of resistant pneumococci is usually limited to those serotypes carried in children. N ew conjugate vaccines may be able to reduce colonization o f these serotypes. This study was undertaken to determine the serotypes and susceptibility o f pneumococci and H. influenzae type b, and the proportion o f healthy children colonized at Zola Community Health Centre (ZCHC) in Soweto.