La batalla decisiva contra la fiebre tifoidea en Santiago de Chile en el siglo XX / The decisive battle against typhoid fever in Santiago de Chile in the twentieth century

Hasta 1983, cuando alcanzaba la increíble tasa de 118 casos por 100.000 habitantes, la fiebre tifoidea era la peor amenaza infecciosa en Santiago, Chile, ciudad que figuraba junto a Ciudad de México, El Cairo y Bombay, como una de las con mayor endemia en el mundo. El Ministerio de Salud respondió formando el Comité de Tifoidea de Chile, con participación de expertos nacionales y del grupo de Myron Levine, de la Universidad de Maryland, que llevó a cabo ingeniosas investigaciones, culpando al río Mapocho, cuyas aguas contaminadas con Salmonella typhi regaban los predios agrícolas vecinos, conformando así un ciclo largo de infección. Las vacunas antitíficas ensayadas (oral Ty21a atenuada y polisacárido capsular Vi inyectable) no mostraron eficacia, los portadores crónicos no se trataron, pero una campaña sanitaria a través de la televisión contribuyó decisivamente a mejorar los hábitos higiénicos de la población, fortalecida por el pánico que causó la llegada del cólera en 1991, y la fiebre tifoidea prácticamente desapareció del escenario.

Until 1983, when reached the incredible frequency of 118 cases for 100.000 habitants, typhoid fever was the worst infectious threat in Santiago, Chile, city that appeared next to Mexico City, Cairo and Bombay, as one of the most endemic in the world. The Ministry of Health responded with the creation of The Chilean Typhoid Committee, with the participation of national experts and Myron Levine's group, which carried out ingenious investigations blaming the Mapocho River, whose waters contaminated with Salmonella typhi irrigated the neighboring farms, thus conforming a long cycle of infection. Typhoid vaccines tested (strain Ty 21a oral and Vi capsular polysaccharide) did not show efficacy, chronic carriers were not treated, but a health campaign on television made a decisive contribution to improving hygiene habits of the population, strengthened by the panic caused by the arrival of cholera in 1991, and typhoid fever practically disappeared from the stage.

Influence of the Polysaccharide Capsule on the Bactericidal Activity of Indolicidin on Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide's bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.

Influence of the Polysaccharide capsule on the bactericidal activity of indolicidin on Streptococcus pneumoniae

Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide’s bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.

Cryptococcus gattii polysaccharide capsule: An insight on fungal-host interactions and vaccine studies.

Cryptococcus neoformans and C. gattii complexes are the main causative agents of cryptococcosis, a neglected disease with high lethality. The capsule, composed predominantly of the capsular polysaccharide (CP) GXM, is the main virulence factor of this pathogen. The role of CP is well described for C. neoformans and; however, there is a scarcity of studies focused on C. gattii, especially in the context of the fungal-host interaction. Understanding how the immune system recognizes C. gattii can generate meaningful information for diagnosing, preventing, and treating cryptococcosis. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2021. 51: 2281-2295], Ueno et al. demonstrate that CP inhibits C. gattii recognition by CD11b. In this commentary, we highlight the importance of deeply understanding the role of C. gattii CP during infection and how this knowledge would influence the strategies to develop new vaccines against cryptococcosis.

Frequency of Staphylococcus aureus virulence genes in milk of cows and goats with mastitis / Frequência de genes fatores de virulência de Staphylococcus aureus em leite de vacas e cabras com mastite

The present study determined the frequency of Staphylococcus aureus virulence genes in 2,253 milk samples of cows (n=1000) and goats (n=1253) raised in three different geographical regions of the state Pernambuco, Brazil. The presence of genes of virulence factors associated to adhesion to host cells (fnbA, fnbB, clfA and clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla and hlb), and capsular polysaccharide (cap5 and cap8) was evaluated by PCR. A total of 123 and 27 S. aureus strains were isolated from cows' and goats' milk, respectively. The sec and tsst genes were detected exclusively in goats' isolates, while the seh gene was only identified in cows' isolates. The number of toxin genes per strain showed that goats' isolates are likely more toxic than bovines' isolates. The cap5 genotype predominated in both host species, especially in strains collected from cows raised in the Agreste region. The cap8 genotype is likely more virulent due to the number of virulence genes per strain. The results of the present study demonstrate that S. aureus may pose a potential threat to human health in Brazil, and, therefore, these results should support actions related to mastitis control programs.(AU)

O presente estudo determinou a frequência de genes de virulência de Staphylococcus aureus em 2253 amostras de leite, sendo de vacas n=1000 e de cabras n=1253, procedentes das três regiões geográficas do estado de Pernambuco, Brasil. A presença de genes de fatores de virulência associados à adesão às células hospedeiras (fnbA, fnbB, clfA e clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla e hlb) e polissacarídeo capsular (cap5 e cap8) foram avaliadas por PCR. Um total de 123 e 27 cepas de S. aureus foram isoladas do leite de vacas e cabras, respectivamente. Os genes sec e tsst foram detectados exclusivamente em isolados de cabras, enquanto o gene seh foi identificado apenas em isolados de vaca. O número de genes de toxina por cepa mostrou que os isolados de cabras são potencialmente mais tóxicos do que os isolados obtidos de bovinos. O genótipo cap5 predominou em ambas as espécies hospedeiras, especialmente em cepas coletadas de vacas criadas na região Agreste. O genótipo cap8 é potencialmente mais virulento devido ao número de genes de virulência por isolado. Os resultados do presente estudo demonstram que S. aureus pode representar uma ameaça potencial para a saúde humana no Brasil e, portanto, estes resultados devem subsidiar ações relacionadas aos programas de controle de mastite.(AU)

Frequency of Staphylococcus aureus virulence genes in milk of cows and goats with mastitis / Frequência de genes fatores de virulência de Staphylococcus aureus em leite de vacas e cabras com mastite

The present study determined the frequency of Staphylococcus aureus virulence genes in 2,253 milk samples of cows (n=1000) and goats (n=1253) raised in three different geographical regions of the state Pernambuco, Brazil. The presence of genes of virulence factors associated to adhesion to host cells (fnbA, fnbB, clfA and clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla and hlb), and capsular polysaccharide (cap5 and cap8) was evaluated by PCR. A total of 123 and 27 S. aureus strains were isolated from cows' and goats' milk, respectively. The sec and tsst genes were detected exclusively in goats' isolates, while the seh gene was only identified in cows' isolates. The number of toxin genes per strain showed that goats' isolates are likely more toxic than bovines' isolates. The cap5 genotype predominated in both host species, especially in strains collected from cows raised in the Agreste region. The cap8 genotype is likely more virulent due to the number of virulence genes per strain. The results of the present study demonstrate that S. aureus may pose a potential threat to human health in Brazil, and, therefore, these results should support actions related to mastitis control programs.(AU)

O presente estudo determinou a frequência de genes de virulência de Staphylococcus aureus em 2253 amostras de leite, sendo de vacas n=1000 e de cabras n=1253, procedentes das três regiões geográficas do estado de Pernambuco, Brasil. A presença de genes de fatores de virulência associados à adesão às células hospedeiras (fnbA, fnbB, clfA e clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla e hlb) e polissacarídeo capsular (cap5 e cap8) foram avaliadas por PCR. Um total de 123 e 27 cepas de S. aureus foram isoladas do leite de vacas e cabras, respectivamente. Os genes sec e tsst foram detectados exclusivamente em isolados de cabras, enquanto o gene seh foi identificado apenas em isolados de vaca. O número de genes de toxina por cepa mostrou que os isolados de cabras são potencialmente mais tóxicos do que os isolados obtidos de bovinos. O genótipo cap5 predominou em ambas as espécies hospedeiras, especialmente em cepas coletadas de vacas criadas na região Agreste. O genótipo cap8 é potencialmente mais virulento devido ao número de genes de virulência por isolado. Os resultados do presente estudo demonstram que S. aureus pode representar uma ameaça potencial para a saúde humana no Brasil e, portanto, estes resultados devem subsidiar ações relacionadas aos programas de controle de mastite.(AU)

A Predicted Mannoprotein Participates in Cryptococcus gattii Capsular Structure.

The yeast-like pathogen Cryptococcus gattii is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from C. gattii, designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that C. gattii Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence in vivoIMPORTANCECryptococcus gattii has the ability to escape from the host's immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall.

Mutation of Agr Is Associated with the Adaptation of Staphylococcus aureus to the Host during Chronic Osteomyelitis.

Selection pressures exerted on Staphylococcus aureus by host factors may lead to the emergence of mutants better adapted to the evolving conditions at the infection site. This study was aimed at identifying the changes that occur in S. aureus exposed to the host defense mechanisms during chronic osteomyelitis and evaluating whether these changes affect the virulence of the organism. Genome assessment of two S. aureus isolates collected 13 months apart (HU-85a and HU-85c) from a host with chronic osteomyelitis was made by whole genome sequencing. Agr functionality was assessed by qRT-PCR. Isolates were tested in a rat model of osteomyelitis and the bacterial load (CFU/tibia) and the morphometric osteomyelitic index (OI) were determined. The ability of the isolates to trigger the release of proinflammatory cytokines was determined on macrophages in culture. Persistence of S. aureus within the host resulted in an agrC frameshift mutation that likely led to the observed phenotype. The capacity to cause bone tissue damage and trigger proinflammatory cytokines by macrophages of the agr-deficient, unencapsulated derivative (HU-85c) was decreased when compared with those of the isogenic CP8-capsulated parental strain (HU-85a). By comparison, no significant differences were found in the bacterial load or the OI from rats challenged with isogenic Reynolds strains [CP5, CP8, and non-typeable (NT)], indicating that lack of CP expression alone was not likely responsible for the reduced capacity to cause tissue damage in HU-85c compared with HU-85a. The production of biofilm was significantly increased in the isogenic derivative HU-85c. Lack of agr-dependent factors makes S. aureus less virulent during chronic osteomyelitis and alteration of the agr functionality seems to permit better adaptation of S. aureus to the chronically infected host.

Comparative genomics reveals diverse capsular polysaccharide synthesis gene clusters in emerging Raoultella planticola

Raoultella planticola is an emerging zoonotic pathogen that is associated with rare but life-threatening cases of bacteremia, biliary tract infections, and urinary tract infections. Moreover, increasing antimicrobial resistance in the organism poses a potential threat to public health. In spite of its importance as a human pathogen, the genome of R. planticola remains largely unexplored and little is known about its virulence factors. Although lipopolysaccharides has been detected in R. planticola and implicated in the virulence in earlier studies, the genetic background is unknown. Here, we report the complete genome and comparative analysis of the multidrug-resistant clinical isolate R. planticola GODA. The complete genome sequence of R. planticola GODA was sequenced using single-molecule real-time DNA sequencing. Comparative genomic analysis reveals distinct capsular polysaccharide synthesis gene clusters in R. planticola GODA. In addition, we found bla TEM-57 and multiple transporters related to multidrug resistance. The availability of genomic data in open databases of this emerging zoonotic pathogen, in tandem with our comparative study, provides better understanding of R. planticola and the basis for future work.

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium.

Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508-1519, 2017.

Capsular polysaccharide production by Streptococcus pneumoniae serotype 1: from strain selection to fed-batch cultivation.

Streptococcus pneumoniae is a human pathogen largely transmitted by aerosols. Vaccines are the main strategy against this pathogen, and the capsular polysaccharide (PS) is its major antigen. S. pneumoniae serotype 1 is associated with large outbreaks and epidemics of invasive diseases. The aims of this work were to screen serotype 1 strains to identify the best PS1 producer, evaluate three peptones for PS1 production, investigate the effects of culture medium components using a design of experiments (DoE), a statistic tool for optimization, and propose a new medium/cultivation strategy. After flask cultivation of nine strains, two that produced high PS1 and biomass values were chosen for further evaluation in the bioreactor, and ST595/01 was chosen as the best PS1 producer strain. Among the peptones tested (Casamino acids, Soytone, and Phytone), the highest PS1 production (298 mg/L) was reached with Phytone. Next, DoE (2(4-1)) was performed to evaluate the effects of yeast extract (YE), Phytone, L-asparagine (Asn), and L-glutamine (Gln), yielding the following results: Phytone presented positive effects (p < 0.05) for maximum production of biomass, PS1, acetate, and lactate; YE showed positive effects for biomass and acid production (p < 0.05); Gln exerted a minor positive effect on PS1 yield factor on glucose (p < 0.1); and Asn presented only an effect on acetate production (p < 0.1). Hence, a new culture medium was formulated based on Phytone, YE, and glucose, and batch and fed-batch cultivations were evaluated. The fed-batch cultivation showed almost 2 times the biomass and 2.5 times the PS1 production as the batch culture, and 8-10 times higher PS1 production than has been previously reported.

Desenvolvimento de meio quimicamente definido para produção de polissacarídeo capsular em cultivo de Streptococcus pneumoniae sorotipo 14 / Development of a chemically defined medium for capsular polysaccharide production by Streptococcus pneumoniae serotype 14

Neste trabalho avaliou-se a influência de fontes de carbono (FC) e composições de meio definido no crescimento celular e na produção do polissacarídeo PS14. Em batelada, testou-se como FC glicose, sacarose e frutose em diferentes concentrações. Testou-se também meios com ausência dos aminoácidos asparagina, ácido aspártico, fenilalanina, serina, alanina, treonina, triptofano, lisina e tirosina, das vitaminas/cofatores ácido fólico, piridoxamina, ácido p-aminobenzóico, b-NAD e riboflavina, além bem como da adição de maiores concentrações de aminoácidos identificados como importantes. Em cultivo contínuo foram avaliadas vazões específicas de alimentação (D) de 0,1h-1a 0,5h-1 e a influência das bases nitrogenadas. O meio com sacarose como FC, retirada dos aminoácidos e vitaminas citados e adição do dobro de glicina isoleucina, leucina, valina e o triplo de glutamina levou à maior produção de PS14 (441mg/L). Obteve-se a maior produtividade com D=0,4h-1e a maior quantidade de PS14 com adenina na concentração original no meio de cultura.

In this work we assessed the influence of different carbon sources (CS) and defined medium compositions on cell growth and polysaccharide PS14 production. In bath, glucose, sucrose and fructose were tested at different concentrations. Also, media were tested with absence of the amino acids: asparagine, aspartic acid, phenylalanine, serine, alanine, threonine, tryptophan, lysine, and tyrosine, and vitamins/cofactors: folic acid, pyridoxamine, p-aminobenzoic acid, riboflavin and b-NAD, besides the addition of higher concentration of amino acids identified as important. In continuous cultivation, dilution rates (D) from 0.1 h-1 to 0.5 h-1 were evaluated as well as the influence of nitrogenous bases. The medium containing sucrose as CS, absence of amino acids and vitamins and addition of twice glycine isoleucine, leucine, valine, and triple glutamine led to higher production of PS14 (441mg/L). D of 0.4 h-1 showed higher productivity and adenine in standard concentration produced greater amounts of PS14.

Cultivo Contínuo de Streptococcus penumoniae sorotipo 14: Metabolismo e Cinética de Produção de Polissacarídeo Capsular / Continous cultivation of Streptococcus penumoniae serotype 14: Metabolism and Kinetics of Capsular Polysaccharide Production

S. pneumoniae, micro- organismo causador de pneumonia, bacterimia, meningite e otite aguda do ouvido médio, é detentor de altas taxas de mortalidade em crianças. Seu principal fator de virulência, o polissacarídeo capsular (PS), é utilizado como antígeno nas vacinas pneumocócicas existentes e é a base para a classificação do pneumococo em mais de 90 sorotipos. Atualmente, o sorotipo 14 é o que mais atinge crianças no Brasil, sendo então escolhido para este estudo. Apesar de sua grande importância, pouco são os trabalhos dedicados ao estudo do metabolismo do pneumococo. O objetivo desta dissertação foi estudar o metabolismo de crescimento, de carbono e de produção de PS em cultivo contínuo em biorreator, uma abordagem inédita para este micro-organismo...

S. pneumoniae, causative agente of pneumonia, bacteremia, meningitis and acute otitis media, is a major cause of children mortality. The capsular polysaccharide (PS) is the most important pneumococcal virulence factor and has been used as antigen in all avalaible pneumococcal vaccines. The PS is also the basis for S. pneumoniae has been devoted to pneumococcal metabolism. The serotype 14 , the most commom pediatric in Brazil, was selected for this study aiming to evaluate the growing, carbon and PS from S. pneumoniae serotype 14 (PS14) and the best producer strain was select among eght strains...