Pneumococcal population genomics changes during the early time period of conjugate vaccine uptake in southern India.

Streptococcus pneumoniae is a major cause of invasive disease of young children in low- and middle-income countries. In southern India, pneumococcal conjugate vaccines (PCVs) that can prevent invasive pneumococcal disease began to be used more frequently after 2015. To characterize pneumococcal evolution during the early time period of PCV uptake in southern India, genomes were sequenced and selected characteristics were determined for 402 invasive isolates collected from children <5 years of age during routine surveillance from 1991 to 2020. Overall, the prevalence and diversity of vaccine type (VT) and non-vaccine type (NVT) isolates did not significantly change post-uptake of PCV. Individually, serotype 1 and global pneumococcal sequence cluster (GPSC or strain lineage) 2 significantly decreased, whereas serotypes 6B, 9V and 19A and GPSCs 1, 6, 10 and 23 significantly increased in proportion post-uptake of PCV. Resistance determinants to penicillin, erythromycin, co-trimoxazole, fluoroquinolones and tetracycline, and multidrug resistance significantly increased in proportion post-uptake of PCV and especially among VT isolates. Co-trimoxazole resistance determinants were common pre- and post-uptake of PCV (85 and 93 %, respectively) and experienced the highest rates of recombination in the genome. Accessory gene frequencies were seen to be changing by small amounts across the frequency spectrum specifically among VT isolates, with the largest changes linked to antimicrobial resistance determinants. In summary, these results indicate that as of 2020 this pneumococcal population was not yet approaching a PCV-induced equilibrium and they highlight changes related to antimicrobial resistance. Augmenting PCV coverage and prudent use of antimicrobials are needed to counter invasive pneumococcal disease in this region.

Contaminants of emerging concern: Occurrence, analytical techniques, and removal with electrochemical advanced oxidation processes with special emphasis in Latin America.

The occurrence of contaminants of emerging concern (CECs) in environmental systems is gradually more studied worldwide. However, in Latin America, the presence of contaminants of emerging concern, together with their environmental and toxicological impacts, has recently been gaining wide interest in the scientific community. This paper presents a critical review about the source, fate, and occurrence of distinct emerging contaminants reported during the last two decades in various countries of Latin America. In recent years, Brazil, Chile, and Colombia are the main countries that have conducted research on the presence of these pollutants in biological and aquatic compartments. Data gathered indicated that pharmaceuticals, pesticides, and personal care products are the most assessed CECs in Latin America, being the most common compounds the followings: atrazine, acenaphthene, caffeine, carbamazepine, ciprofloxacin, diclofenac, diuron, estrone, losartan, sulfamethoxazole, and trimethoprim. Most common analytical methodologies for identifying these compounds were HPLC and GC coupled with mass spectrometry with the potential to characterize and quantify complex substances in the environment at low concentrations. Most CECs' monitoring and detection were observed near to urban areas which confirm the out-of-date wastewater treatment plants and sanitization infrastructures limiting the removal of these pollutants. Therefore, the implementation of tertiary treatment should be required. In this tenor, this review also summarizes some studies of CECs removal using electrochemical advanced oxidation processes that showed satisfactory performance. Finally, challenges, recommendations, and future perspectives are discussed.

The healing effect of Pseudomonas Quinolone Signal (PQS) with co-infection of Staphylococcus aureus and Pseudomonas aeruginosa: A preclinical animal co-infection model.

BACKGROUND: Because of the rise in antibiotic resistance and the control of pathogenicity, polymicrobial bacterial biofilms exacerbate wound infections. Since bacterial quorum sensing (QS) signals can dysregulate biofilm development, they are interesting therapeutic treatments. In this study, Pseudomonas Quinolone Signal (PQS) was used to treat an animal model of a wound that had both Staphylococcus aureus and Pseudomonas aeruginosa co-infection. METHODS: S. aureus and P. aeruginosa mono- and co-infection models were developed in vitro on the L-929 cell line and in an animal model of wound infection. Moreover, PQS was extracted and purified using liquid chromatography. Then, the mono- and co-infection models were treated by PQS in vitro and in vivo. RT-PCR analysis was used to look into changes in biofilm, QS, tissue regeneration, and apoptosis genes after the treatment. RESULTS: PQS significantly disrupted established biofilm up to 90% in both in vitro and in vivo models. Moreover, a 93% reduction in the viability of S. aureus and P. aeruginosa was detected during the 10 days of treatment in comparison to control groups. In addition, the biofilm-encoding and QS-regulating genes were down-regulated to 75% in both microorganisms. Also, fewer epithelial cells died when treated with PQS compared to control groups in both mono- and co-infection groups. CONCLUSION: According to this study, PQS may facilitate wound healing by stimulating the immune system and reducing apoptosis. It seems to be a potential medication to use in conjunction with antibiotics to treat infections that are difficult to treat.

Oxidation of hemoproteins by Streptococcus pneumoniae collapses the cell cytoskeleton and disrupts mitochondrial respiration leading to the cytotoxicity of human lung cells.

IMPORTANCE: Streptococcus pneumoniae (Spn) colonizes the lungs, killing millions every year. During its metabolism, Spn produces abundant amounts of hydrogen peroxide. When produced in the lung parenchyma, Spn-hydrogen peroxide (H2O2) causes the death of lung cells, and details of the mechanism are studied here. We found that Spn-H2O2 targets intracellular proteins, resulting in the contraction of the cell cytoskeleton and disruption of mitochondrial function, ultimately contributing to cell death. Intracellular proteins targeted by Spn-H2O2 included cytochrome c and, surprisingly, a protein of the cell cytoskeleton, beta-tubulin. To study the details of oxidative reactions, we used, as a surrogate model, the oxidation of another hemoprotein, hemoglobin. Using the surrogate model, we specifically identified a highly reactive radical whose creation was catalyzed by Spn-H2O2. In sum, we demonstrated that the oxidation of intracellular targets by Spn-H2O2 plays an important role in the cytotoxicity caused by Spn, thus providing new targets for interventions.

Endogenously produced H2O2 is intimately involved in iron metabolism in Streptococcus pneumoniae.

IMPORTANCE: Heme degradation provides pathogens with growth essential iron, leveraging on the host heme reservoir. Bacteria typically import and degrade heme enzymatically, and here, we demonstrated a significant deviation from this dogma. We found that Streptococcus pneumoniae liberates iron from met-hemoglobin extracellularly, in a hydrogen peroxide (H2O2)- and cell-dependent manner; this activity serves as a major iron acquisition mechanism for S. pneumoniae. Inhabiting oxygen-rich environments is a major part of pneumococcal biology, and hence, H2O2-mediated heme degradation likely supplies iron during infection. Moreover, H2O2 reaction with ferrous hemoglobin but not with met-hemoglobin is known to result in heme breakdown. Therefore, the ability of pneumococci to degrade heme from met-hemoglobin is a new paradigm. Lastly, this study will inform other research as it demonstrates that extracellular degradation must be considered in the interpretations of experiments in which H2O2-producing bacteria are given heme or hemoproteins as an iron source.

Oxidation of hemoglobin in the lung parenchyma facilitates the differentiation of pneumococci into encapsulated bacteria.

Pneumococcal pneumonia causes cytotoxicity in the lung parenchyma but the underlying mechanism involves multiple factors contributing to cell death. Here, we discovered that hydrogen peroxide produced by Streptococcus pneumoniae (Spn-H 2 O 2 ) plays a pivotal role by oxidizing hemoglobin, leading to its polymerization and subsequent release of labile heme. At physiologically relevant levels, heme selected a population of encapsulated pneumococci. In the absence of capsule and Spn-H 2 O 2 , host intracellular heme exhibited toxicity towards pneumococci, thus acting as an antibacterial mechanism. Further investigation revealed that heme-mediated toxicity required the ABC transporter GlnPQ. In vivo experiments demonstrated that pneumococci release H 2 O 2 to cause cytotoxicity in bronchi and alveoli through the non-proteolytic degradation of intracellular proteins such as actin, tubulin and GAPDH. Overall, our findings uncover a mechanism of lung toxicity mediated by oxidative stress that favor the growth of encapsulated pneumococci suggesting a therapeutic potential by targeting oxidative reactions. Highlights: Oxidation of hemoglobin by Streptococcus pneumoniae facilitates differentiation to encapsulated pneumococci in vivo Differentiated S. pneumoniae produces capsule and hydrogen peroxide (Spn-H 2 O 2 ) as defense mechanism against host heme-mediated toxicity. Spn-H 2 O 2 -induced lung toxicity causes the oxidation and non-proteolytic degradation of intracellular proteins tubulin, actin, and GAPDH. The ABC transporter GlnPQ is a heme-binding complex that makes Spn susceptible to heme toxicity.

Limited protection of pneumococcal vaccines against emergent Streptococcus pneumoniae serotype 14/ST876 strains.

PURPOSE: Streptococcus pneumoniae (Spn) is a major cause of child death. We investigated the epidemiology of S. pneumoniae in a pediatric fever clinic and explored the genomics basis of the limited vaccine response of serotype 14 strains worldwide. METHODS: Febrile disease and pneumonia were diagnosed following criteria from the WHO at the end of 2019 at a tertiary children's hospital. Spn was isolated by culture from nasopharyngeal (NP) swabs. The density was determined by lytA-base qPCR. Isolates were serotyped by Quellung and underwent antimicrobial susceptibility testing. Whole-genome sequencing was employed for molecular serotyping, MLST, antibiotic gene determination, SNP calling, recombination prediction, and phylogenetic analysis. RESULTS: The presence of pneumococcus in the nasopharynx (87.5%, 7/8, p = 0.0227) and a high carriage (100%, 7/7, p = 0.0123) were significantly associated with pneumonia development. Living with siblings (73.7%, 14/19, p = 0.0125) and non-vaccination (56.0%, 28/50, p = 0.0377) contributed significantly to the Spn carriage. Serotype 14 was the most prevalent strain (16.67%, 5/30). The genome analysis of 1497 serotype 14 strains indicated S14/ST876 strains were only prevalent in China, presented limited vaccine responses with higher recombination activities within its cps locus, and unique variation patterns in the genes wzg and lrp. CONCLUSION: With the lifting of the one-child policy, it will be crucial for families with multiple children to get PCV vaccinations in China. Due to the highly variant cps locus and distinctive variation patterns in capsule shedding and binding proteins genes, the prevalent S14/ST876 strains have shown poor response to current vaccines. It is necessary to continue monitoring the molecular epidemiology of this vaccine escape clone.

Association between nasopharyngeal colonization with multiple pneumococcal serotypes and total pneumococcal colonization density in young Peruvian children.

OBJECTIVES: We examined the association of nasopharyngeal (NP) pneumococcal co-colonization (>1 pneumococcal serotype) and pneumococcal density in young Peruvian children enrolled in a prospective cohort study. METHODS: NP swabs collected monthly from children aged <3 years during both asymptomatic and acute respiratory illness (ARI) periods underwent culture-enriched microarray for pneumococcal detection and serotyping and lytA polymerase chain reaction for density assessment. We examined the serotypes commonly associated with co-colonization and the distribution of densities by co-colonization, age, current ARI, and other covariates. The association of co-colonization and pneumococcal density was assessed using a multivariable mixed-effects linear regression model, accounting for repeated measures and relevant covariates. RESULTS: A total of 27 children contributed 575 monthly NP samples. Pneumococcus was detected in 302 of 575 (53%) samples, and co-colonization was detected in 61 of these 302 (20%). The total densities were higher during ARI than non-ARI periods and lowest among the youngest children, increasing with age. In the multivariable analysis, there was no significant association between pneumococcal density and co-colonization (coefficient estimate 0.22, 95% confidence interval 0.11-0.55; reference: single-serotype detections). Serotypes 23B and 19F were detected significantly more frequently as single isolates. CONCLUSION: Pneumococcal co-colonization was common and not associated with increased pneumococcal density. Differential propensity for co-colonization was observed among individual serotypes.

Oxidation of hemoproteins by Streptococcus pneumoniae collapses the cell cytoskeleton and disrupts mitochondrial respiration leading to cytotoxicity of human lung cells.

Streptococcus pneumoniae (Spn) causes pneumonia that kills millions through acute toxicity and invasion of the lung parenchyma. During aerobic respiration, Spn releases hydrogen peroxide (Spn-H 2 O 2 ), as a by-product of enzymes SpxB and LctO, and causes cell death with signs of both apoptosis and pyroptosis by oxidizing unknown cell targets. Hemoproteins are molecules essential for life and prone to oxidation by H 2 O 2 . We recently demonstrated that during infection-mimicking conditions, Spn-H 2 O 2 oxidizes the hemoprotein hemoglobin (Hb), releasing toxic heme. In this study, we investigated details of the molecular mechanism(s) by which the oxidation of hemoproteins by Spn-H 2 O 2 causes human lung cell death. Spn strains, but not H 2 O 2 -deficient SpnΔ spxB Δ lctO strains caused time-dependent cell cytotoxicity characterized by the rearrangement of the actin, the loss of the microtubule cytoskeleton and nuclear contraction. Disruption of the cell cytoskeleton correlated with the presence of invasive pneumococci and an increase of intracellular reactive oxygen species. In cell culture, the oxidation of Hb or cytochrome c (Cyt c ) caused DNA degradation and mitochondrial dysfunction from inhibition of complex I-driven respiration, which was cytotoxic to human alveolar cells. Oxidation of hemoproteins resulted in the creation of a radical, which was identified as a protein derived side chain tyrosyl radical by using electron paramagnetic resonance (EPR). Thus, we demonstrate that Spn invades lung cells, releasing H 2 O 2 that oxidizes hemoproteins, including Cyt c , catalyzing the formation of a tyrosyl side chain radical on Hb and causing mitochondrial disruption, that ultimately leads to the collapse of the cell cytoskeleton.

Comparative analyses of holder pasteurization vs. HTST pasteurization for donor milk: a cost-minimization study applicable to human milk banks.

BACKGROUND: High-temperature short-time (HTST) pasteurization (72-75 °C, 15 s) is an alternative treatment to traditional Holder pasteurization (HoP) (62ºC, 30 min) for donor milk. HTST pasteurization guarantees the milk's microbiological safety and retains more of its biologically and nutritionally active compounds, but the cost of implementing this technology for a human milk bank is unknown. METHODS: A cost-minimization study was carried out on the facilities of a regional human milk bank in a public hospital. Total production costs (fixed plus variables) were quantified using HTST pasteurization and HoP in three hypothetical scenarios: (1) costs of the first 10 L of pasteurized milk in a newly opened milk bank; (2) costs of the first 10 L of pasteurized milk in an active milk bank; and (3) costs using the maximum production capacity of both technologies in the first two years of operation. The following costs were analyzed: health care professionals, equipment and software, external services, and consumables. RESULTS: In scenario 1, the total production costs were € 228,097.00 for the HTST method versus € 154,064.00 for the HoP method. In scenario 2, these costs were similar (€ 6,594.00 for HTST pasteurization versus € 5,912.00 for HoP). The cost of healthcare professionals was reduced by more than half when pasteurization was carried out by the HTST method versus the Holder method (€ 84.00 and € 191.00, respectively). In scenario 3, the unit cost of milk pasteurized by the HTST method decreased from the first to the second year by 43.5%, while for the HoP method, it decreased by 30%. CONCLUSIONS: HTST pasteurization requires a high initial investment in equipment; however, it provides a significant minimization of production costs in the long term, pasteurizes large quantities of donor milk per working day and achieves a more efficient management of the time of the health care professionals in charge of the bank's operation compared to HoP.

Effect of surfactants on the sorption-desorption, degradation, and transport of chlorothalonil and hydroxy-chlorothalonil in agricultural soils.

The fungicide chlorothalonil (CTL) and its metabolite hydroxy chlorothalonil (OH-CTL) constitute a risk of soil and water contamination, highlighting the need to find suitable soil remediation methods for these compounds. Surfactants can promote the bioavailability of organic compounds for enhanced microbial degradation, but the performance depends on soil and surfactant properties, sorption-desorption equilibria of contaminants and surfactants, and possible adverse effects of surfactants on microorganisms. This study investigated the influence of five surfactants [e.g., Triton X-100 (TX-100), sodium dodecyl sulphate (SDS), hexadecyltrimethylammonium bromide (HDTMA), Aerosol 22 and Tween 80] on the sorption-desorption, degradation, and mobility of CTL and OH-CTL in two volcanic and one non-volcanic soil. Sorption and desorption of fungicides depended on the sorption of surfactants on soils, surfactants' capacity to neutralize the net negative charge of soils, surfactants' critical micellar concentration, and pH of soils. HDTMA was strongly adsorbed on soils, which shifted the fungicide sorption equilibria by increasing the distribution coefficient (Kd) values. Contrarily, SDS and TX-100 lowered CTL and OH-CTL sorption on soils by decreasing the Kd values, which resulted in an efficient extraction of the fungicide compounds from soil. SDS increased the degradation of CTL, especially in the non-volcanic soil (DT50 values were 14 and 7 days in natural and amended soils, with final residues <7% of the initial dose), whereas TX-100 enabled an early start and sustenance of OH-CTL degradation in all soils. CTL and OH-CTL stimulated soil microbial activities without noticeable deleterious effects of the surfactants. SDS and TX-100 also reduced the vertical transport of OH-CTL in soils. Results of this study could be extended to soils in other regions of the world because the tested soils represent widely different physical, chemical, and biological properties.

Complex post-traumatic stress disorder (CPTSD) of ICD-11 in youths with childhood maltreatment: Associations with age of exposure and clinical outcomes.

BACKGROUND: Exposure to childhood maltreatment (CM) increases the risk of psychiatric morbidity in youths. The new Complex Post-Traumatic Stress Disorder (CPTSD) diagnosis captures the heterogeneity and complexity of clinical outcomes observed in youths exposed to CM. This study explores CPTSD symptomatology and its association with clinical outcomes, considering the impact of CM subtypes and age of exposure. METHODS: Exposure to CM and clinical outcomes were evaluated in 187 youths aged 7-17 (116 with psychiatric disorder; 71 healthy controls) following the Tools for Assessing the Severity of Situations in which Children are Vulnerable (TASSCV) structured interview criteria. CPTSD symptomatology was explored by confirmatory factor analysis, considering four subdomains: post-traumatic stress symptoms, emotion dysregulation, negative self-concept and interpersonal problems. RESULTS: Youths exposed to CM (with or without psychiatric disorders) showed greater internalizing, externalizing and other symptomatology, worse premorbid adjustment and poorer overall functioning. Youth with psychiatric disorder and exposed to CM reported more CPTSD symptomatology, psychiatric comorbidity and polypharmacy and earlier onset of cannabis use. Different subtypes of CM and the developmental stage of exposure differentially impact CPTSD subdomains. LIMITATIONS: Small percentage of resilient youths was studied. It was not possible to explore specific interactions between diagnostic categories and CM. Direct inference cannot be assumed. CONCLUSIONS: Gathering information on type and age of exposure to CM is clinically useful to understand the complexity of psychiatric symptoms observed in youths. Inclusion of the CPTSD diagnosis should increase the implementation of early specific interventions, improving youths' functioning and reducing the severity of clinical outcomes.

Diferencias en la duración del reflejo rectoanal inhibitorio en pacientes con constipación funcional refractaria y mielomeningocele / Differences in rectoanal inhibitory reflex duration between patients with refractory functional constipation and myelomeningocele

Introducción. Habitualmente, durante la manometría anorrectal, en lo correspondiente al reflejo rectoanal inhibitorio (RRAI) solo se pesquisa su presencia o ausencia. Estudios han reportado que su análisis detallado puede brindar datos de interés. Nuestra hipótesis es que la medición del RRAI puede dar información para reconocer causas orgánicas (médula anclada, lipoma, etc.) en pacientes en los que previamente se consideró como de causa funcional. Objetivos. Comparar la duración del reflejo rectoanal inhibitorio en la manometría anorrectal de pacientes con constipación funcional refractaria (CFR) y mielomeningocele (MMC). Población y métodos. Estudio observacional, transversal, analítico (2004-2019). Pacientes constipados crónicos con incontinencia fecal funcional y orgánica (mielomeningocele). Se les realizó manometría anorrectal con sistema de perfusión de agua y se midió la duración del RRAI con diferentes volúmenes (20, 40 y 60 cc). Grupo 1 (G1): 81 CFR. Grupo 2 (G2): 54 MMC. Se excluyeron pacientes con retraso madurativo, esfínter anal complaciente, agenesia sacra y aquellos no colaboradores. Resultados. Se incluyeron 135 sujetos (62 varones). La mediana de edad fue G1:9,57 años; G2: 9,63 años. Duración promedio G1 vs. G2 con 20 cc: 8,89 vs. 15,21 segundos; con 40 cc: 11.41 vs. 21,12 segundos; con 60 cc: 14,15 vs. 26,02 segundos. La diferencia de duración del RRAI entre ambos grupos con diferentes volúmenes fue estadísticamente significativa (p = 0,0001). Conclusión. La duración del RRAI aumenta a mayor volumen de insuflación del balón en ambas poblaciones. Pacientes con MMC tuvieron mayor duración del RRAI que aquellos con CFR. En los pacientes con RRAI prolongado, debe descartarse lesión medular.

Introduction. Usually, during anorectal manometry, only the presence or absence of rectoanal inhibitory reflex (RAIR) is investigated. Studies have reported that a detailed analysis may provide data of interest. Our hypothesis is that RAIR measurement may provide information to detect organic causes (tethered cord, lipoma, etc.) in patients in whom a functional cause had been previously considered. Objectives. To compare RAIR duration in anorectal manometry between patients with refractory functional constipation (RFC) and myelomeningocele (MMC). Population and methods. Observational, analytical, cross-sectional study (2004­2019). Patients with chronic constipation and functional and organic fecal incontinence (myelomeningocele). The anorectal manometry was performed with a water-perfused system, and the duration of RAIR was measured with different volumes (20, 40, and 60 cc). Group 1 (G1): 81 RFC. Group 2 (G2): 54 MMC. Patients with developmental delay, compliant anal sphincter, sacral agenesis and non-cooperative patients were excluded. Results. A total of 135 individuals were included (62 were male). Their median age was 9.57 years in G1 and 9.63 years in G2. Average duration in G1 versus G2 with 20 cc: 8.89 versus 15.21 seconds; 40 cc: 11.41 versus 21.12 seconds; 60 cc: 14.15 versus 26.02 seconds. The difference in RAIR duration with the varying volumes was statistically significant (p = 0.0001). Conclusion. RAIR duration was longer with increasing balloon inflation volumes in both populations. RAIR duration was longer in patients with MMC than in those with RFC. Spinal injury should be ruled out in patients with prolonged RAIR.

Dissemination of Tn916-Related Integrative and Conjugative Elements in Streptococcus pneumoniae Occurs by Transformation and Homologous Recombination in Nasopharyngeal Biofilms.

Multidrug resistance in Streptococcus pneumoniae (or pneumococcus) continues to be a global challenge. An important class of antibiotic resistance determinants disseminating in S. pneumoniae are >20-kb Tn916-related integrative and conjugative elements (ICEs), such as Tn2009, Tn6002, and Tn2010. Although conjugation has been implicated as the transfer mechanism for ICEs in several bacteria, including S. pneumoniae, the molecular basis for widespread dissemination of pneumococcal Tn916-related ICEs remains to be fully elucidated. We found that Tn2009 acquisition was not detectable via in vitro transformation nor conjugative mating with donor GA16833, yielding a transfer frequency of <10-7. GA16833 Tn2009 conjugative gene expression was not significantly induced, and ICE circular intermediate formation was not detected in biofilms. Consistently, Tn2009 transfer efficiency in biofilms was not affected by deletion of the ICE conjugative gene ftsK. However, GA16833 Tn2009 transfer occurred efficiently at a recombination frequency (rF) of 10-4 in dual-strain biofilms formed in a human nasopharyngeal cell bioreactor. DNase I addition and deletions of the early competence gene comE or transformation apparatus genes comEA and comEC in the D39 recipient strain prevented Tn2009 acquisition (rF of <10-7). Genome sequencing and single nucleotide polymorphism analyses of independent recombinants of recipient genotype identified ~33- to ~55-kb donor DNAs containing intact Tn2009, supporting homologous recombination. Additional pneumococcal donor and recipient combinations were demonstrated to efficiently transfer Tn916-related ICEs at a rF of 10-4 in the biofilms. Tn916-related ICEs horizontally disseminate at high frequency in human nasopharyngeal S. pneumoniae biofilms by transformation and homologous recombination of >30-kb DNA fragments into the pneumococcal genome. IMPORTANCE The World Health Organization has designated Streptococcus pneumoniae as a priority pathogen for research and development of new drug treatments due to extensive multidrug resistance. Multiple strains of S. pneumoniae colonize and form mixed biofilms in the human nasopharynx, which could enable exchange of antibiotic resistance determinants. Tn916-related integrative and conjugative elements (ICEs) are largely responsible for the widespread presence of macrolide and tetracycline resistance in S. pneumoniae. Utilizing a system that simulates colonization of donor and recipient S. pneumoniae strains in the human nasopharynx, efficient transfer of Tn916-related ICEs occurred in human nasopharyngeal biofilms, in contrast to in vitro conditions of planktonic cells with exogenous DNA. This high-frequency Tn916-related ICE transfer between S. pneumoniae strains in biofilms was due to transformation and homologous recombination, not conjugation. Understanding the molecular mechanism for dissemination of Tn916-related ICEs can facilitate the design of new strategies to combat antibiotic resistance.

Oligopeptide Transporters of Nonencapsulated Streptococcus pneumoniae Regulate CbpAC and PspA Expression and Reduce Complement-Mediated Clearance.

Streptococcus pneumoniae colonizes the human nasopharynx and causes several diseases. Pneumococcal vaccines target the polysaccharide capsule and prevent most serious disease, but there has been an increase in the prevalence of nonencapsulated S. pneumoniae (NESp). Previously, it was thought that a capsule was necessary to cause invasive disease. NESp strains expressing the oligopeptide transporters AliC and AliD have been isolated from patients with invasive disease. The AliC and AliD oligopeptide transporters regulate the expression of several genes, including choline binding protein AC (CbpAC) (a homolog of PspA), which aids in reducing C3b deposition. It is hypothesized that by altering CbpAC expression, AliC and AliD provide protection from classical complement-mediated clearance by reducing C-reactive protein (CRP) binding. Our study demonstrates that AliC and AliD regulate CbpAC expression in NESp and that AliD found in certain serotypes of encapsulated strains regulates PspA expression. C3b deposition was increased in the NESp ΔaliD and encapsulated mutants in comparison to the wild type. NESp strains expressing AliC and AliD have a significant decrease in C1q and CRP deposition in comparison to the ΔaliC ΔaliD mutant. The complement protein C1q is required for NESp clearance in a murine model and increases opsonophagocytosis. By regulating CbpAC expression, NESp inhibits CRP binding to the bacterial surface and blocks classical complement activation, leading to greater systemic survival and virulence. Due to the increase in the prevalence of NESp, it is important to gain a better understanding of NESp virulence mechanisms that aid in establishing disease and persistence within a host by avoiding clearance by the immune system. IMPORTANCE Streptococcus pneumoniae (pneumococcus) can cause a range of diseases. Although there is a robust pneumococcal vaccination program that reduces invasive pneumococcal disease by targeting various polysaccharide capsules, there has been an increase in the isolation of nonvaccine serotypes and nonencapsulated S. pneumoniae (NESp) strains. While most studies of pneumococcal pathogenesis have focused on encapsulated strains, there is little understanding of how NESp causes disease. NESp lacks a protective capsule but contains novel genes, such as aliC and aliD, which have been shown to regulate the expression of numerous genes and to be required for NESp virulence and immune evasion. Furthermore, NESp strains have high transformation efficiencies and harbor resistance to multiple drugs. This could be deleterious to current treatment strategies employed for pneumococcal disease as NESp can be a reservoir of drug resistance genes. Therefore, deciphering how NESp survives within a host and facilitates disease is a necessity that will allow the fabrication of improved, broad-spectrum treatments and preventatives against pneumococcal disease. Our study provides a better understanding of NESp virulence mechanisms during host-pathogen interactions through the examination of genes directly regulated by the NESp proteins AliC and AliD.

The quorum sensing com system regulates pneumococcal colonisation and invasive disease in a pseudo-stratified airway tissue model.

BACKGROUND: The effects of the com quorum sensing system during colonisation and invasion of Streptococcus pneumoniae (Spn) are poorly understood. METHODS: We developed an ex vivo model of differentiated human airway epithelial (HAE) cells with beating ciliae, mucus production and tight junctions to study Spn colonisation and translocation. HAE cells were inoculated with Spn wild-type TIGR4 (wtSpn) or its isogenic ΔcomC quorum sensing-deficient mutant. RESULTS: Colonisation density of ΔcomC mutant was lower after 6 h but higher at 19 h and 30 h compared to wtSpn. Translocation correlated inversely with colonisation density. Transepithelial electric resistance (TEER) decreased after pneumococcal inoculation and correlated with increased translocation. Confocal imaging illustrated prominent microcolony formation with wtSpn but disintegration of microcolony structures with ΔcomC mutant. ΔcomC mutant showed greater cytotoxicity than wtSpn, suggesting that cytotoxicity was likely not the mechanism leading to translocation. There was greater density- and time-dependent increase of inflammatory cytokines including NLRP3 inflammasome-related IL-18 after infection with ΔcomC compared with wtSpn. ComC inactivation was associated with increased pneumolysin expression. CONCLUSIONS: ComC system allows a higher organisational level of population structure resulting in microcolony formation, increased early colonisation and subsequent translocation. We propose that ComC inactivation unleashes a very different and possibly more virulent phenotype that merits further investigation.

Synergistic Protection against Secondary Pneumococcal Infection by Human Monoclonal Antibodies Targeting Distinct Epitopes.

Streptococcus pneumoniae persists as a leading cause of bacterial pneumonia despite the widespread use of polysaccharide-based vaccines. The limited serotype coverage of current vaccines has led to increased incidence of nonvaccine serotypes, as well as an increase in antibiotic resistance among these serotypes. Pneumococcal infection often follows a primary viral infection such as influenza virus, which hinders host defense and results in bacterial spread to the lungs. We previously isolated human monoclonal Abs (mAbs) against the conserved surface Ag pneumococcal histidine triad protein D (PhtD), and we demonstrated that mAbs to this Ag are protective against lethal pneumococcal challenge prophylactically and therapeutically. In this study, we elucidated the mechanism of protection of a protective anti-pneumococcal human mAb, PhtD3, which is mediated by the presence of complement and macrophages in a mouse model of pneumococcal infection. Treatment with mAb PhtD3 reduced blood and lung bacterial burden in mice, and mAb PhtD3 is able to bind to bacteria in the presence of the capsular polysaccharide, indicating exposure of surface PhtD on encapsulated bacteria. In a mouse model of secondary pneumococcal infection, protection mediated by mAb PhtD3 and another mAb targeting a different epitope, PhtD7, was reduced; however, robust protection was restored by combining mAb PhtD3 with mAb PhtD7, indicating a synergistic effect. Overall, these studies provide new insights into anti-pneumococcal mAb protection and demonstrate, to our knowledge, for the first time, that mAbs to pneumococcal surface proteins can protect against secondary pneumococcal infection in the mouse model.

Differences in rectoanal inhibitory reflex duration between patients with refractory functional constipationand myelomeningocele. / Diferencias en la duración del reflejo rectoanal inhibitorio en pacientes con constipación funcional refractaria y mielomeningocele.

Introduction. Usually, during anorectal manometry, only the presence or absence of rectoanal inhibitory reflex (RAIR) is investigated. Studies have reported that a detailed analysis may provide data of interest. Our hypothesis is that RAIR measurement may provide information to detect organic causes (tethered cord, lipoma, etc.) in patients in whom a functional cause had been previously considered. Objectives. To compare RAIR duration in anorectal manometry between patients with refractory functional constipation (RFC) and myelomeningocele (MMC). Population and methods. Observational, analytical, cross-sectional study (2004-2019). Patients with chronic constipation and functional and organic fecal incontinence (myelomeningocele). The anorectal manometry was performed with a water-perfused system, and the duration of RAIR was measured with different volumes (20, 40, and 60 cc). Group 1 (G1): 81 RFC. Group 2 (G2): 54 MMC. Patients with developmental delay, compliant anal sphincter, sacral agenesis and non-cooperative patients were excluded. Results. A total of 135 individuals were included (62 were male). Their median age was 9.57 years in G1 and 9.63 years in G2. Average duration in G1 versus G2 with 20 cc: 8.89 versus 15.21 seconds; 40 cc: 11.41 versus 21.12 seconds; 60 cc: 14.15 versus 26.02 seconds. The difference in RAIR duration with the varying volumes was statistically significant (p = 0.0001). Conclusion. RAIR duration was longer with increasing balloon inflation volumes in both populations. RAIR duration was longer in patients with MMC than in those with RFC. Spinal injury should be ruled out in patients with prolonged RAIR.

Introducción. Habitualmente, durante la manometría anorrectal, en lo correspondiente al reflejo rectoanal inhibitorio (RRAI) solo se pesquisa su presencia o ausencia. Estudios han reportado que su análisis detallado puede brindar datos de interés. Nuestra hipótesis es que la medición del RRAI puede dar información para reconocer causas orgánicas (médula anclada, lipoma, etc.) en pacientes en los que previamente se consideró como de causa funcional. Objetivos. Comparar la duración del reflejo rectoanal inhibitorio en la manometría anorrectal de pacientes con constipación funcional refractaria (CFR) y mielomeningocele (MMC). Población y métodos. Estudio observacional, transversal, analítico (2004-2019). Pacientes constipados crónicos con incontinencia fecal funcional y orgánica (mielomeningocele). Se les realizó manometría anorrectal con sistema de perfusión de agua y se midió la duración del RRAI con diferentes volúmenes (20, 40 y 60 cc). Grupo 1 (G1): 81 CFR. Grupo 2 (G2): 54 MMC. Se excluyeron pacientes con retraso madurativo, esfínter anal complaciente, agenesia sacra y aquellos no colaboradores. Resultados. Se incluyeron 135 sujetos (62 varones). La mediana de edad fue G1:9,57 años; G2: 9,63 años. Duración promedio G1 vs. G2 con 20 cc: 8,89 vs. 15,21 segundos; con 40 cc: 11.41 vs. 21,12 segundos; con 60 cc: 14,15 vs. 26,02 segundos. La diferencia de duración del RRAI entre ambos grupos con diferentes volúmenes fue estadísticamente significativa (p = 0,0001). Conclusión. La duración del RRAI aumenta a mayor volumen de insuflación del balón en ambas poblaciones. Pacientes con MMC tuvieron mayor duración del RRAI que aquellos con CFR. En los pacientes con RRAI prolongado, debe descartarse lesión medular.

Hacia un proceso de formación del residente en Gastroenterología con enfoque creativo y tecnológico / Towards a training process for residents in Gastroenterology with a creative and technological approach

Introducción: la formación de los residentes deviene proceso fundamental para garantizar especialistas con una sólida preparación profesional. Objetivo: exponer un conjunto de ideas, posicionamientos teóricos y metodológicos orientados a elevar la calidad de la formación del residente en Gastroenterología desde un enfoque creativo y tecnológico. Métodos: se realizó una revisión bibliográfica sistemática para desarrollar un análisis del contenido de documentos, tesis de doctorado, maestría, artículos originales y de revisión publicados entre 1981 y 2022 en español. Se consultaron 47 artículos y se seleccionaron 28. La búsqueda fue realizada en las bases de datos SciELO y Google académico de julio a septiembre de 2022. Tras la identificación de los estudios preseleccionados, se llevó a cabo la lectura de los títulos, resumen y palabras claves para comprobar su pertinencia con el estudio. Desarrollo: la revisión documental permitió esclarecer una serie de dificultades presentes en el proceso de formación de los especialistas en Gastroenterología y hacer propuestas que pueden contribuir en este sentido desde las nuevas concepciones de la educación como la creatividad, el trabajo colaborativo y la enseñanza problémica, usando las ventajas de las tecnologías de la información y las comunicaciones. Conclusiones: el proceso de formación de los residentes en Gastroenterología necesita adaptarse a las nuevas exigencias de la educación. Potencializar la creatividad desde la perspectiva de la enseñanza problémica y el trabajo colaborativo mediante el uso de las herramientas que brindan las tecnologías de la información y la comunicación, debe ser la directriz, en este propósito.

Introduction: The training of residents becomes a fundamental process to guarantee specialists with a solid professional preparation. Objective: To present a set of ideas, theoretical and methodological positions that guide how to raise the quality of the resident's training in Gastroenterology from a creative and technological approach. Method: A systematic bibliographic review was carried out to develop an analysis of the content of documents, doctoral theses, master's degrees, original and review articles published from 1981 to 2022 in Spanish. We consulted 47 articles and selected 26. The search was carried out in the SciELO and Google scholar databases from July to September 2022, after the identification of the pre-selected studies, the titles, abstract and keywords were read to check their relevance to the study. Development: The documentary review allowed to clarify a series of difficulties in the training process of specialists in gastroenterology and make proposals that can contribute in this sense from the new conceptions of education, such as creativity, collaborative work, problem-solving teaching using the advantages of information and communication technologies. Conclusions: The training process of residents in gastroenterology needs to adapt to the new demands of education. Potentiating creativity from the perspective of probability teaching and collaborative work through the use of the tools provided by information and communication technologies, should be the guideline, in this purpose.