The landscape of antimicrobial resistance in the neonatal and multi-host pathogen group B Streptococcus: review from a One Health perspective.

Group B Streptococcus (GBS) stands out as a major agent of pediatric disease in humans, being responsible for 392,000 invasive disease cases and 91,000 deaths in infants each year across the world. Moreover, GBS, also known as Streptococcus agalactiae, is an important agent of infections in animal hosts, notably cattle and fish. GBS population structure is composed of multiple clades that differ in virulence, antimicrobial resistance (AMR), and niche adaptation; however, there is growing evidence of interspecies transmission, both from evolutionary analysis and from disease investigations. The prevention of GBS infections through vaccination is desirable in humans as well as animals because it reduces the burden of GBS disease and reduces our reliance on antimicrobials, and the risk of adverse reactions or selection for AMR. In this perspective article, we navigate through the landscape of AMR in the pediatric and multi-host pathogen GBS under the One Health perspective and discuss the use of antimicrobials to control GBS disease, the evolution of AMR in the GBS population, and the future perspectives of resistant GBS infections in the post-pandemic era.

Yolk corticosterone and progesterone levels in Greater Rhea (Rhea americana) eggs vary in a changing social environment.

Maternal hormones in avian egg yolks may signal and prepare offspring for the prevailing conditions. However, this adjustment requires some degree of flexibility in regulating yolk hormone deposition. The Greater Rhea (Rhea americana) has a particular mating system that combines mixed polygyny and polyandry, communal nesting, and exclusive paternal care of chicks. In this species, we previously found that yolk hormone deposition varies among eggs of different captive populations and could influence chicks' physiology and behavior. However, it is still unknown whether females can modify yolk hormone deposition in a changing social environment. Using a captive population of Greater Rheas, in this study, we quantified yolk hormone levels before and after a reduction in the number of females present in the population. We found that females deposited on average higher yolk corticosterone and lower yolk progesterone after the change in their social environment. Since corticosterone deposited into the yolk comes exclusively from the female's plasma, our results suggest that females had, on average, higher plasma corticosterone levels. The change in the number of females may increase the events of male-male competitions, courtships, and matings, leading to an increase of corticosterone in the females' plasma and then into their eggs. Since we previously found that higher yolk corticosterone and lower yolk progesterone were associated with the production of chicks that have an attenuated stress response, the present study results suggest that yolk hormone deposition is mediated by flexible mechanisms that could adjust development to the prevailing conditions.

Localized surface plasmon resonance-based biosensor on gold nanoparticles for Taenia solium detection.

This paper describes, for the first time to our knowledge, a fast-response and specific biosensor for detection of Taenia solium, a parasite responsible for neurocysticercosis disease that affects the central nervous system. The biosensor is based on the localized surface plasmon resonance (LSPR) technique on gold nanoparticles (AuNPs) in colloidal suspension that were functionalized and activated with antibodies to perform an immuno-capture effect. The AuNPs were synthetized by Turkevich and seed-mediated growth methods. A variety of concentrations of T. solium antigen were added to test the detection and the dose-response profile. Small antigen concentrations were detected indicating that the limit of detection is lower than 0.1 µg/mL of antigen. The results demonstrate the potential of the AuNPs LSPR biosensor as a clinical tool for neurocysticercosis diagnostic.

Description of optochin-resistant Streptococcus pneumoniae due to an uncommon mutation in the atpA gene and comparison with previously identified atpC mutants from Brazil.

Optochin susceptibility testing is a major assay used for presumptive identification of Streptococcus pneumoniae. Still, atypical optochin-resistant (Optr) pneumococci have been reported and this phenotype has been attributed to nucleotide substitutions in the genes coding for the F0F1ATPase. While substitutions in the atpC gene (c-subunit of ATPase) are more common and better characterized, data on mutations in the atpA (a-subunit) are still limited. We have characterized five Optr isolates presenting alterations in the atpA (Trp206Cys in four isolates and Trp206Ser in one isolate), constituting the first report of such mutations in Brazil. Most of the Optr isolates consisted of heterogeneous populations. Except for Opt MICs and the nucleotide changes in the atpA gene, Optr and Opts subpopulations originating from the same culture had identical characteristics. In addition, we compared phenotypic and genetic characteristics of these atpA mutants with those of atpC mutants previously identified in Brazil. No structural alterations were detected among predicted proteins, regardless of mutations in the coding gene, suggesting that, despite the occurrence of mutations, protein structures tend to be highly conserved, ensuring their functionalities. Phylogenetic analysis revealed that atypical Optr strains are true pneumococci and Opt resistance does not represent any apparent selective advantage for clinical isolates.

Epigenetic characterization of the H19/IGF2 locus in calf clones placenta / Caracterização epigenética do locus H19/IGF2 na placenta de bezerros clones

Somatic Cell Nuclear Transfer (SCNT-Cloning) is a promising technique in many areas and is based on genetically identical individuals. However, its efficiency is low. Studies suggest that the leading cause is inadequate epigenetic reprogramming. This study aimed to characterize the methylation pattern of the exon 10 regions of the IGF2 gene and the Imprinting Control Region (ICR) of the H19 gene in the placenta of cloned calves. For this study, female and male cloned calves presenting different phenotypes were used. Genomic DNA from these animals' placenta was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region, controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.(AU)

A Transferência Nuclear de Células Somáticas (TNCS-Clonagem) é uma técnica promissora em várias áreas, e se baseia na produção de indivíduos geneticamente idênticos. No entanto, sua eficiência é baixa. Estudos sugerem que a principal causa seja uma reprogramação epigenética inadequada. O objetivo desse trabalho é caracterizar o padrão de metilação da região éxon 10 do gene IGF2 e da Região Controladora de Imprinting (ICR) do gene H19 na placenta de bezerros clonados. Para a execução do trabalho foram selecionados clones bovinos fêmeas e machos, apresentando diferentes fenótipos. O DNA da placenta desses animais foi extraído, e em seguida foi tratado com bissulfito de sódio e amplificado para os loci ICR/H19 e IGF2. Os produtos da PCR foram clonados em bactérias competentes e, por fim, sequenciados. Foi encontrada uma diferença significativa entre os controles e os clones com fenótipos saudáveis para a região da ICR/H19. Nesta região, os controles tiveram um padrão hemimetilado, como previsto pela literatura, devido essa região ser imprinted. Enquanto os clones encontravam-se menos metilados. Para a região do éxon 10 do IGF2, não foi encontrada diferença significativa entre controles e clones. Estes resultados sugerem que as diferentes regiões do genoma podem se reprogramar independente umas das outras e que falhas na reprogramação do padrão de metilação do DNA de genes imprinted podem ser uma das causas da baixa eficiência da TNCS.(AU)

Virulence-Associated Characteristics of Serotype 14 and Serogroup 9 Streptococcus pneumoniae Clones Circulating in Brazil: Association of Penicillin Non-susceptibility With Transparent Colony Phenotype Variants.

Streptococcus pneumoniae remains a major agent of invasive diseases, especially in children and the elderly. The presence of pneumococcal capsule, pneumococcal surface protein A (PspA), and pilus type 1 (PI-1) and the ability of colony phase variation are assumed to play important roles in the virulence potential of this microorganism. Differences in the capsular polysaccharide allow the characterization of more than 90 pneumococcal serotypes; among them, serotype 14 and serogroup 9 stand out due to their prevalence in the pre- pneumococcal conjugate vaccine era and frequent association with penicillin non-susceptibility. Here we investigated the distribution of PI-1 and pspA genes and colony phase variants among 315 S. pneumoniae isolates belonging to serotype 14 and serogroup 9, recovered over 20 years in Brazil, and correlated these characteristics with penicillin susceptibility and genotype as determined by multilocus sequence typing. All strains were shown to carry pspA genes, with those of family 2 (pspA2) being the most common, and nearly half of the strains harbored P1-1 genes. The pspA gene family and the presence of PI-1 genes were conserved features among strains belonging to a given clone. A trend for increasing the occurrence of pspA2 and PI-1 genes over the period of investigation was observed, and it coincided with the dissemination of CC156 (Spain9V -3) clone in Brazil, suggesting a role for these virulence attributes in the establishment and the persistence of this successful clone. Opaque variant was the colony phenotype most frequently observed, regardless of clonal type. On the other hand, the transparent variant was more commonly associated with penicillin-non-susceptible pneumococci and with strains presenting evidence of recombination events involving the genes coding for polysaccharide capsule and PspA, suggesting that pneumococcal transparent variants may present a higher ability to acquire exogenous DNA. The results bring to light new information about the virulence potentials of serotype 14 and serogroup 9 S. pneumoniae isolates representing the major clones that have been associated with the emergence and the dissemination of antimicrobial resistance in our setting since the late 1980s.

Effect of maternal environment on yolk immunoreactive corticosterone and its influence on adrenocortical and behavioral activity in chicks of Greater Rhea (Rhea americana).

Maternal corticosterone in avian eggs may modify offspring phenotype in order to increase survival in poor environments. In the Greater Rhea (Rhea americana), we previously found that yolk immunoreactive corticosterone is influenced by the quality of the maternal environment: eggs laid by females of the intensive rearing system (IRS), living in poor captive conditions, had higher yolk immunoreactive corticosterone than those produced by females of the semi-extensive rearing system (SRS), living in better conditions. Here, we evaluate if these different hormone levels are associated with the production of different phenotypes. We collected eggs from the IRS and SRS for hormonal quantification and artificial incubation. Then, half of the chicks selected from each environment were exposed to a capture and restraint protocol, and the rest remained undisturbed and were used as controls. In the IRS, we found that higher yolk immunoreactive corticosterone was associated with the production of chicks that had reduced hatchability, lower hatchling mass and higher baseline fecal glucocorticoid metabolites (FGM) than those produced by SRS females. Moreover, after capture and restraint, IRS chicks did not modify their FGM nor their behaviors compared to their controls, while SRS chicks increased their FGM and spent more time ambulating and less time pecking, compared to their controls. These results indicate that yolk immunoreactive corticosterone could modify offspring phenotype. Although future studies are needed to elucidate their implications for fitness, our results suggest that yolk corticosterone could be mediating an adaptive maternal effect that allows individuals to better cope with poor conditions.

State of the art knowledge in adrenocortical and behavioral responses to environmental challenges in a threatened South American ratite: Implications to in situ and ex-situ conservation.

The Greater Rhea (Rhea americana) is an endemic ratite to South America, whose wild populations have undergone a remarkable decrease due to habitat degradation and fragmentation by the expansion of the agricultural frontier, poaching and predation by dogs. Anthropogenic perturbations in wild environments, as well as the management in captivity, can generate different stress responses in this species, thus, the monitoring of adrenocortical and behavioral activities are considered primary assessment tools with both conservation and welfare implications. In this review we analyze and integrate the different measurements of glucocorticoids (in plasma, feces, and yolk) carried out in different captive and wild populations, taking into account the diverse predictable and unpredictable conditions to which the Greater Rhea responds in each of those environments. In addition, the translocation of this bird is presented as an application of stress physiology in field ecology for conservation purposes, in which we evaluated how this species responds when it is released into a novel environment. Our results indicate that this ratite has a striking high sensitivity of the hypothalamic-pituitary-adrenal (HPA) axis compared to that of other bird species and shows a wide variety of adrenocortical responses depending on the environment in which it lives. This suggests that its HPA axis has a phenotypic plasticity that enables the rhea to cope with the environmental challenges. In this sense, we propose that one of the routes of this plasticity could be mediated by the maternal transfer of steroid hormones to the egg. Finally, we discuss the importance of integrating the monitoring of the adrenocortical response along with the environmental variables that define the life history of the species, in management and conservation programs ex-situ and in situ.

Potential of MALDI-TOF MS as an alternative approach for capsular typing Streptococcus pneumoniae isolates.

Streptococcus pneumoniae can be classified in more than 90 capsular types, as traditionally determined by serological methods and more recently by PCR-based techniques. Such methods, however, can be expensive, laborious or unable to accurately discriminate among certain serotypes. Therefore, determination of capsular types, although extremely important for epidemiological purposes and for estimating the impact of pneumococcal conjugate vaccines, is mainly restricted to research laboratories, being rarely performed in the clinical setting. In the present study, MALDI-TOF MS was evaluated as an alternative tool to characterize 416 pneumococcal isolates belonging to serotypes 6A, 6B, 6C, 9N, 9V or 14. For MALDI-TOF MS analysis, each isolate was submitted to an extraction protocol using formic acid and acetonitrile. Measurements were performed with a Bruker Microflex LT mass spectrometer using default parameters and generating spectra in the range of 2,000-20,000 m/z. Spectra were analyzed with the BioNumerics software v7.6. Isolates were mainly distributed according to the capsular type in a Neighbor Joining tree and serotypes investigated were successfully discriminated by the presence/absence of 14 selected biomarkers. The results suggest that MALDI-TOF MS is a promising alternative for typing pneumococcal strains, highlighting its usefulness for rapid and cost-effective routine application in clinical laboratories.

Recreational use of marijuana during pregnancy and negative gestational and fetal outcomes: An experimental study in mice.

The prevalence of marijuana use among pregnant women is high. However, the effects on gestation and fetal development are not well known. Epidemiological and experimental studies present conflicting results because of the route of administration, dose, time of exposure, species used, and how Cannabis toxicity is tested (prepared extracts, specific components, or by pyrolysis). In this study, we experimentally investigated the effects of maternal inhalation of Cannabis sativa smoke representing as nearly as possible real world conditions of human marijuana use. Pregnant mice (n=20) were exposed (nose-only) daily for 5min to marijuana smoke (0.2g of Cannabis) from gestational day (GD) 5.5 to GD17.5 or filtered air. Food intake and maternal weight gain were recorded. Ultrasound biomicroscopy was performed on 10.5 and 16.5dpc.On GD18.5, half of the dams were euthanized for the evaluation of term fetus, placenta, and resorptions. Gestation length, parturition, and neonatal outcomes were evaluated in the other half. Five minutes of daily (low dose) exposure during pregnancy resulted in reduced birthweight, and litter size was not altered; however, the number of male pups per litter was higher. Besides, placental wet weight was increased and fetal to placental weight ratio was decreased in male fetuses, showing a sex-specific effect. At the end of gestation, females from the Cannabis group presented reduced maternal net body weight gain, despite a slight increase in their daily food intake compared to the control group. In conclusion, our results indicate that smoking marijuana during pregnancy even at low doses can be embryotoxic and fetotoxic.

MLVA Typing of Streptococcus pneumoniae Isolates with Emphasis on Serotypes 14, 9N and 9V: Comparison of Previously Described Panels and Proposal of a Novel 7 VNTR Loci-Based Simplified Scheme.

Streptococcus pneumoniae remains as an important cause of community-acquired bacterial infections, and the nasopharynx of asymptomatic carriers is the major reservoir of this microorganism. Pneumococcal strains of serotype 14 and serogroup 9 are among the most frequently isolated from both asymptomatic carriers and patients with invasive disease living in Brazil. Internationally disseminated clones belonging to such serotypes have been associated with the emergence and spread of antimicrobial resistance in our setting, highlighting the need for epidemiological tracking of these isolates. In this scenario, Multiple Loci VNTR Analysis (MLVA) has emerged as an alternative tool for the molecular characterization of pneumococci, in addition to more traditional techniques such as Multi-Locus Sequence Typing (MLST) and Pulsed-Field Gel Electrophoresis (PFGE). In the present study, 18 VNTR loci, as well as other previously described reduced MLVA panels (7 VNTR loci), were evaluated as tools to characterize pneumococcal strains of serotypes 14, 9N and 9V belonging to international and regional clones isolated in Brazil. The 18 VNTR loci panel was highly congruent with MLST and PFGE, being also useful for indicating the genetic relationship with international clones and for discriminating among strains with indistinguishable STs and PFGE profiles. Analysis of the results also allowed deducing a novel shorter 7 VNTR loci panel, keeping a high discriminatory power for isolates of the serotypes investigated and a high congruence level with MLST and PFGE. The newly proposed simplified panel was then evaluated for typing pneumococcal strains of other commonly isolated serotypes. The results indicate that MLVA is a faster and easier to perform, reliable approach for the molecular characterization of S. pneumoniae isolates, with potential for cost-effective application, especially in resource-limited countries.

Influence of the rearing system on yolk corticosterone concentration in captive Greater Rheas (Rhea americana).

Many environmental conditions elevate plasma corticosterone in laying birds, leading to elevated hormone accumulation in the egg. We investigated whether maternal yolk corticosterone levels in Greater Rheas differ between fresh eggs collected from an intensive (IRS) and a semi-extensive (SRS) rearing system. After HPLC validation, yolk corticosterone was measured using a corticosterone (125) I radio-immunoassay kit. Results (mean ± SE) showed that eggs collected from the IRS exhibited a significantly higher corticosterone concentration than eggs from SRS (89.88 ± 8.93 vs. 45.41 ± 5.48 ng/g yolk, respectively). Our findings suggest that rearing conditions under an intensive scheme (e.g., small pens with bare ground, no direct foraging and handling) might be perceived as more stressful for Greater Rhea females than semi-extensive rearing conditions (e.g., low animal density distributed in extensive areas and direct foraging), which would result in the transfer of higher yolk corticosterone levels. A better understanding of environmental conditions and female traits that affect yolk corticosterone deposition provides a background for future studies concerning the roles of maternal corticosterone on offspring development. Zoo Biol. 35:246-250, 2016. © 2016 Wiley Periodicals, Inc.

Circadian control of the pupillary light responses in an avian model of blindness, the GUCY1* chickens.

PURPOSE: The vertebrate inner retina has a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the nonvisual photopigment melanopsin. The intrinsically photosensitive retinal ganglion cells send light information from the environment to the brain to control, among other parameters, the amount of energy entering the eyes through the pupillary light reflex (PLR). A daily variation in the PLR in both mice and humans has recently been shown, indicating circadian control of this response. In a previous work involving the sensitivity spectra for the PLR, we showed that blind chickens (GUCY1*) display the highest sensitivity to light of 480 nm. The aim of the present study was to evaluate the potential circadian control of PLRs in blind birds under scotopic conditions. METHODS: Circadian PLR was performed on GUCY1* chickens with lights of different wavelengths (white or blue light of 475 nm) under scotopic conditions. RESULTS: We found a significant daily variation in the PLRs of chickens exposed to white or blue light of 475 nm, with increased sensitivity at circadian time 6 during the subjective day. CONCLUSIONS: Our observations clearly point to circadian control of PLRs even in blindness, strongly indicating that both the entry of light into the eyes and its quality are differentially regulated during the day in diurnal animals.

Seasonal changes in plasma levels of sex hormones in the greater Rhea (Rhea americana), a South American Ratite with a complex mating system.

Seasonal rhythm in sex hormones has been extensively studied in birds, as well as its relationship with the type of mating system. The Greater Rhea (Rhea americana), a South American ratite species, reproduces seasonally and has a complex mating system: female-defense polygyny and sequential polyandry. The present study aimed at analyzing the endocrine basis of reproduction in this species and its relationship with its mating system. We used HPLC and electrochemiluminescence techniques to identify and measure plasma testosterone and estradiol levels. Annual oscillations in sex hormones, testosterone and estradiol, in adult males and females were observed. Lower levels of these hormones were exhibited during the non reproductive season (February to July), whereas their maximum values were reached in September for males and November-December for females. These fluctuations reflect the seasonal changes in gonadal function. By contrast, no significant sex hormones oscillations were observed in juvenile males and females (negative control of seasonal changes). Greater rheas maintain high testosterone and estradiol levels throughout the reproductive period. The high testosterone levels during incubation and chick rearing did not inhibit parental behavior in males, which appears not to conform to the "Challenge Hypothesis". In females, the high estradiol levels throughout the reproductive season would be needed to sustain their long egg-laying period.

Phenotypic and molecular characterization of optochin-resistant Streptococcus pneumoniae isolates from Brazil, with description of five novel mutations in the ATPC gene.

Optochin (Opt) susceptibility is used largely for the identification of Streptococcus pneumoniae in diagnostic laboratories. Opt-resistant (Opt(r)) S. pneumoniae isolates have been reported, however, indicating the potential for misidentification of this important pathogen. Point mutations in the atpC gene have been associated with the emergence of Opt(r) S. pneumoniae, but data on the characterization of such atypical variants of S. pneumoniae are still limited. The present report describes the results of a polyphasic approach to identifying and characterizing 26 Opt(r) S. pneumoniae isolates recovered from patients or carriers living in Brazil. Sixteen isolates consisted of heterogeneous populations, and 10 isolates were homogeneously Opt(r). The isolates had different serotypes and antimicrobial susceptibility profiles. They also presented diverse genetic characteristics, as indicated by pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem-repeat analysis (MLVA), and pspA gene typing. Except for Opt MICs (4- to 64-fold higher among Opt(r) variants), Opt(r) and Opt-susceptible (Opt(s)) subpopulations originating from the same culture had identical characteristics. Sequencing of the atpC gene of the Opt(r) variants revealed 13 different nucleotide changes distributed among eight different codons. Changes in codon 49 were the most frequent, suggesting that this might be a hot spot for optochin resistance-conferring mutations. On the other hand, five novel types of mutations in the atpC gene (Met13Ile, Gly18Ser, Gly20Ala, Ala31Val, and Ala49Gly) were identified. In silico prediction modeling indicated that the atpC gene mutations corresponded to alterations in the transmembrane region of the ATPase, leading to a higher hydrophobicity profile in α-helix 1 and to a lower hydrophobicity profile in α-helix 2.