Assessment of spillover of antimicrobial resistance to untreated children 7 to 12 years old after mass drug administration of azithromycin for child survival in Niger: a secondary analysis of the MORDOR cluster-randomized trial.

BACKGROUND: The risk of antibiotic resistance is complicated by the potential for spillover effects from one treated population to another. Azithromycin mass drug administration programs report higher rates of antibiotic resistance among treatment arms in targeted groups. This study aims to understand the risk of spillover of antibiotic resistance to non-target groups in these programs. METHODS: Data was used from a cluster-randomized trial comparing the effect of biannual azithromycin and placebo distribution to children 1-59 months on child mortality. Nasopharyngeal samples from untreated children 7-12 years old were tested for genetic determinants of macrolide resistance (primary outcome) and resistance to other antibiotic classes (secondary outcomes). Linear regression was used to compare the community-level mean difference in prevalence by arm at the 24-month timepoint adjusting for baseline prevalence. RESULTS: 1,103 children 7-12 years old in 30 communities were included in the analysis (15 azithromycin, 15 placebo). Adjusted mean differences in prevalence of resistance determinants for macrolides, beta-lactams and tetracyclines were 3.4% (95% CI -4.1% to 10.8%, P-value 0.37), -1.2% (95% CI -7.9% to 5.5%, P-value 0.72), and -3.3% (95% CI -9.5% to 2.8%, P-value 0.61), respectively. CONCLUSIONS: We were unable to demonstrate a statistically significant increase in macrolide resistance determinants in untreated groups in an azithromycin mass drug administration program. While the result might be consistent with a small spillover effect, this study was not powered to detect such a small difference. Larger studies are warranted to better understand the potential for spillover effects within these programs.

Microbiome toxicology - bacterial activation and detoxification of insecticidal compounds.

Insect gut bacteria have been implicated in a myriad of physiological processes from nutrient supplementation to pathogen protection. In fact, symbiont-mediated insecticide degradation has helped explain sudden control failure in the field to a range of active ingredients. The mechanisms behind the loss of susceptibility are varied based on host, symbiont, and insecticide identity. However, while some symbionts directly break down pesticides, others modulate endogenous host detoxification pathways or involve reciprocal degradation of insecticidal and bactericidal compounds both inspiring new questions and requiring the reexamination of past conclusions. Good steward of the chemical pesticide arsenal requires consideration of these ecological interactions from development to deployment.

Comparison of door-to-door and fixed-point delivery of azithromycin distribution for child survival in Niger: A cluster-randomized trial.

Recent evidence indicates mass azithromycin distribution reduces under-5 mortality. This intervention is being considered for child survival programs in high mortality sub-Saharan African settings. The delivery approach used in prior studies required a full-time census and distribution team, which is not feasible for most programs. To determine the optimal programmatic approach to delivery, this study aimed to compare treatment coverage, costs, and acceptability of different delivery approaches with existing community health workers (CHWs). This cluster-randomized trial included rural and peri-urban communities in Dosso, Niger (clinicaltrials.gov, NCT04774991). A random sample of 80 eligible communities was randomized 1:1 to biannual door-to-door or fixed-point delivery of oral azithromycin to children 1-59 months old over 1 year. Data analysts alone were masked given the nature of the intervention. The primary outcome was community-level treatment coverage defined as the number of children treated recorded by CHWs divided by the number of eligible children determined using a post-distribution census. Costs were monitored through routine administrative data collection and micro-costing. The census included survey questions on intervention acceptability among caregivers, community leaders, and CHWs. After randomization, 1 community was excluded due to inaccuracies in available administrative data, resulting in 39 communities receiving door-to-door delivery. At the second distribution, community-level mean treatment coverage was 105% (SD 44%) in the door-to-door arm and 92% (SD 20%) in the fixed-point arm (Mean difference 13%, 95% CI -2% to 28%, P-value = 0.08). The total cost per dose delivered was $1.91 in the door-to-door arm and $2.51 in the fixed-point arm. Indicators of acceptability were similar across stakeholder groups in both arms, with most respondents in each group indicating a preference for door-to-door. Overall, door-to-door delivery is the preferred approach to azithromycin distribution in this setting and might reach more children at a lower cost per dose delivered than fixed-point. Trial Registration: clinicaltrials.gov NCT04774991.

Comparison of Population-Based Census versus Birth History for the Estimation of Under-5 Mortality in Niger.

The WHO guidelines on mass distribution of azithromycin for child survival recommend monitoring of mortality to evaluate effectiveness. Trials that contributed evidence to these guidelines used a population-based census to monitor vital status, requiring census workers to visit each household biannually (twice yearly). Birth history is an alternative to the census approach that may be more feasible because it decreases the time and labor needed for mortality monitoring. This study aimed to compare the population-based census (reference standard) and birth history (index test) approaches to estimating mortality among children 1 to 59 months old using data from the Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance (MORDOR) trial. Sixteen communities that received 5 years of biannual census in the MORDOR trial were selected randomly also to receive birth history surveys. The census approach recorded more participants and households than birth history, with correlations more than 0.94 for each. The correlation between number of deaths in each community was 0.84 (95% CI, 0.59-0.94). A comparison of the mortality incidence rate estimated from the census against the under-5 mortality rate estimated from the birth history resulted in a correlation of 0.60 (95% CI, 0.15-0.84). Of the 47% of children who were linked individually to compare vital status from each method, the death status of children had a sensitivity of 80% (95% CI, 73-89) and a specificity of 98% (95% CI, 98-99), comparing birth history to census. Overall birth histories were found to be a reasonable alternative to biannual census for tracking vital status.

In vitro assays reveal inherently insecticide-tolerant termite symbionts.

Introduction: Termite symbionts are well known for conferring a myriad of benefits to their hosts. Bacterial symbionts are repeatedly associated with increased fitness, nutritional supplementation, pathogen protection, and proper development across insect taxa. In addition, several recent studies link bacterial symbionts to reduced insecticide efficacy. This has important implications both in pest control management and environmental bioremediation efforts. Insects' guts may be a valuable resource for microbes with broad application given their unique niches and metabolic diversity. Though insecticide resistance in termites is considered unlikely due to their life history, the close association of termites with a multitude of bacteria raises the question: is there potential for symbiont-mediated pesticide tolerance in termites? Methods and results: We identified a candidate that could grow in minimal medium containing formulated pesticide. This bacterial isolate was then subjected to continuous culture and subsequently demonstrated improved performance in the presence of pesticide. Isolates subjected to continuous culture were then grown at a range of concentrations from 1-10X the formulation rate. After constant exposure for several generations, isolates grew significantly better. Conclusion: Here we demonstrate that naïve insect hosts can harbor symbionts with inherent insecticide tolerance capable of rapid adaptation to increasing insecticide concentrations overtime. This has broad implications for both pest control and environmental cleanup of residual pesticides.

Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform.

Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta, and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1-BA.5) of Omicron. In silico, analytical and clinical testing of the variant panel indicate that the assays exhibit high sensitivity and specificity. This panel can help fulfill the need for rapid identification of variants in samples, leading to quick decision making with respect to public health measures, as well as treatment options for individuals. Compared to sequencing, these genotyping PCR assays allow much faster turn-around time from sample to results-just a couple hours instead of days or weeks.

A Century of Synergy in Termite Symbiosis Research: Linking the Past with New Genomic Insights.

Termites have long been studied for their symbiotic associations with gut microbes. In the late nineteenth century, this relationship was poorly understood and captured the interest of parasitologists such as Joseph Leidy; this research led to that of twentieth-century biologists and entomologists including Cleveland, Hungate, Trager, and Lüscher. Early insights came via microscopy, organismal, and defaunation studies, which led to descriptions of microbes present, descriptions of the roles of symbionts in lignocellulose digestion, and early insights into energy gas utilization by the host termite. Focus then progressed to culture-dependent microbiology and biochemical studies of host-symbiont complementarity, which revealed specific microhabitat requirements for symbionts and noncellulosic mechanisms of symbiosis (e.g., N2 fixation). Today, knowledge on termite symbiosis has accrued exponentially thanks to omic technologies that reveal symbiont identities, functions, and interdependence, as well as intricacies of host-symbiont complementarity. Moving forward, the merging of classical twentieth-century approaches with evolving omic tools should provide even deeper insights into host-symbiont interplay.

Symbiont-Mediated Insecticide Detoxification as an Emerging Problem in Insect Pests.

Pesticide use is prevalent with applications from the backyard gardener to large-scale agriculture and combatting pests in homes and industrial settings. Alongside the need to control unwanted pests comes the selective pressure generated by sustained pesticide use has become a concern leading to environmental contamination, pest resistance, and, thus, reduced pesticide efficacy. Despite efforts to improve the environmental impact and reduce off-target effects, chemical pesticides are relied on and control failures are costly. Though pesticide resistance mechanisms vary, one pattern that has recently emerged is symbiont-mediated detoxification within insect pests. The localization within the insect host, the identity of the symbiotic partner, and the stability of the associations across different systems vary. The diversity of insects and ecological settings linked to this phenomenon are broad. In this mini-review, we summarize the recent trend of insecticide detoxification modulated by symbiotic associations between bacteria and insects, as well as highlight the implications for pesticide development, pest management strategies, and pesticide bioremediation.

In vivo efficacy of a unique first-in-class antibiofilm antibiotic for biofilm-related wound infections caused by Acinetobacter baumannii.

Wounds complicated by biofilms challenge even the best clinical care and can delay a return to duty for service members. A major component of treatment in wounded warriors includes infected wound management. Yet, all antibiotic therapy options have been optimized against planktonic bacteria, leaving an important gap in biofilm-related wound care. We tested the efficacy of a unique compound (CZ-01179) specifically synthesized to eradicate biofilms. CZ-01179 was formulated as the active agent in a hydrogel, and tested in vitro and in vivo in a pig excision wound model for its ability to treat and prevent biofilm-related wound infection caused by Acinetobacter baumannii. Data indicated that compared to a clinical standard-silver sulfadiazine-CZ-01179 was much more effective at eradicating biofilms of A. baumannii in vitro and up to 6 days faster at eradicating biofilms in vivo. CZ-01179 belongs to a broader class of newly-synthesized antibiofilm agents (referred to as CZ compounds) with reduced risk of resistance development, specific efficacy against biofilms, and promising formulation potential for clinical applications. Given its broad spectrum and biofilm-specific nature, CZ-01179 gel may be a promising agent to increase the pipeline of products to treat and prevent biofilm-related wound infections.

Dialectical Tensions Associated with Health Advocacy.

In this piece I narrate the lived dialectical tensions I experienced and embodied as a health advocate for my daughter during her stay in the pediatric intensive care unit: emotionality-rationality and presence-absence. Ultimately, I argue that "metacommunication" (Tracy, 2004) is one strategy that can help health advocates recast these tensions as complementary rather than contradictory in nature, with the aim of empowering these advocates to live more productively within them.

Mild thermal stress affects Steinernema carpocapsae infective juvenile survival but not protein content.

Steinernema nematodes and their Xenorhabdus symbionts are a malleable model system to study mutualistic relations. One of the advantages they possess is their ability to be disassociated under in vitro rearing conditions. Various in vitro methods have been developed to produce symbiont colonized and aposymbiotic (symbiont-free) nematodes. Until now, there has been no investigation on how in vitro rearing conditions may have an impact on the storage ability and the protein content of the infective juvenile at different storage temperatures. Thus, in this study, we investigated how infective juvenile longevity and protein content are impacted when the nematodes were reared with two in vitro methods (lipid and liver kidney agar) considering colonized and uncolonized nematodes, and under two different temperatures: 15 °C and 20 °C (mild stress). Infective juveniles reared in vitro (with or without their symbionts) had lower 8-week survival rates. No in vitro reared, colonized IJs survived to the desired 16-week time point. Survival of infective juveniles stored under mild stress temperature (20 °C) was lower than that observed at 15 °C. However, when comparing the interaction between rearing condition and storage temperature, there were not significant differences. With respect to protein content, in vivo, colonized infective juveniles maintained a static protein content over time, suggesting symbiont colonization may influence protein metabolism and/or turnover in infective juveniles.

Ecological characterization of Heterorhabditis sonorensis (Caborca strain) (Nematoda: Heterorhabditidae), an entomopathogenic nematode from the Sonoran Desert.

Heterorhabditis nematodes are parasites of a wide range of soil-dwelling insect species. Although these nematodes have been exploited as biological control agents since the last half of the 20th century, much research remains to be done to understand how these organisms function in agricultural and other ecosystems. In this study, we present some ecological traits of Heterorhabditis sonorensis, a natural parasite of the cicada Diceroprocta ornea, from the Sonoran Desert. Specifically, we evaluated its infectivity across a diverse panel of insect groups and assessed its fitness (infectivity and reproduction) considering different temperatures, and soil moisture levels. Three other Heterorhabditis species served as points of comparison for temperature and soil moisture assays. Host range experiments indicate that H. sonorensis, although isolated from seasonal cicada nymphs, is more virulent and reproductively fit in the lepidopteran hosts tested. This nematode has an optimum temperature range at 25-30 °C but can also successfully reproduce at temperatures ranging from 15 to 35 °C. Additionally, this nematode is adapted to a variety of soil moisture conditions with successful infections across the tested moisture range (3%-20%). Finally, we demonstrate that H. sonorensis infective juveniles have a high survival rate (over 80%) at various storage temperatures (10-25 °C) after 24 weeks of storage and remain infective as revealed by the post-storage infection assays.

Growth substrate may influence biofilm susceptibility to antibiotics.

The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.

Courtesy Stigma and Social Support: An Exploration of Fathers' Buffering Strategies and Blocking Rationalizations.

This study extends scholarship on stigma management communication and social support by exploring the experiences of fathers of children living with a rare health condition, Sturge-Weber Syndrome. Findings from this interview-based interpretive study reveal that fathers assuaged the negative effects of stigma on their children-and courtesy stigma on themselves-by employing buffering strategies, including reactive and preemptive information sharing, preparatory conversations, and support blocking. Further, fathers offered three rationalizations for their blocking behaviors-reasoning that to accept support would violate social norms, as well as privacy expectations and that accepting support was not worth the effort (social exchange). These findings encourage scholars to continue to upend predominant constructions of masculinity and also call to question prevailing assumptions about the relationship between technology and privacy.

Screening of 57 Candidate Double-Stranded RNAs for Insecticidal Activity Against the Pest Termite Reticulitermes flavipes (Isoptera: Rhinotermitidae).

RNA interference insecticides have received increasing attention in recent years due to their classification as a reduced-risk biopesticide and their proposed faster path to registration compared with conventional synthetic insecticides. The goal of this study was to synthesize and compare efficacy of 62 double-stranded RNAs (dsRNAs) from 31 target genes against the pest termite species, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae). Fifty-seven dsRNAs of ~125 base pairs each were successfully synthesized. First-tier screens using a combination immersion/feeding assay revealed 10 top candidates and also that dsRNAs coming from synthesis reactions with 80-90× yields were the most effective. Follow-up studies using uptake enhancers in combination with top candidate dsRNAs were unsuccessful. Subsequent concentration range feeding assays on the top candidates revealed two lead termiticidal dsRNAs (3' Hexamerin-2 and 3' Glycosyl Hydrolase Family [GHF] 9-2 cellulase) and another that enhanced feeding (5' GHF9-2 cellulase). Testing a matrix of combinations of these three dsRNAs revealed ultimately that the most consistently effective dsRNA combination was the 3' Hexamerin-2 + 3' GHF9-2 cellulase dsRNA combination. These results provide new information on candidate termiticidal dsRNAs and some apparent factors that have a bearing on their efficacy. Despite these successes, further research and development will be necessary to move dsRNA termiticides from pest management theory to real-world application.

Metatranscriptomic Techniques for Identifying Cellulases in Termites and their Symbionts.

Characterizing symbiotic communities, like that of the termite hindgut, is essential for understanding their functionality and capabilities. However, the same complexity that allows termites to digest wood so efficiently also makes them difficult to study. With the expansion in technology and sequencing strategies the feasibility of sequencing entire consortiums or microecosystems is now possible. Here we present an adapted library preparation strategy which allows for the detection and measurement of expressed genes from all three domains of life in a single sample simultaneously. This technique effectively captures the transcriptome contributions by the various members of the consortium regardless of their taxonomic identity, which can then be annotated using custom-built databases and reciprocal BLASTing. Joining the universality of this library prep strategy with the power of bioinformatics allows for the identification of cellulases and other genes encoding carbohydrate active enzymes from complex communities using metatranscriptomics.

Fitness costs of symbiont switching using entomopathogenic nematodes as a model.

BACKGROUND: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. RESULTS: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. CONCLUSIONS: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

Metatranscriptome analysis reveals bacterial symbiont contributions to lower termite physiology and potential immune functions.

BACKGROUND: Symbioses throughout the animal kingdom are known to extend physiological and ecological capabilities to hosts. Insect-microbe associations are extremely common and are often related to novel niche exploitation, fitness advantages, and even speciation events. These phenomena include expansions in host diet, detoxification of insecticides and toxins, and increased defense against pathogens. However, dissecting the contributions of individual groups of symbionts at the molecular level is often underexplored due to methodological and analytical limitations. Termites are one of the best studied systems for physiological collaborations between host and symbiota; however, most work in lower termites (those with bacterial and protist symbionts) focuses on the eukaryotic members of this symbiotic consortium. Here we present a metatranscriptomic analysis which provides novel insights into bacterial contributions to the holobiont of the eastern subterranean termite, Reticulitermes flavipes, in the presence and absence of a fungal pathogen. RESULTS: Using a customized ribodepletion strategy, a metatranscriptome assembly was obtained representing the host termite as well as bacterial and protist symbiota. Sequence data provide new insights into biosynthesis, catabolism, and transport of major organic molecules and ions by the gut consortium, and corroborate previous findings suggesting that bacteria play direct roles in nitrogen fixation, amino acid biosynthesis, and lignocellulose digestion. With regard to fungal pathogen challenge, a total of 563 differentially expressed candidate host and symbiont contigs were identified (162 up- and 401 downregulated; α/FDR = 0.05) including an upregulated bacterial amidohydrolase. CONCLUSIONS: This study presents the most complete bacterial metatranscriptome from a lower termite and provides a framework on which to build a more complete model of termite-symbiont interactions including, but not limited to, digestion and pathogen defense.

Lower Termite Associations with Microbes: Synergy, Protection, and Interplay.

Lower-termites are one of the best studied symbiotic systems in insects. Their ability to feed on a nitrogen-poor, wood-based diet with help from symbiotic microbes has been under investigation for almost a century. A unique microbial consortium living in the guts of lower termites is essential for wood-feeding. Host and symbiont cellulolytic enzymes synergize each other in the termite gut to increase digestive efficiency. Because of their critical role in digestion, gut microbiota are driving forces in all aspects of termite biology. Social living also comes with risks for termites. The combination of group living and a microbe-rich habitat makes termites potentially vulnerable to pathogenic infections. However, the use of entomopathogens for termite control has been largely unsuccessful. One mechanism for this failure may be symbiotic collaboration; i.e., one of the very reasons termites have thrived in the first place. Symbiont contributions are thought to neutralize fungal spores as they pass through the termite gut. Also, when the symbiont community is disrupted pathogen susceptibility increases. These recent discoveries have shed light on novel interactions for symbiotic microbes both within the termite host and with pathogenic invaders. Lower termite biology is therefore tightly linked to symbiotic associations and their resulting physiological collaborations.

Unique features of a global human ectoparasite identified through sequencing of the bed bug genome.

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.