Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea.

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.

Distribution of blaCTX-M-gene variants in E. coli from different origins in Ecuador.

The increasing abundance of extended spectrum (ß-lactamase (ESBL) genes in E. coli, and other commensal and pathogenic bacteria, endangers the utility of third or more recent generation cephalosporins, which are major tools for fighting deadly infections. The role of domestic animals in the transmission of ESBL carrying bacteria has been recognized, especially in low- and middle-income countries, however the horizontal gene transfer of these genes is difficult to assess. Here we investigate blaCTX-M gene diversity (and flanking nucleotide sequences) in E. coli from chicken and humans, in an Ecuadorian rural community and from chickens in another location in Ecuador. The blaCTX-M associated sequences in isolates from humans and chickens in the same remote community showed greater similarity than those found in E. coli in a chicken industrial operation 200 km away. Our study may provide evidence of blaCTX-M transfer between chickens and humans in the community.

Removal of antimicrobial prophylaxis and its effect on swine carriage of antimicrobial-resistant coliforms.

The use of antimicrobials in the food animal industry has caused an increased prevalence of antimicrobial-resistant bacteria and antimicrobial resistance genes, which can be transferred to the microbiota of humans through the food chain or the environment. To reduce the development and spread of antimicrobial resistance, restrictions on antimicrobial use in food animals have been implemented in different countries. We investigated the impact of an antimicrobial restriction intervention during two generations of pigs. Fecal samples were collected in five growth phases. The frequency of antimicrobial-resistant coliforms and antimicrobial-resistant bacteria or antimicrobial resistance genes was analyzed. No differences in the richness or abundance of antimicrobial-resistant coliforms or antimicrobial resistance genes were found when animals fed with or without prophylactic antimicrobials were compared. Withholding antimicrobial supplementation did not negatively affect weight gain in pigs. Withdrawal of prophylactic antimicrobial consumption during two generations of pigs was not enough to reduce the prevalence of antimicrobial resistance genes, as measured by richness and abundance markers. This study indicates that the fitness costs associated with bacterial carriage of some antimicrobial resistance genes are low.

Mollicellins S-U, three new depsidones from Chaetomium brasiliense SD-596 with anti-MRSA activities.

Fungi are important resources for drug development, as they have a diversity of genes, that can produce novel secondary metabolites with effective bioactivities. Here, five depsidone-based analogs were isolated from the rice media of Chaetomium brasiliense SD-596. Their structures were elucidated using NMR and mass spectrometry analysis. Five compounds, including three new depsidone analogs, mollicellin S (1), mollicellin T (2), and mollicellin U (3), and two known compounds, mollicellin D (4) and mollicellin H (5), exhibited significant inhibition against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), with MIC values ranging from 6.25 to 12.5 µg ml-1. Herein, we identified the predicted plausible biosynthetic cluster of the compounds and discussed the structure-activity relationship. Finally, we found that the introduction of aldehyde and methoxyl groups provide marked improvement for the inhibition against MRSA.

Spatial Exposure of Agricultural Antimicrobial Resistance in Relation to Free-Ranging Domestic Chicken Movement Patterns among Agricultural Communities in Ecuador.

The use of antimicrobial growth promoters in chicken farming has been commonly associated with high levels of antimicrobial resistance (AMR) in humans. Most of this work, however, has been focused on intensive large-scale operations. Intensive small-scale farming that regularly uses antibiotics is increasing worldwide and has different exposure pathways compared with large-scale farming, most notably the spatial connection between chickens and households. In these communities, free-ranging backyard chickens (not fed antibiotics) can roam freely, whereas broiler chickens (fed antibiotics) are reared in the same husbandry environment but confined to coops. We conducted an observational field study to better understand the spatial distribution of AMR in communities that conduct small-scale farming in northwestern Ecuador. We analyzed phenotypic resistance of Escherichia coli sampled from humans and backyard chickens to 12 antibiotics in relation to the distance to the nearest small-scale farming operation within their community. We did not find a statistically significant relationship between the distance of a household to small-scale farming and antibiotic-resistant E. coli isolated from chicken or human samples. To help explain this result, we monitored the movement of backyard chickens and found they were on average 17 m (min-max: 0-59 m) from their household at any given time. These backyard chickens on average ranged further than the average distance from any study household to its closest neighbor. This level of connectivity provides a viable mechanism for the spread of antimicrobial-resistant bacteria and genes throughout the community.

High Prevalence of Extended-Spectrum Beta-Lactamase CTX-M-Producing Escherichia coli in Small-Scale Poultry Farming in Rural Ecuador.

Small-scale farming may have large impacts on the selection and spread of antimicrobial resistance to humans. We conducted an observational study to evaluate antibiotic-resistant Escherichia coli populations from poultry and humans in rural northwestern Esmeraldas, Ecuador. Our study site is a remote region with historically low resistance levels of third-generation antibiotics such cefotaxime (CTX), a clinically relevant antibiotic, in both poultry and humans. Our study revealed 1) high CTX resistance (66.1%) in farmed broiler chickens, 2) an increase in CTX resistance over time in backyard chicken not fed antibiotics (2.3-17.9%), and 3) identical bla CTX-M sequences from human and chicken bacteria, suggesting a spillover event. These findings provide evidence that small-scale meat production operations have direct impacts on the spread and selection of clinically important antibiotics among underdeveloped settings.

Antibiotic Resistome Associated with Small-Scale Poultry Production in Rural Ecuador.

Small-scale poultry farming is common in rural communities across the developing world. To examine the extent to which small-scale poultry farming serves as a reservoir for resistance determinants, the resistome of fecal samples was compared between production chickens that received antibiotics and free-ranging household chickens that received no antibiotics from a rural village in northern Ecuador. A qPCR array was used to quantify antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) using 248 primer pairs; and the microbiome structure was analyzed via 16S rRNA gene sequencing. A large number of ARGs (148) and MGEs (29) were detected. The ARG richness in production chickens was significantly higher than that of household chickens with an average of 15 more genes detected ( p < 0.01). Moreover, ARGs and MGEs were much more abundant in production chickens than in household chickens (up to a 157-fold difference). Production chicken samples had significantly lower taxonomic diversity and were more abundant in Gammaproteobacteria, Betaproteobacteria, and Flavobacteria. The high abundance and diversity of ARGs and MGEs found in small-scale poultry farming was comparable to the levels previously found in large scale animal production, suggesting that these chickens could act as a local reservoir for spreading ARGs into rural communities.

The Role of Mobile Genetic Elements in the Spread of Antimicrobial-Resistant Escherichia coli From Chickens to Humans in Small-Scale Production Poultry Operations in Rural Ecuador.

Small-scale production poultry operations are increasingly common worldwide. To investigate how these operations influence antimicrobial resistance and mobile genetic elements (MGEs), Escherichia coli isolates were sampled from small-scale production birds (raised in confined spaces with antibiotics in feed), household birds (no movement constraints; fed on scraps), and humans associated with these birds in rural Ecuador (2010-2012). Isolates were screened for genes associated with MGEs as well as phenotypic resistance to 12 antibiotics. Isolates from small-scale production birds had significantly elevated odds of resistance to 7 antibiotics and presence of MGE genes compared with household birds (adjusted odds ratio (OR) range = 2.2-87.9). Isolates from humans associated with small-scale production birds had elevated odds of carrying an integron (adjusted OR = 2.0; 95% confidence interval (CI): 1.06, 3.83) compared with humans associated with household birds, as well as resistance to sulfisoxazole (adjusted OR = 1.9; 95% CI: 1.01, 3.60) and trimethoprim/sulfamethoxazole (adjusted OR = 2.1; 95% CI: 1.13, 3.95). Stratifying by the presence of MGEs revealed antibiotic groups that are explained by biological links to MGEs; in particular, resistance to sulfisoxazole, trimethoprim/sulfamethoxazole, or tetracycline was highest among birds and humans when MGE exposures were present. Small-scale production poultry operations might select for isolates carrying MGEs, contributing to elevated levels of resistance in this setting.

Antibiotic Resistance in Animal and Environmental Samples Associated with Small-Scale Poultry Farming in Northwestern Ecuador.

The effects of animal agriculture on the spread of antibiotic resistance (AR) are cross-cutting and thus require a multidisciplinary perspective. Here we use ecological, epidemiological, and ethnographic methods to examine populations of Escherichia coli circulating in the production poultry farming environment versus the domestic environment in rural Ecuador, where small-scale poultry production employing nontherapeutic antibiotics is increasingly common. We sampled 262 "production birds" (commercially raised broiler chickens and laying hens) and 455 "household birds" (raised for domestic use) and household and coop environmental samples from 17 villages between 2010 and 2013. We analyzed data on zones of inhibition from Kirby-Bauer tests, rather than established clinical breakpoints for AR, to distinguish between populations of organisms. We saw significantly higher levels of AR in bacteria from production versus household birds; resistance to either amoxicillin-clavulanate, cephalothin, cefotaxime, and gentamicin was found in 52.8% of production bird isolates and 16% of household ones. A strain jointly resistant to the 4 drugs was exclusive to a subset of isolates from production birds (7.6%) and coop surfaces (6.5%) and was associated with a particular purchase site. The prevalence of AR in production birds declined with bird age (P < 0.01 for all antibiotics tested except tetracycline, sulfisoxazole, and trimethoprim-sulfamethoxazole). Farming status did not impact AR in domestic environments at the household or village level. Our results suggest that AR associated with small-scale poultry farming is present in the immediate production environment and likely originates from sources outside the study area. These outside sources might be a better place to target control efforts than local management practices. IMPORTANCE In developing countries, small-scale poultry farming employing antibiotics as growth promoters is being advanced as an inexpensive source of protein and income. Here, we present the results of a large ecoepidemiological study examining patterns of antibiotic resistance (AR) in E. coli isolates from small-scale poultry production environments versus domestic environments in rural Ecuador, where such backyard poultry operations have become established over the past decade. Our previous research in the region suggests that introduction of AR bacteria through travel and commerce may be an important source of AR in villages of this region. This report extends the prior analysis by examining small-scale production chicken farming as a potential source of resistant strains. Our results suggest that AR strains associated with poultry production likely originate from sources outside the study area and that these outside sources might be a better place to target control efforts than local management practices.

Distribution of Enteroinvasive and Enterotoxigenic Escherichia coli Across Space and Time in Northwestern Ecuador.

Although Escherichia coli infections are common throughout the developing world, their prevalence patterns in space and over time are not well characterized. We used serial case control data collected from 16 communities in northwestern Ecuador between 2004 and 2010, to examine the prevalence of enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC). At its peak, the regional prevalence of EIEC was 8.3 infections/100 persons but this decreased to 1 infection/1,000 persons. The regional prevalence of ETEC ranged from 8 infections/1,000 persons to 3.7 infections/100 persons. The prevalence pattern of EIEC resembled that of a large epidemic whereas the prevalence of ETEC was more stable over time. Here, we provide community-based evidence for temporal shifts in the dominant E. coli pathotype from EIEC to ETEC over a multi-year time period. Furthermore, genotype analysis suggests that a given strain of EIEC and ETEC can persist in this region for long periods, up to 24 and 55 months, respectively.

Unexpected distribution of the fluoroquinolone-resistance gene qnrB in Escherichia coli isolates from different human and poultry origins in Ecuador.

Fluoroquinolone resistance can be conferred through chromosomal mutations or by the acquisition of plasmids carrying genes such as the quinolone resistance gene (qnr). In this study, 3,309 strains of commensal Escherichia coli were isolated in Ecuador from: (i) humans and chickens in a rural northern coastal area (n = 2368, 71.5%) and (ii) chickens from an industrial poultry operation (n = 827, 25%). In addition, 114 fluoroquinolone-resistant strains from patients with urinary tract infections who were treated at three urban hospitals in Quito, Ecuador were analyzed. All of the isolates were subjected to antibiotic susceptibility screening. Fluoroquinolone-resistant isolates (FRIs) were then screened for the presence of qnrB genes. A significantly higher phenotypic resistance to fluoroquinolones was determined in E. coli strains from chickens in both the rural area (22%) and the industrial operation (10%) than in strains isolated from humans in the rural communities (3%). However, the rates of qnrB genes in E. coli isolates from healthy humans in the rural communities (11 of 35 isolates, 31%) was higher than in chickens from either the industrial operations (3 of 81 isolates, 6%) or the rural communities (7 of 251 isolates, 2.8%). The occurrence of qnrB genes in human FRIs obtained from urban hospitals was low (1 of 114 isolates, 0.9%). These results suggested that the qnrB gene is more widely distributed in rural settings, where antibiotic usage is low, than in urban hospitals and industrial poultry operations. The role of qnrB in clinical resistance to fluoroquinolones is thus far unknown.