basicsynbio and the BASIC SEVA collection: Software and vectors for an established DNA assembly method

Standardized DNA assembly methods utilizing modular components provide a powerful framework to explore design spaces and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use, while enabling simple hierarchical assemblies through an idempotent format. These attributes facilitate various applications including pathway engineering, ribosome binding site tuning, fusion protein synthesis and multiplex gRNA expression. In this work we present basicsynbio, an open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio). With basicsynbio, users can access commonly used BASIC parts and linkers while robustly designing new parts and assemblies with exception handling for common design errors. Furthermore, users can export sequence data and create build instructions for manual or automated workflows. The generation of build instructions relies on the BasicBuild Open Standard which is easily parsed for bespoke workflows and is serialised in Java Script Object Notation for transfer and storage. We demonstrate basicsynbio by assembling a collection of 30 BASIC-compatible vectors using various sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA collection encompasses plasmids containing six antibiotic resistance markers and five origins of replication from different compatibility groups, including a temperature-sensitive variant. We deposit the collection on Addgene under an OpenMTA agreement, making them available. Furthermore, these sequences are accessible from within the basicsynbio application programming interface along with other collections of parts and linkers, providing an ideal environment to design BASIC DNA assemblies for bioengineering applications.

The Omics Hunt for Novel Molecular Markers of Resistance to Phytophthora infestans

Wild Solanum accessions are a treasured source of resistance against pathogens, including oomycete Phytophthora infestans, causing late blight disease. Here, Solanum pinnatisectum, Solanum tuberosum, and the somatic hybrid between these two lines were analyzed, representing resistant, susceptible, and moderately resistant genotypes, respectively. Proteome and metabolome analyses showed that the infection had the highest impact on leaves of the resistant plant and indicated, among others, an extensive remodeling of the leaf lipidome. The lipidome profiling confirmed an accumulation of glycerolipids, a depletion in the total pool of glycerophospholipids, and showed considerable differences between the lipidome composition of resistant and susceptible genotypes. The analysis of putative resistance markers pinpointed more than 100 molecules that positively correlated with resistance including phenolics and cysteamine, a compound with known antimicrobial activity. Putative resistance protein markers were targeted in an additional 12 genotypes with contrasting resistance to P. infestans. At least 27 proteins showed a negative correlation with the susceptibility including HSP70-2, endochitinase B, WPP domain-containing protein, and cyclase 3. In summary, these findings provide insights into molecular mechanisms of resistance against P. infestans and present novel targets for selective breeding.

Temporal evolution of the resistance genotypes of Plasmodium falciparum in isolates from Equatorial Guinea during 20 years (1999 to 2019)

Background Malaria is one of the deadliest diseases in the world, particularly in Africa. As such, resistance to anti-malarial drugs is one of the most important problems in terms of global malaria control. This study assesses the evolution of the different resistance markers over time and the possible influence of interventions and treatment changes that have been made in Equatorial Guinea. Methods A total of 1223 biological samples obtained in the period 1999 to 2019 were included in the study. Screening for mutations in the pfdhfr, pfdhps, pfmdr1, and pfcrt genes was carried out by nested PCR and restriction-fragment length polymorphisms (RFLPs), and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations. Results The partially and fully resistant haplotypes (pfdhfr + pfdhps) were found to increase over time. Moreover, in 2019, the fully resistant haplotype was found to be increasing, although its super-resistant counterpart remains much less prevalent. A continued decline in pfmdr1 and pfcrt gene mutations over time was also found. The number of mutations detected in pfk13 has increased since 2008, when artemisinin-based combination therapy (ACT) were first introduced, with more mutations being observed in 2019, with two synonymous and five non-synonymous mutations being detected, although these are not related to resistance to ACT. In addition, the non-synonymous A578S mutation, which is the most frequent on the African continent, was detected in 2013, although not in the following years. Conclusions Withdrawal of the use of chloroquine (CQ) as a treatment in Equatorial Guinea has been shown to be effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in sulfadoxine-pyrimethamine (SP) resistance markers suggest its misuse, either alone or in combination with artesunate (AS) or amodiaquine (AQ), in some areas of the country, as was found in a previous study conducted by this group, which allows selective pressure from SP to continue. Single nucleotide polymorphisms (SNPs) 540E and 581G do not exceed the limit of 50 and 10%, respectively, thus meaning that SP is still effective as an intermittent preventive treatment (IPT) in this country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACT. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008. Graphical Abstract

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfcrt and pfmdr1 genes in isolates from Honduras and Nicaragua, 2018–2021

Background Central America and the island of Hispaniola have set out to eliminate malaria by 2030. However, since 2014 a notable upturn in the number of cases has been reported in the Mosquitia region shared by Nicaragua and Honduras. In addition, the proportion of Plasmodium falciparum malaria cases has increased significantly relative to vivax malaria. Chloroquine continues to be the first-line drug to treat uncomplicated malaria in the region. The objective of this study was to evaluate the emergence of chloroquine resistant strains of P. falciparum using a genetic approach. Plasmodium vivax populations are not analysed in this study. Methods 205 blood samples from patients infected with P. falciparum between 2018 and 2021 were analysed. The pfcrt gene fragment encompassing codons 72–76 was analysed. Likewise, three fragments of the pfmdr1 gene were analysed in 51 samples by nested PCR and sequencing. Results All samples revealed the CVMNK wild phenotype for the pfcrt gene and the N86, Y184F, S1034C, N1042D, D1246 phenotype for the pfmdr1 gene. Conclusions The increase in falciparum malaria cases in Nicaragua and Honduras cannot be attributed to the emergence of chloroquine-resistant mutants. Other possibilities should be investigated further. This is the first study to report the genotype of pfmdr1 for five loci of interest in Central America.

Prevalence of potential mediators of artemisinin resistance in African isolates of Plasmodium falciparum

Background The devastating public health impact of malaria has prompted the need for effective interventions. Malaria control gained traction after the introduction of artemisinin-based combination therapy (ACT). However, the emergence of artemisinin (ART) partial resistance in Southeast Asia and emerging reports of delayed parasite sensitivity to ACT in African parasites signal a gradual trend towards treatment failure. Monitoring the prevalence of mutations associated with artemisinin resistance in African populations is necessary to stop resistance in its tracks. Mutations in Plasmodium falciparum genes pfk13, pfcoronin and pfatpase6 have been linked with ART partial resistance. Methods Findings from published research articles on the prevalence of pfk13, pfcoronin and pfatpase6 polymorphisms in Africa were collated. PubMed, Embase and Google Scholar were searched for relevant articles reporting polymorphisms in these genes across Africa from 2014 to August 2021, for pfk13 and pfcoronin. For pfatpase6, relevant articles between 2003 and August 2021 were retrieved. Results Eighty-seven studies passed the inclusion criteria for this analysis and reported 742 single nucleotide polymorphisms in 37,864 P. falciparum isolates from 29 African countries. Five validated-pfk13 partial resistance markers were identified in Africa: R561H in Rwanda and Tanzania, M476I in Tanzania, F446I in Mali, C580Y in Ghana, and P553L in an Angolan isolate. In Tanzania, three (L263E, E431K, S769N) of the four mutations (L263E, E431K, A623E, S769N) in pfatpase6 gene associated with high in vitro IC50 were reported. pfcoronin polymorphisms were reported in Senegal, Gabon, Ghana, Kenya, and Congo, with P76S being the most prevalent mutation. Conclusions This meta-analysis provides an overview of the prevalence and widespread distribution of pfk13, pfcoronin and pfatpase6 mutations in Africa. Understanding the phenotypic consequences of these mutations can provide information on the efficacy status of artemisinin-based treatment of malaria across the continent. Graphical Abstract

Distribution of ermB, ermF, tet(W), and tet(M) Resistance Genes in the Vaginal Ecosystem of Women during Pregnancy and Puerperium

The inhabitants of the vaginal ecosystem can harbor genetic determinants conferring antimicrobial resistance. However, detailed data about the distribution of resistance genes in the vaginal microbiome of pregnant women are still lacking. Therefore, we assessed the presence of macrolide (i.e., erm genes) and tetracycline (i.e., tet genes) resistance markers in the vaginal environment of Caucasian women at different gestational ages. Furthermore, the detection of resistance genes was related to the composition of the vaginal microbiota. A total of 228 vaginal samples, collected at different trimesters of pregnancy or during the puerperium, were tested for the presence of ermB, ermF, tet(W), and tet(M) by in-house end-point PCR assays. The composition of the vaginal microbiota was assessed through a microscopic evaluation (i.e., Nugent score) and by means of sequencing V3–V4 hypervariable regions of the bacterial 16 rRNA gene. Overall, the most detected resistance gene was tet(M) (76.7%), followed by ermB (55.2%). In 17% of women, mainly with a ‘normal’ vaginal microbiota, no resistance genes were found. Except for tet(W), a significant correlation between the positivity of resistance genes and a dysbiotic vaginal status (i.e., bacterial vaginosis (BV)) was noticed. Indeed, samples positive for at least one resistance determinant were characterized by a decrease in Lactobacillus spp. and an increase of BV-related genera (Prevotella, Gardnerella, Atopobium, Sneathia). A high predominance of vaginal Lactobacillus spp. (>85%) was associated with a lower risk of tet(W) gene detection, whereas the presence of Megasphaera (>1%) increased the risk of positivity for all analyzed genes. Different types of vaginal microbiota are associated with peculiar resistance profiles, being a lactobacilli-dominated ecosystem poor in or free of resistance genes. These data could open new perspectives for promoting maternal and neonatal health.

Contribution of markers of adiposopathy and adipose cell size in predicting insulin resistance markers in women of varying age and adiposity

Markers of adipose tissue (AT) dysfunctions, such as adipocyte hypertrophy, macrophage infiltration and secretory adiposopathy (low plasma adiponectin/leptin, A/L, ratio), associate with metabolic disorders. However, no study has compared the relative contribution of these markers to cardiometabolic risk in women of varying age and adiposity. Body composition, regional AT distribution, lipid-lipoprotein profile, glucose homeostasis and plasma A and L levels were determined in 67 women (age: 40-62 years; BMI: 17-41 kg/m2). Expression of macrophage infiltration marker CD68 and adipocyte size were measured from subcutaneous abdominal (SCABD) and omental (OME) fat samples. AT dysfunction markers were correlated with most lipid-lipoprotein levels such as TAGs (-0.36<rho<0.46; 0.0005<p<0.05), except OME CD68 expression which was negatively related to HDL-cholesterol. The A/L ratio was negatively associated with fasting insulinemia and HOMA-IR (-0.60<rho<-0.63; p<0.0001), while SCABD or OME adipocyte size and SCABD CD68 expression were positively related to these variables (0.39<rho<0.59; p<0.01). Multiple regression analyses including these markers and triacylglycerol (TAG) levels revealed that the A/L ratio was the only predictor of fasting insulinemia and HOMA-IR (partial R2=0.31-0.33; 0.000<p<0.005) in the model with TAGs and CD68 expression. However, the contribution of the A/L ratio was supplanted by adipose cell size in the model where the latter replaced TAGs. Combination of tertiles of largest adipocyte size and lowest A/L ratio showed the highest HOMA-IR. Taken together, our results show that adipocyte hypertrophy combined with reduced A/L ratio was related to increased IR, suggesting an independent contribution of both markers to the variance of cardiometabolic risk.

654. Performance of the T2Resistance Panel in Detecting Antibiotic Resistant Bacteria Directly in Whole Blood, and Implications for Improving Appropriate Therapy of Bloodstream Infections

Background Appropriate antibiotic (Ab) therapy of bloodstream infections (BSI) is often delayed by time to blood culture (BC) positivity, species (sp) identification and Ab sensitivity (sensi). The T2Resistance (T2R) Panel is a direct-from-blood (culture-independent) diagnostic that detects 13 genetic markers associated with methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus (VRE), ESBL- and carbapenemase-producing Enterobacteriaceae (E). We assessed T2R performance in detecting these resistant bacteria in whole blood (WB) and analyzed possible impact on time to appropriate Ab. Methods We performed T2R using WB samples obtained from patients (pts) on the same day as BCs from July 2019-2020. Receipt of appropriate Ab was assessed at time of empiric, Gram stain-directed, MALDI-directed (sp identification) and sensi-directed therapy. T2R results were not available to care teams. Teams were notified of positive BCs. Stewardship optimized Abs based on sensi. Results BC from 103 pts grew 114 bacterial sp: E (n=54; 16 ESBL-, 1 KPC-producer), S. aureus (n=29, 22 MRSA), Enterococcus (n=21, 16 VRE), P. aeruginosa and others (n=10). 12 ESBL-E produced CTX-M 14/15. T2R sensitivity and specificity was 78% and 99%, respectively, compared to sequencing of resistance markers. Sensitivity was excellent for vanA/B, KPC (100% each), and CTX-M14/15 (92%); sensitivity was 58% for mecA/C. T2R detected resistance determinants in 3-7h. Median time to appropriate Ab was 16.3h, which was significantly longer for VRE (25.6h) and ESBL- or KPC-E (50.9h) BSIs than for T2R marker-negative bacteria (6.7h; p=0.04). Pts with VRE or ESBL-/KPC-E BSI were less likely to received appropriate empiric Ab (18% and 30%, respectively) than pts with T2R marker-negative BSI (63%; p=0.02; Fig.1). Median times to achieve ≥80% appropriate Ab therapy of marker-negative, VRE and CTX-M/KPC-E BSIs were 15.5h (after Gram stain), 43.9h (after MALDI) and 63.5h (after sensi), respectively. Antibiotic Therapy Conclusion There was a significant delay in appropriate Ab therapy of BSIs, especially in pts infected with VRE and ESBL/KPC-E. T2R rapidly and accurately detected BSI caused by VRE and ESBL/KPC-E, and has the potential to significantly shorten time to appropriate Ab. Disclosures Cornelius J. Clancy, MD, Merck (Grant/Research Support) Ryan K. Shields, PharmD, MS, Shionogi (Consultant, Research Grant or Support) Minh-Hong Nguyen, MD, Merck (Grant/Research Support)

Conjugation-based genome engineering in Deinococcus radiodurans

D. radiodurans has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based transformation system from E. coli to D. radiodurans allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers. Using this method with nonreplicating plasmids, we developed a protocol for creating sequential gene deletions in D. radiodurans by target-ing restriction-modification system genes. Importantly, we demonstrated a conjugation-based method for cloning the large (178 kb), high G+C content MP1 megaplasmid from D. radiodurans in E. coli. The conjugation-based tools described here will facili-tate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications.

Prevalence of Mycoplasma genitalium fluoroquinolone-resistance markers, and dual-class-resistance markers, in asymptomatic men who have sex with men

Introduction. Failure of fluoroquinolones, the principal treatment option for macrolide-resistant Mycoplasma genitalium infections, has recently emerged. This is of particular concern for men who have sex with men (MSM), who have high proportions of macrolide-resistant M. genitalium infections. Treatment failure with moxifloxacin is likely the result of single nucleotide polymorphisms (SNPs) in parC, whilst concurrent gyrA mutations may play a role. Gap Statement. The levels of fluoroquinolone resistance and dual-class (i.e. macrolide and fluoroquinolone) resistance in M. genitalium among asymptomatic MSM is unknown. Aim. To (i) determine the proportion of fluoroquinolone resistance and dual-class resistance in M. genitalium infections among asymptomatic MSM, (ii) explore any clinical and behavioural associations with fluoroquinolone resistance, and (iii) determine the distribution of antibiotic resistance among M. genitalium mgpB sequence types (STs). Methodology. M. genitalium positive samples (N=94) were obtained from 1001 asymptomatic MSM enrolled in a study at Melbourne Sexual Health Centre (Carlton, Australia) between August 2016 and September 2017. Sanger sequencing was performed to determine the proportion of M. genitalium infections with SNPs in parC that have previously been associated with failure of moxifloxacin (corresponding to amino changes S83I, D83R, D87Y and D87N) and in gyrA (corresponding to amino acid changes M95I, D99N, D99Y and D99G). Associations between clinical/behavioural factors and parC SNPs were examined. Strain typing was performed by sequencing a portion of the mgpB gene. Results. The proportion of MSM with infections harbouring parC and gyrA SNPs was 13.0 % [95 % confidence interval (CI): 6.8–23.2 %] and 4.7 % (95 % CI: 1.1–13.4 %), respectively; dual-class resistance was 13.0 %. No significant clinical/behavioural associations were found. Antibiotic resistance was not restricted to specific mgpB STs. Conclusion. One in eight (13 %) of asymptomatic MSM with M. genitalium had an infection with dual-class-resistance mutations. Typing by mgpB sequence suggested fluoroquinolone resistance is arising from independent mutation events. This study illustrates that asymptomatic MSM may act as a reservoir for antibiotic-resistant M. genitalium .