Designing a Genetic Screen to Identify Factors Required for Meiotic Nuclear Rejuvenation

During the natural cycle of life, most eukaryotic organisms grow old, age, and die. A common natural mechanism by which organisms “reset” their lifespan is through sexual reproduction; however, how this rejuvenation takes place remains unknown. My lab has found that meiosis in budding yeast, the developmental program that forms sex cells, eliminates age-induced damage. This involves the formation of a novel nuclear compartment, the Gametogenesis Uninherited Nuclear Compartment (GUNC), which acts as a trash can for accumulated age-induced damage. To understand the molecular details of this process, I worked on designing a screen for genes involved in GUNC formation. My mentor and I fused three different proteins targeted to the GUNC and a protein that is able to bind to a drug-resistance plasmid, in order to couple the inheritance of a selectable DNA marker with the elimination of age-induced damage. Initial testing of these three fusion proteins suggested that they were unable to successfully target the plasmid to the GUNC; as such, testing of additional candidate proteins is necessary. We plan to eventually use this system to identify mutations that disrupt GUNC formation and cause inheritance of the drug-resistance plasmid. By identifying and perturbing proteins involved in GUNC formation, we are hoping to be able to drive the inheritance of specific types of age-induced damage, allowing for the determination of what a symptom versus a cause of aging is.

Chicken gut microbiome members limit the spread of an antimicrobial resistance plasmid in Escherichia coli

Plasmid-mediated antimicrobial resistance is a major contributor to the spread of resistance genes within bacterial communities. Successful plasmid spread depends upon a balance between plasmid fitness effects on the host and rates of horizontal transmission. While these key parameters are readily quantified in vitro , the influence of interactions with other microbiome members is largely unknown. Here, we investigated the influence of three genera of lactic acid bacteria (LAB) derived from the chicken gastrointestinal microbiome on the spread of an epidemic narrow-range ESBL resistance plasmid, IncI1 carrying bla CTX-M-1 , in mixed cultures of isogenic Escherichia coli strains. Secreted products of LAB decreased E. coli growth rates in a genus-specific manner but did not affect plasmid transfer rates. Importantly, we quantified plasmid transfer rates by controlling for density-dependent mating opportunities. Parametrization of a mathematical model with our in vitro estimates illustrated that small fitness costs of plasmid carriage may tip the balance towards plasmid loss under growth conditions in the gastrointestinal tract. This work shows that microbial interactions can influence plasmid success and provides an experimental-theoretical framework for further study of plasmid transfer in a microbiome context.

Complete Genome Sequence of Escherichia coli Sequence Type 1193 Isolate AVS0096, Recovered from River Water in Switzerland

Escherichia coli sequence type 1193 (ST1193) is an important cause of multidrug-resistant extraintestinal infections. Here, we report the complete genome sequence of strain AVS0096, isolated from river water in Switzerland in 2020. The genome consists of a chromosome (4.9 Mbp), a multidrug resistance plasmid (101 kb), and two small plasmids.

In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid

FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.

Transfer of Antibiotic Resistance Plasmid from Commensal E. coli Towards Human Intestinal Microbiota in the M-SHIME: Effect of E. coli dosis, Human Individual and Antibiotic Use

Along with (in) direct contact with animals and a contaminated environment, humans are exposed to antibiotic-resistant bacteria by consumption of food. The implications of ingesting antibiotic-resistant commensal bacteria are unknown, as dose-response data on resistance transfer and spreading in our gut is lacking. In this study, transfer of a resistance plasmid (IncF), harbouring several antibiotic resistance genes, from a commensal E. coli strain towards human intestinal microbiota was assessed using a Mucosal Simulator of the Human Intestinal Ecosystem (M-SHIME). More specifically, the effect of the initial E. coli plasmid donor concentration (105 and 107 CFU/meal), antibiotic treatment (cefotaxime) and human individual (n = 6) on plasmid transfer towards lumen coliforms and anaerobes was determined. Transfer of the resistance plasmid to luminal coliforms and anaerobes was observed shortly after the donor strain arrived in the colon and was independent of the ingested dose. Transfer occurred in all six simulated colons and despite their unique microbial community composition, no differences could be detected in antibiotic resistance transfer rates between the simulated human colons. After 72 h, resistant coliform transconjugants levels ranged from 7.6 × 104 to 7.9 × 106 CFUcefotaxime resistant/Ml colon lumen. Presence of the resistance plasmid was confirmed and quantified by PCR and qPCR. Cefotaxime treatment led to a significant reduction (85%) in resistant coliforms, however no significant effect on the total number of cultivable coliforms and anaerobes was observed.

Contagious Antibiotic Resistance: Plasmid Transfer among Bacterial Residents of the Zebrafish Gut

ABSTRACTBy characterizing the trajectories of antibiotic resistance gene transfer in bacterial communities such as the gut microbiome, we will better understand the factors that influence this spread of resistance. Our aim was to investigate the host network of a multidrug resistance broad-host-range plasmid in the culturable gut microbiome of zebrafish. This was done throughin vitroandin vivoconjugation experiments withEscherichia colias the donor of the plasmid pB10::gfp. When this donor was mixed with the extracted gut microbiome, only transconjugants ofAeromonas veroniiwere detected. In separate matings between the same donor and four prominent isolates from the gut microbiome, the plasmid transferred to two of these four isolates,A. veroniiandPlesiomonas shigelloides, but not toShewanella putrefaciensandVibrio mimicus. When theseA. veroniiandP. shigelloidestransconjugants were the donors in matings with the same four isolates, the plasmid now also transferred fromA. veroniitoS. putrefaciens.P. shigelloideswas unable to donate the plasmid, andV. mimicuswas unable to acquire it. Finally, when theE. colidonor was addedin vivoto zebrafish through their food, plasmid transfer was observed in the gut, but only toAchromobacter, a rare member of the gut microbiome. This work shows that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome depends on the donor-recipient species combinations and therefore their spatial arrangement. It also suggests that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food.IMPORTANCETo understand how antibiotic resistance plasmids end up in human pathogens, it is crucial to learn how, where, and when they are transferred and maintained in members of bacterial communities such as the gut microbiome. To gain insight into the network of plasmid-mediated antibiotic resistance sharing in the gut microbiome, we investigated the transferability and maintenance of a multidrug resistance plasmid among the culturable bacteria of the zebrafish gut. We show that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome can depend on which species are involved, as some are important nodes in the plasmid-host network and others are dead ends. Our findings also suggest that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food.

Genomic Portrait of Community-Associated Methicillin-Resistant Staphylococcus Aureus ST772-SCCmec V Lineage From India

BackgroundSignificant changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognised with the emergence of community-associated methicillin-resistant Staphylococcus aureus. However, studies on the molecular epidemiology and the genomic investigation of MRSA are limited in India. The aim of the study was to understand the molecular epidemiology of MRSA causing bloodstream infection and also to investigate the origin and evolution of ST772 S. aureus isolated from India.ResultsSCCmec V (42%) was the predominant gene followed by SCCmec III (27%), SCCmec IV (13%), SCCmec I (7%) and SCCmec II (3%). We demonstrate the presence of multiple SCCmec types in 18 isolates. MLST analysis revealed ten different clonal complexes and three singletons. ST772 (27%), ST22 (19%) and ST239 (16%) were the predominant MRSA genotypes in causing bloodstream infection. The spa types were highly diverse. Phylogenetic analysis revealed that nearly three-fourth of the Indian STT72-SCCmec V isolates belongs to dominant (ST772-A2) and emerging subgroups (ST772-A3). A prophage Φ IND772 carrying PVL toxin and a staphylococcal enterotoxin A was noticed in all isolates, except two. In addition, three distinct genomic islands (vSa-alpha, beta and gamma) were universally seen in all ST772 S. aureus genomes. A pattern of increasing antimicrobial resistance was noticed in the dominant and emerging subgroups. An integrated resistance plasmid encoding resistance clusters for beta-lactam (blaZ), macrolides (mphC, msrA), and aminoglycoside resistance (aphA-III, sat-4, aadE) was identified in all isolates, except four basal strains. ST772-SCCmec V was emerged on the Indian subcontinent in 1964 and diverged into a dominant subgroup in 1991. Furthermore, the expansion is likely to be associated with the acqusition of mobile genetic elements such as integrated resistance plasmid and SCCmec V (5C2) as well as the fixation of double serine mutation (S84L, S80Y) in the quinolone resistance determining region. ConclusionsST772-SCCmec V has the multi-drug resistance trait of hospital-associated (HA) MRSA and the epidemiological characteristics of CA-MRSA. ST772 S. aureus have consistent virulence and resistance determinants which may results in successful survival in both community and hospital settings.