Modeling bacterial resistance to antibiotics: bacterial conjugation and drug effects

Antibiotic resistance is a major burden in many hospital settings as it drastically reduces the successful probability of treating bacterial infections. Generally, resistance is associated with bacterial fitness reduction and selection pressure from antibiotic usage. Here, we investigate the effects of bacterial conjugation, plasmid loss, and drug responses on the population dynamics of sensitive and resistant bacteria by using a mathematical model. Two types of drugs are considered here: antibiotic M that kills only sensitive bacteria and antibiotic N that kills both bacteria. Our results highlight that larger dose and longer dosing interval of antibiotic M may result in the higher prevalence of resistant bacteria while they do the opposite for antibiotic N. When delays in administering initial and second doses are incorporated, the results demonstrate that the delays may lead to the higher prevalence of resistant bacteria when antibiotic M or N is administered with the longer time of bacteria remaining at the lower prevalence of the latter. Our results highlight that switching antibiotic agents during a treatment course and different bacterial strain characteristics result in a significant impact on the prevalence of resistant bacteria.

Progress towards an inducible, replication-proficient transposon delivery vector for Chlamydia trachomatis

Background Genetic systems have been developed for Chlamydia but the extremely low transformation frequency remains a significant bottleneck. Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction. Methods We made E. coli/ C. trachomatis shuttle vectors bearing the Himar1 C9 transposase under control of the tet promoter and a novel rearrangement of the Himar1 transposon with the β-lactamase gene. Activity of the transposase was monitored by immunoblot and by DNA sequencing. Results We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as a self-replicating vector carrying both the Himar1 C9 transposase under tet promoter control and its transposon. However, we were unable to recover this plasmid in C. trachomatis following multiple attempts at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the Himar1 C9 transposase (under tet promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon. We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in E. coli. Both these plasmids could be independently recovered in C. trachomatis. We attempted to perform lateral gene transfer by transformation and mixed infection with C. trachomatis strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase). Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. Conclusions We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9 transposase under tet promoter control. Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis. Based on selected deletions and phenotypic analyses we conclude that low level expression from the tet inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.

Dissemination and Stability of the blaNDM-5-Carrying IncX3-Type Plasmid among Multiclonal Klebsiella pneumoniae Isolates

ABSTRACT NDM-5 carbapenemase was mainly identified in Escherichia coli, while the rapid transmission of blaNDM-5 among Enterobacteriaceae has raised serious public attention. This study identified 14 NDM-5-producing Klebsiella pneumoniae isolates from 107 carbapenem-resistant K. pneumoniae isolates, recovered from blood, urine, and normally sterile body fluids of pediatric patients from January 2016 to December 2018. All NDM-5-producing isolates were highly resistant to β-lactams, while tigecycline and polymyxin B exhibited excellent antimicrobial activity. These 14 strains belonged to 9 different sequence types (STs) and displayed various pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they were not clonally related. S1-PFGE followed by Southern blotting showed that the blaNDM-5 gene was located on an ∼46-kb IncX3 plasmid in all strains. All blaNDM-5-carrying plasmids were successfully transferred into recipient E. coli J53. PCR-based sequencing demonstrated that all of the blaNDM-5-carrying plasmids shared highly similar backbones, with nucleotide sequence identity of >99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 blaNDM-5-carrying plasmids, with dynamic changes observed only in blaNDM-5-surrounding elements. Interestingly, the IncX3 blaNDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% blaNDM-5-carrying plasmid loss after coculture with linoleic acid for 5 days. These results showed that the IncX3 plasmid facilitated the dissemination of blaNDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniae. IMPORTANCE The emergence and spread of New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 blaNDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of blaNDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 blaNDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 blaNDM-5-carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 blaNDM-5-carrying plasmids.

A natural variant of the essential host gene MMS21 restricts the parasitic 2-micron plasmid in Saccharomyces cerevisiae

Antagonistic coevolution with selfish genetic elements (SGEs) can drive evolution of host resistance. Here, we investigated host suppression of 2-micron (2μ) plasmids, multicopy nuclear parasites that have co-evolved with budding yeasts. We developed SCAMPR (Single-Cell Assay for Measuring Plasmid Retention) to measure copy number heterogeneity and 2μ plasmid loss in live cells. We identified three S. cerevisiae strains that lack endogenous 2μ plasmids and reproducibly inhibit mitotic plasmid stability. Focusing on the Y9 ragi strain, we determined that plasmid restriction is heritable and dominant. Using bulk segregant analysis, we identified a high-confidence Quantitative Trait Locus (QTL) with a single variant of MMS21 associated with increased 2μ instability. MMS21 encodes a SUMO E3 ligase and an essential component of the Smc5/6 complex, involved in sister chromatid cohesion, chromosome segregation, and DNA repair. Our analyses leverage natural variation to uncover a novel means by which budding yeasts can overcome highly successful genetic parasites.

The role of toxin:antitoxin systems and insertion sequences in the loss of virulence in Shigella sonnei

SUMMARYThe Shigella plasmid, pINV, contains a 30 kb pathogenicity island (PAI) encoding a Type III secretion system (T3SS) which is essential for virulence. During growth in the laboratory, avirulent colonies of Shigella (which do not express a T3SS) arise spontaneously. Avirulence in Shigella flexneri mostly follows loss of the PAI, following recombination between insertion sequences (ISs) on pINV; toxin:antitoxin (TA) systems on pINV promote its retention through post-segregational killing (PSK). We show that avirulence in Shigella sonnei mainly results from plasmid loss, consistent with previous findings; IS-mediated PAI deletions can occur in S. sonnei, but through different ISs than in S. flexneri. We investigated the molecular basis for frequent loss of the S. sonnei plasmid, pINVSsonn. Introduction into pINVSsonn of CcdAB and GmvAT, toxin:antitoxin TA systems in pINV from S. flexneri but not S. sonnei, reduced plasmid loss and the emergence of avirulent bacteria. However, plasmid loss remained the leading cause of avirulence. We show that a single amino acid difference in the VapC toxin of the VapBC TA system in pINV also contributes to high frequency plasmid loss in S. sonnei compared to S. flexneri. Our findings demonstrate that the repertoire of ISs, complement of TA systems, and polymorphisms in TA systems influence plasmid dynamics and virulence loss in S. sonnei. Understanding the impact of polymorphisms should be informative about how TA systems contribute to PSK, and could be exploited for generating strains with stable plasmids.

Molecular genetics of the virulence plasmids of pathogenic Escherichia coli O104:H4

In 2011 a large outbreak of enterohemorrhagic gastroenteritis and haemolytic uremic syndrome (HUS) throughout Europe resulted in almost 4,000 infections, 845 cases of HUS and 54 fatalities. This was due to a dangerous exchange of mobile genetic elements (MGE) resulting in a hybrid strain of E. coli O104:H4. This strain carried an unusual combination of EAEC- and STEC-associated virulence factors on a plasmid and phage respectively. In vitro the virulence plasmid has exhibited unusual stability under a wide range of environmental stresses, contrasting with rapid plasmid loss in the human gut. This project will characterise plasmid encoded maintenance systems responsible for its unique stability. Current investigations focus on toxin-antitoxin (TA) systems involved in postsegregationalkilling which contribute to plasmid maintenance therefore resulting in increased virulence. By inducing expression of putative TA genes cloned onto lab-made plasmid vectors, we have analysed their function in the cell. Once characterised we will further investigate the effects of various environmental conditions able to disrupt these TA systems, ultimately resulting in plasmid loss. This atypical strain displayed heightened pathogenicity and providedun foreseen treatment challenges. We aim to further our understanding of MGE carriage in O104:H4 as a model to predict and combat future outbreaks of hybrid pathovars.

Modeling slow-processing of toxin messenger RNAs in type-I Toxin-Antitoxin systems: post-segregational killing and noise filtering

In type-I toxin-antitoxin (TA) systems, the action of growth-inhibiting toxin proteins is counteracted by the antitoxin small RNAs (sRNAs) that prevent the translation of toxin messenger RNAs (mRNAs). When a TA module is encoded on a plasmid, the short lifetime of antitoxin sRNA compared to toxin mRNAs mediates post-segregational killing (PSK) that contribute the plasmid maintenance, while some of the chromosomal encoded TA loci have been reported to contribute to persister formation in response to a specific upstream signal. Some of the well studied type-I TA systems such ashok/sokare known to have a rather complex regulatory mechanism. Transcribed full-length toxin mRNAs fold such that the ribosome binding site is not accessible and hence cannot be translated. The mRNAs are slowly processed by RNases, and the truncated mRNAs can be either translated or bound by antitoxin sRNA to be quickly degraded. We analyze the role of this extra processing by a mathematical model. We first consider the PSK scenario, and demonstrate that the extra processing compatibly ensures the high toxin expression upon complete plasmid loss, without inducing toxin expression upon acquisition of a plasmid or decrease of plasmid number to a non-zero number. We further show that the extra processing help filtering the transcription noise, avoiding random activation of toxins in transcriptionally regulated TA systems as seen in chromosomal ones. The present model highlights impacts of the slow processing reaction, offering insights on why the slow processing reactions are commonly identified in multiple type-I TA systems.

Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay

ABSTRACTCRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression ofcasgenes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich forPseudomonas aeruginosastrains with new spacer acquisition. We used this assay to demonstrate thatP. aeruginosarapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for theP. aeruginosatype I-F system, thecas1gene is required for CRISPR adaptation,recGcontributes to (but is not required for) primed CRISPR adaptation,recDis dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer.IMPORTANCEOur understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants ofEscherichia coli, orcas1-cas2overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allowsP. aeruginosato rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system ofP. aeruginosa. Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that manyP. aeruginosastrains maintain a CRISPR-Cas system.