The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

The expression of virulence genes increases membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium.

Virulence gene expression can represent a substantial fitness cost to pathogenic bacteria. In the model entero-pathogen Salmonella Typhimurium (S.Tm), such cost favors emergence of attenuated variants during infections that harbor mutations in transcriptional activators of virulence genes (e.g., hilD and hilC). Therefore, understanding the cost of virulence and how it relates to virulence regulation could allow the identification and modulation of ecological factors to drive the evolution of S.Tm toward attenuation. In this study, investigations of membrane status and stress resistance demonstrate that the wild-type (WT) expression level of virulence factors embedded in the envelope increases membrane permeability and sensitizes S.Tm to membrane stress. This is independent from a previously described growth defect associated with virulence gene expression in S.Tm. Pretreating the bacteria with sublethal stress inhibited virulence expression and increased stress resistance. This trade-off between virulence and stress resistance could explain the repression of virulence expression in response to harsh environments in S.Tm. Moreover, we show that virulence-associated stress sensitivity is a burden during infection in mice, contributing to the inherent instability of S.Tm virulence. As most bacterial pathogens critically rely on deploying virulence factors in their membrane, our findings could have a broad impact toward the development of antivirulence strategies.

Impact of horizontal gene transfer on emergence and stability of cooperative virulence in Salmonella Typhimurium.

Intestinal inflammation fuels the transmission of Salmonella Typhimurium (S.Tm). However, a substantial fitness cost is associated with virulence expression. Mutations inactivating transcriptional virulence regulators generate attenuated variants profiting from inflammation without enduring virulence cost. Such variants interfere with the transmission of fully virulent clones. Horizontal transfer of functional regulatory genes (HGT) into attenuated variants could nevertheless favor virulence evolution. To address this hypothesis, we cloned hilD, coding for the master regulator of virulence, into a conjugative plasmid that is highly transferrable during intestinal colonization. The resulting mobile hilD allele allows virulence to emerge from avirulent populations, and to be restored in attenuated mutants competing against virulent clones within-host. However, mutations inactivating the mobile hilD allele quickly arise. The stability of virulence mediated by HGT is strongly limited by its cost, which depends on the hilD expression level, and by the timing of transmission. We conclude that robust evolution of costly virulence expression requires additional selective forces such as narrow population bottlenecks during transmission.

Component Parts of Bacteriophage Virions Accurately Defined by a Machine-Learning Approach Built on Evolutionary Features.

Antimicrobial resistance (AMR) continues to evolve as a major threat to human health, and new strategies are required for the treatment of AMR infections. Bacteriophages (phages) that kill bacterial pathogens are being identified for use in phage therapies, with the intention to apply these bactericidal viruses directly into the infection sites in bespoke phage cocktails. Despite the great unsampled phage diversity for this purpose, an issue hampering the roll out of phage therapy is the poor quality annotation of many of the phage genomes, particularly for those from infrequently sampled environmental sources. We developed a computational tool called STEP3 to use the "evolutionary features" that can be recognized in genome sequences of diverse phages. These features, when integrated into an ensemble framework, achieved a stable and robust prediction performance when benchmarked against other prediction tools using phages from diverse sources. Validation of the prediction accuracy of STEP3 was conducted with high-resolution mass spectrometry analysis of two novel phages, isolated from a watercourse in the Southern Hemisphere. STEP3 provides a robust computational approach to distinguish specific and universal features in phages to improve the quality of phage cocktails and is available for use at http://step3.erc.monash.edu/. IMPORTANCE In response to the global problem of antimicrobial resistance, there are moves to use bacteriophages (phages) as therapeutic agents. Selecting which phages will be effective therapeutics relies on interpreting features contributing to shelf-life and applicability to diagnosed infections. However, the protein components of the phage virions that dictate these properties vary so much in sequence that best estimates suggest failure to recognize up to 90% of them. We have utilized this diversity in evolutionary features as an advantage, to apply machine learning for prediction accuracy for diverse components in phage virions. We benchmark this new tool showing the accurate recognition and evaluation of phage component parts using genome sequence data of phages from undersampled environments, where the richest diversity of phage still lies.

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

Forensic genomics of a novel Klebsiella quasipneumoniae type from a neonatal intensive care unit in China reveals patterns of colonization, evolution and epidemiology.

During March 2017, a neonatal patient with severe diarrhoea subsequently developed septicaemia and died, with Klebsiella isolated as the causative microorganism. In keeping with infection control protocols, the coincident illness of an attending staff member and three other neonates with Klebsiella infection triggered an outbreak response, leading to microbiological assessment of isolates collected from the staff member and all 21 co-housed neonates. Multilocus sequence typing and genomic sequencing identified that the isolates from the 21 neonates were of a new Klebsiella sequence type, ST2727, and taxonomically belonged to K. quasipneumoniae subsp. similipneumoniae (formerly referred to as KpIIB). Genomic characterization showed that the isolated ST2727 strains had diverged from other K. quasipneumoniae subsp. similipneumoniae strains at least 90 years ago, whereas the neonatal samples were highly similar with a genomic divergence of 3.6 months. There was no relationship to the Klebsiella isolate from the staff member. This demonstrates that no transmission occurred from staff to patient or between patients. Rather, the data suggest that ST2727 colonized each neonate from a common hospital source. Sequence-based analysis of the genomes revealed several genes for antimicrobial resistance and some virulence features, but suggest that ST2727 is neither extremely-drug resistant nor hypervirulent. Our results highlight the clinical significance and genomic properties of ST2727 and urge genome-based measures be implemented for diagnostics and surveillance within hospital environments. Additionally, the present study demonstrates the need to scale the power of genomic analysis in retrospective studies where relatively few samples are available.

Dynamic Epidemiology and Virulence Characteristics of Carbapenem-Resistant Klebsiella pneumoniae in Wenzhou, China from 2003 to 2016.

PURPOSE: To investigate transitions in resistance mechanisms, virulence characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) during 2003-2016 in a major Eastern Chinese medical center. PATIENTS AND METHODS: From a total of 2299 K. pneumoniae clinical strains collected from 2003 to 2016, 214 were found to be CRKP isolates and were selected for further study. Characterization of these was conducted by molecular detection of antibiotic resistance markers and virulence determinants, modified carbapenem inactivation method and multilocus sequence typing (MLST). RESULTS: In this study, the prevalence of CRKP was increasing over the 14-year period, mirroring a national trend. These CRKP strains were resistant to most of the tested, clinically relevant drugs. The majority of these CRKP strains were positive for carbapenemases, with the Klebsiella pneumoniae carbapenemase (KPC) found to be the dominant type (207/210, 98.6%). The carrier rates of virulence genes uge, entB, fimH, mrkD and ureA increased in 2016, while the ybtA, iucA and irp2 showed a relatively constant trend. From MLST data, ST11 (88.8%, 190/214) was the preponderant sequence type (ST), followed by ST15 (1.9%, 4/214) and ST656 (1.4%, 3/214). Several strains with less common STs (ST690, ST895, ST1823 and ST1384) were also detected, and these too showed high levels of antimicrobial resistance. CONCLUSION: The average national rise in CRKP across China is mirrored in this in-depth analysis of a single hospital, while the prevalence of hypervirulent CRKP (such as ST15) was relatively low as of 2016. Continuous monitoring is necessary to keep track of CRKP and should include the prospect of newly emerging strains with less common STs and the prospect of detecting carbapenem-resistant, carbapenemase-negative Klebsiella pneumoniae.

Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in Klebsiella pneumoniae.

In Gram-negative bacteria, the permeability of the outer membrane governs rates of antibiotic uptake and thus the efficacy of antimicrobial treatment. Hydrophilic drugs like ß-lactam antibiotics depend on diffusion through pore-forming outer membrane proteins to reach their intracellular targets. In this study, we investigated the distribution of porin genes in more than 2,700 Klebsiella isolates and found a widespread loss of OmpK35 functionality, particularly in those strains isolated from clinical environments. Using a defined set of outer-membrane-remodeled mutants, the major porin OmpK35 was shown to be largely responsible for ß-lactam permeation. Sequence similarity network analysis characterized the porin protein subfamilies and led to discovery of a new porin family member, OmpK38. Structure-based comparisons of OmpK35, OmpK36, OmpK37, OmpK38, and PhoE showed near-identical pore frameworks but defining differences in the sequence characteristics of the extracellular loops. Antibiotic sensitivity profiles of isogenic Klebsiella pneumoniae strains, each expressing a different porin as its dominant pore, revealed striking differences in the antibiotic permeability characteristics of each channel in a physiological context. Since K. pneumoniae is a nosocomial pathogen with high rates of antimicrobial resistance and concurrent mortality, these experiments elucidate the role of porins in conferring specific drug-resistant phenotypes in a global context, informing future research to combat antimicrobial resistance in K. pneumoniaeIMPORTANCEKlebsiella pneumoniae is a pathogen of humans with high rates of mortality and a recognized global rise in incidence of carbapenem-resistant K. pneumoniae (CRKP). The outer membrane of K. pneumoniae forms a permeability barrier that modulates the ability of antibiotics to reach their intracellular target. OmpK35, OmpK36, OmpK37, OmpK38, PhoE, and OmpK26 are porins in the outer membrane of K. pneumoniae, demonstrated here to have a causative relationship to drug resistance phenotypes in a physiological context. The data highlight that currently trialed combination treatments with a carbapenem and ß-lactamase inhibitors could be effective on porin-deficient K. pneumoniae Together with structural data, the results reveal the role of outer membrane proteome remodeling in antimicrobial resistance of K. pneumoniae and point to the role of extracellular loops, not channel parameters, in drug permeation. This significant finding warrants care in the development of phage therapies for K. pneumoniae infections, given the way porin expression will be modulated to confer phage-resistant-and collateral drug-resistant-phenotypes in K. pneumoniae.

An Outbreak of Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae in an Intensive Care Unit of a Major Teaching Hospital in Wenzhou, China.

Carbapenem-resistant, hypervirulent Klebsiella pneumoniae (CR-hvKP) has recently emerged as a significant threat to public health. In this study, 29 K. pneumoniae isolates were isolated from eight patients admitted to the intensive care unit (ICU) of a comprehensive teaching hospital located in China from March 2017 to January 2018. Clinical information of patients was the basis for the further analyses of the isolates including antimicrobial susceptibility tests, identification of antibiotic resistance and virulence gene determinants, multilocus sequence typing (MLST), XbaI-macrorestriction by pulsed-field gel electrophoresis (PFGE). Selected isolates representing distinct resistance profiles and virulence phenotypes were screened for hypervirulence in a Galleria mellonella larvae infection model. In the course of the outbreak, the overall mortality rate of patients was 100% (n = 8) attributed to complications arising from CR-hvKP infections. All isolates except one (28/29, 96.6%) were resistant to multiple antimicrobial agents, and harbored diverse resistance determinants that included the globally prevalent carbapenemase bla KPC-2. Most isolates had hypervirulent genotypes being positive for 19 virulence-associated genes, including iutA (25/29, 86.2%), rmpA (27/29, 93.1%), ybtA (27/29, 93.1%), entB (29/29, 100%), fimH (29/29, 100%), and mrkD (29/29, 100%). MLST revealed ST11 for the majority of isolates (26/29, 89,7%). Infection assays demonstrated high mortality in the Galleria mellonella model with the highest LD50 values for three isolates (<105 CFU/mL) demonstrating the degree of hypervirulence of these CR-hvKP isolates, and is discussed relative to previous outbreaks of CR-hvKP.

The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis.

Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.

Bastion6: a bioinformatics approach for accurate prediction of type VI secreted effectors.

Motivation: Many Gram-negative bacteria use type VI secretion systems (T6SS) to export effector proteins into adjacent target cells. These secreted effectors (T6SEs) play vital roles in the competitive survival in bacterial populations, as well as pathogenesis of bacteria. Although various computational analyses have been previously applied to identify effectors secreted by certain bacterial species, there is no universal method available to accurately predict T6SS effector proteins from the growing tide of bacterial genome sequence data. Results: We extracted a wide range of features from T6SE protein sequences and comprehensively analyzed the prediction performance of these features through unsupervised and supervised learning. By integrating these features, we subsequently developed a two-layer SVM-based ensemble model with fine-grain optimized parameters, to identify potential T6SEs. We further validated the predictive model using an independent dataset, which showed that the proposed model achieved an impressive performance in terms of ACC (0.943), F-value (0.946), MCC (0.892) and AUC (0.976). To demonstrate applicability, we employed this method to correctly identify two very recently validated T6SE proteins, which represent challenging prediction targets because they significantly differed from previously known T6SEs in terms of their sequence similarity and cellular function. Furthermore, a genome-wide prediction across 12 bacterial species, involving in total 54 212 protein sequences, was carried out to distinguish 94 putative T6SE candidates. We envisage both this information and our publicly accessible web server will facilitate future discoveries of novel T6SEs. Availability and implementation: http://bastion6.erc.monash.edu/. Supplementary information: Supplementary data are available at Bioinformatics online.

3'-NADP and 3'-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1.

An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), are substantially different from the ubiquitous co-enzyme 2'-NADP and the calcium mobilizer 2'-NAADP. Interestingly, 3'-NADP and 3'-NAADP have previously been used as inhibitors or signaling molecules but were regarded as "artificial" compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3'-phosphorylated NAD derivatives.

Type II toxin: antitoxin systems. More than small selfish entities?

Toxin-antitoxin (TA) modules regulate metabolism and viability of bacteria and archaea. In type II TA systems these functions are generally thought to be performed by two small proteins. However, evidence is increasing that the toxins are much more diverse and can form multi-domain proteins. Recently, we published a novel type II TA system in which toxin and antitoxin are covalently linked into a single polypeptide chain. In this review we summarize the current knowledge on these elongated toxin homologs and provide perspectives for future study.

A cis-acting antitoxin domain within the chromosomal toxin-antitoxin module EzeT of Escherichia coli quenches toxin activity.

Toxin-antitoxin (TA) systems are widespread genetic modules in the genomes of bacteria and archaea emerging as key players that modulate bacterial physiology. They consist of two parts, a toxic component that blocks an essential cellular process and an antitoxin that inhibits this toxic activity during normal growth. According to the nature of the antitoxin and the mode of inhibition, TA systems are subdivided into different types. Here, we describe the characterization of a type II-like TA system in Escherichia coli called EzeT. While in conventional type II systems the antitoxin is expressed in trans to form an inactive protein-protein complex, EzeT consists of two domains combining toxin and cis-acting antitoxin functionalities in a single polypeptide chain. We show that the C-terminal domain of EzeT is homologous to zeta toxins and is toxic in vivo. The lytic phenotype could be attributed to UDP-N-acetylglucosamine phosphorylation, so far only described for type II epsilon/zeta systems from Gram-positive streptococci. Presence of the N-terminal domain inhibits toxicity in vivo and strongly attenuates kinase activity. Autoinhibition by a cis-acting antitoxin as described here for EzeT-type TA systems can explain the occurrence of single or unusually large toxins, further expanding our understanding of the TA system network.

Single mimivirus particles intercepted and imaged with an X-ray laser.

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

Nucleosome accessibility governed by the dimer/tetramer interface.

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A-H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3-H4)(2) tetramer/(H2A-H2B) dimer interface, followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)(2) tetramer removal. We estimate that the open intermediate state is populated at 0.2-3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery.