The intersection of capsule gene expression, hypermucoviscosity and hypervirulence in Klebsiella pneumoniae.
Walker Kimberly A,Miller Virginia L
Current opinion in microbiology
For ∼30 years, two distinct groups of clinical isolates of Klebsiella pneumoniae have been recognized. Classical strains (cKp) are typically isolated from patients with some degree of immunocompromise and are not virulent in mouse models of infection whereas hypervirulent strains (hvKp) are associated with community acquired invasive infections and are highly virulent in mouse models of infection. Hyperproduction of capsule and a hypermucoviscous colony phenotype have been strongly associated with the hypervirulence of hvKp strains. Recent studies have begun to elucidate the relationship between capsule gene expression, hypermucoviscosity and hypervirulence. Additionally, genes associated with hyperproduction of capsule and hypermucoviscosity in hvKp strains have been identified in a few cKp isolates. However, it is not clear how the acquisition of these genes impacts the virulence of cKp isolates. A better understanding of the potential risks of these strains is particularly important given that many of them are resistant to multiple antibiotics, including carbapenems.
Pathogenesis of Gram-Negative Bacteremia.
Holmes Caitlyn L,Anderson Mark T,Mobley Harry L T,Bachman Michael A
Clinical microbiology reviews
Gram-negative bacteremia is a devastating public health threat, with high mortality in vulnerable populations and significant costs to the global economy. Concerningly, rates of both Gram-negative bacteremia and antimicrobial resistance in the causative species are increasing. Gram-negative bacteremia develops in three phases. First, bacteria invade or colonize initial sites of infection. Second, bacteria overcome host barriers, such as immune responses, and disseminate from initial body sites to the bloodstream. Third, bacteria adapt to survive in the blood and blood-filtering organs. To develop new therapies, it is critical to define species-specific and multispecies fitness factors required for bacteremia in model systems that are relevant to human infection. A small subset of species is responsible for the majority of Gram-negative bacteremia cases, including , , , and The few bacteremia fitness factors identified in these prominent Gram-negative species demonstrate shared and unique pathogenic mechanisms at each phase of bacteremia progression. Capsule production, adhesins, and metabolic flexibility are common mediators, whereas only some species utilize toxins. This review provides an overview of Gram-negative bacteremia, compares animal models for bacteremia, and discusses prevalent Gram-negative bacteremia species.
Innate Host Defense against Klebsiella pneumoniae and the Outlook for Development of Immunotherapies.
Journal of innate immunity
Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative commensal bacterium and opportunistic pathogen. In healthy individuals, the innate immune system is adept at protecting against K. pneumoniae infection. Notably, the serum complement system and phagocytic leukocytes (e.g., neutrophils) are highly effective at eliminating K. pneumoniae and thereby preventing severe disease. On the other hand, the microbe is a major cause of healthcare-associated infections, especially in individuals with underlying susceptibility factors, such as pre-existing severe illness or immune suppression. The burden of K. pneumoniae infections in hospitals is compounded by antibiotic resistance. Treatment of these infections is often difficult largely because the microbes are usually resistant to multiple antibiotics (multidrug resistant [MDR]). There are a limited number of treatment options for these infections and new therapies, and preventative measures are needed. Here, we review host defense against K. pneumoniae and discuss recent therapeutic measures and vaccine approaches directed to treat and prevent severe disease caused by MDR K. pneumoniae.
Impact of bacterial persisters on their host.
Moldoveanu Ana L,Rycroft Julian A,Helaine Sophie
Current opinion in microbiology
The rise of antibiotic failure poses a severe threat to global health. There is growing concern that this failure is not solely driven by stable antibiotic resistance but also by a subpopulation of transiently non-growing, antibiotic tolerant bacteria. These 'persisters' have been proposed to seed relapsing infections, an important clinical outcome of treatment failure - although definitive evidence for this direct link remains elusive. Recent advances in the field have revealed the complex nature of intra-host persisters which drive their high adaptability through biosynthetic activity. These features of persisters contribute to evolution of antimicrobial resistance and modulation of host immune responses, despite clinically efficacious treatment.
Campylobacter jejuni transcriptional and genetic adaptation during human infection.
Crofts Alexander A,Poly Frédéric M,Ewing Cheryl P,Kuroiwa Janelle M,Rimmer Joanna E,Harro Clayton,Sack David,Talaat Kawsar R,Porter Chad K,Gutierrez Ramiro L,DeNearing Barbara,Brubaker Jessica,Laird Renée M,Maue Alexander C,Jaep Kayla,Alcala Ashley,Tribble David R,Riddle Mark S,Ramakrishnan Amritha,McCoy Andrea J,Davies Bryan W,Guerry Patricia,Trent M Stephen
Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.
Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-Negatives: the Klebsiella pneumoniae Paradigm.
Ramirez Maria S,Traglia German M,Lin David L,Tran Tung,Tolmasky Marcelo E
Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment. Together with other genetic elements such as integrons and transposons, and using a variety of mechanisms, plasmids participate in the dissemination of these traits, resulting in the virtual elimination of barriers among different kinds of bacteria. In this article we review the current information about the physiology of plasmids and their role in virulence and antibiotic resistance from the Gram-negative opportunistic pathogen Klebsiella pneumoniae. This bacterium has acquired multidrug resistance and is the causative agent of serious community- and hospital-acquired infections. It is also included in the recently defined ESKAPE group of bacteria that cause most U.S. hospital infections.
Klebsiella pneumoniae infection biology: living to counteract host defences.
Bengoechea José A,Sa Pessoa Joana
FEMS microbiology reviews
Klebsiella species cause a wide range of diseases including pneumonia, urinary tract infections (UTIs), bloodstream infections and sepsis. These infections are particularly a problem among neonates, elderly and immunocompromised individuals. Klebsiella is also responsible for a significant number of community-acquired infections. A defining feature of these infections is their morbidity and mortality, and the Klebsiella strains associated with them are considered hypervirulent. The increasing isolation of multidrug-resistant strains has significantly narrowed, or in some settings completely removed, the therapeutic options for the treatment of Klebsiella infections. Not surprisingly, this pathogen has then been singled out as an 'urgent threat to human health' by several organisations. This review summarises the tremendous progress that has been made to uncover the sophisticated immune evasion strategies of K. pneumoniae. The co-evolution of Klebsiella in response to the challenge of an activated immune has made Klebsiella a formidable pathogen exploiting stealth strategies and actively suppressing innate immune defences to overcome host responses to survive in the tissues. A better understanding of Klebsiella immune evasion strategies in the context of the host-pathogen interactions is pivotal to develop new therapeutics, which can be based on antagonising the anti-immune strategies of this pathogen.
Targeting the Sugary Armor of Species.
Patro L Ponoop Prasad,Rathinavelan Thenmalarchelvi
Frontiers in cellular and infection microbiology
The emergence of multidrug-resistant strains of Gram-negative species is an urgent global threat. The World Health Organization has listed as one of the global priority pathogens in critical need of next-generation antibiotics. Compared to other Gram-negative pathogens, accumulates a greater diversity of antimicrobial-resistant genes at a higher frequency. The evolution of a hypervirulent phenotype of is yet another concern. It has a broad ecological distribution affecting humans, agricultural animals, plants, and aquatic animals. Extracellular polysaccharides of , such as lipopolysaccharides, capsular polysaccharides, and exopolysaccharides, play crucial roles in conferring resistance against the host immune response, as well as in colonization, surface adhesion, and for protection against antibiotics and bacteriophages. These extracellular polysaccharides are major virulent determinants and are highly divergent with respect to their antigenic properties. Wzx/Wzy-, ABC-, and synthase-dependent proteinaceous nano-machineries are involved in the biosynthesis, transport, and cell surface expression of these sugar molecules. Although the proteins involved in the biosynthesis and surface expression of these sugar molecules represent potential drug targets, variation in the amino acid sequences of some of these proteins, in combination with diversity in their sugar composition, poses a major challenge to the design of a universal drug for infections. This review discusses the challenges in universal vaccine and drug development from the perspective of antigen sugar compositions and the proteins involved in extracellular antigen transport.
Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study.
Cuénod Aline,Wüthrich Daniel,Seth-Smith Helena M B,Ott Chantal,Gehringer Christian,Foucault Frédéric,Mouchet Roxanne,Kassim Ali,Revathi Gunturu,Vogt Deborah R,von Felten Stefanie,Bassetti Stefano,Tschudin-Sutter Sarah,Hettich Timm,Schlotterbeck Götz,Homberger Christina,Casanova Carlo,Moran-Gilad Jacob,Sagi Orli,Rodríguez-Sánchez Belén,Müller Franco,Aerni Martina,Gaia Valeria,van Dessel Helke,Kampinga Greetje A,Müller Claudia,Daubenberger Claudia,Pflüger Valentin,Egli Adrian
BACKGROUND:Klebsiella spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new Klebsiella species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore Klebsiella group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence. METHODS:We analysed whole-genome sequence data of a diverse selection of Klebsiella spp. isolates and identified resistance and virulence factors. Using the genomes of 3594 Klebsiella isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from Klebsiella species were performed using generalized linear mixed-effects models. RESULTS:Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight Klebsiella species can be distinguished using MALDI-TOF MS. We identified K. pneumoniae (71.2%; n = 12,523), K. quasipneumoniae (3.3%; n = 575), K. variicola (9.8%; n = 1717), "K. quasivariicola" (0.3%; n = 52), K. oxytoca (8.2%; n = 1445), K. michiganensis (4.8%; n = 836), K. grimontii (2.4%; n = 425) and K. huaxensis (0.1%; n = 12). Isolates belonging to the K. oxytoca group, which includes the species K. oxytoca, K. michiganensis and K. grimontii, were less often resistant to 4th-generation cephalosporins than isolates of the K. pneumoniae group, which includes the species K. pneumoniae, K. quasipneumoniae, K. variicola and "K. quasivariicola" (odds ratio = 0.17, p < 0.001, 95% confidence interval [0.09,0.28]). Within the K. pneumoniae group, isolates identified as K. pneumoniae were more often resistant to 4th-generation cephalosporins than K. variicola isolates (odds ratio = 2.61, p = 0.003, 95% confidence interval [1.38,5.06]). K. oxytoca group isolates were found to be more likely associated with invasive infection to primary sterile sites than K. pneumoniae group isolates (odds ratio = 2.39, p = 0.0044, 95% confidence interval [1.05,5.53]). CONCLUSIONS:Currently misdiagnosed Klebsiella spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS. Klebsiella groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options.
Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae.
Xu Yanping,Zhang Jianfeng,Wang Meng,Liu Meng,Liu Guitian,Qu Hongping,Liu Jialin,Deng Zixin,Sun Jingyong,Ou Hong-Yu,Qu Jieming
BACKGROUND:Klebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear. METHODS:A total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids. RESULTS:A nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site. CONCLUSIONS:The nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.
Concurrent Host-Pathogen Transcriptional Responses in a Murine Myonecrosis Infection.
Low Lee-Yean,Harrison Paul F,Gould Jodee,Powell David R,Choo Jocelyn M,Forster Samuel C,Chapman Ross,Gearing Linden J,Cheung Jackie K,Hertzog Paul,Rood Julian I
To obtain an insight into host-pathogen interactions in clostridial myonecrosis, we carried out comparative transcriptome analysis of both the bacterium and the host in a murine infection model, which is the first time that such an investigation has been conducted. Analysis of the host transcriptome from infected muscle tissues indicated that many genes were upregulated compared to the results seen with mock-infected mice. These genes were enriched for host defense pathways, including Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling components. Real-time PCR confirmed that host TLR2 and NLRP3 inflammasome genes were induced in response to infection. Comparison of the transcriptome of cells from the infected tissues with that from broth cultures showed that host selective pressure induced a global change in gene expression. A total of 33% (923) of genes were differentially regulated, including 10 potential virulence genes that were upregulated relative to their expression These genes encoded putative proteins that may be involved in the synthesis of cell wall-associated macromolecules, in adhesion to host cells, or in protection from host cationic antimicrobial peptides. This report presents the first successful expression profiling of coregulated transcriptomes of bacterial and host genes during a clostridial myonecrosis infection and provides new insights into disease pathogenesis and host-pathogen interactions. is the causative agent of traumatic clostridial myonecrosis, or gas gangrene. In this study, we carried out transcriptional analysis of both the host and the bacterial pathogen in a mouse myonecrosis infection. The results showed that in comparison to mock-infected control tissues, muscle tissues from -infected mice had a significantly altered gene expression profile. In particular, the expression of many genes involved in the innate immune system was upregulated. Comparison of the expression profiles of cells isolated from the infected tissues with those from equivalent broth cultures identified many potential virulence genes that were significantly upregulated These studies have provided a new understanding of the range of factors involved in host-pathogen interactions in a myonecrosis infection.
An in vivo atlas of host-pathogen transcriptomes during colonization and disease.
D'Mello Adonis,Riegler Ashleigh N,Martínez Eriel,Beno Sarah M,Ricketts Tiffany D,Foxman Ellen F,Orihuela Carlos J,Tettelin Hervé
Proceedings of the National Academy of Sciences of the United States of America
() colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three strains. We identified genes highly expressed at all anatomical sites and in an organ-specific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of and identify therapeutic targets.
Analysis of host-pathogen gene association networks reveals patient-specific response to streptococcal and polymicrobial necrotising soft tissue infections.
BACKGROUND:Necrotising soft tissue infections (NSTIs) are rapidly progressing bacterial infections usually caused by either several pathogens in unison (polymicrobial infections) or Streptococcus pyogenes (mono-microbial infection). These infections are rare and are associated with high mortality rates. However, the underlying pathogenic mechanisms in this heterogeneous group remain elusive. METHODS:In this study, we built interactomes at both the population and individual levels consisting of host-pathogen interactions inferred from dual RNA-Seq gene transcriptomic profiles of the biopsies from NSTI patients. RESULTS:NSTI type-specific responses in the host were uncovered. The S. pyogenes mono-microbial subnetwork was enriched with host genes annotated with involved in cytokine production and regulation of response to stress. The polymicrobial network consisted of several significant associations between different species (S. pyogenes, Porphyromonas asaccharolytica and Escherichia coli) and host genes. The host genes associated with S. pyogenes in this subnetwork were characterised by cellular response to cytokines. We further found several virulence factors including hyaluronan synthase, Sic1, Isp, SagF, SagG, ScfAB-operon, Fba and genes upstream and downstream of EndoS along with bacterial housekeeping genes interacting with the human stress and immune response in various subnetworks between host and pathogen. CONCLUSIONS:At the population level, we found aetiology-dependent responses showing the potential modes of entry and immune evasion strategies employed by S. pyogenes, congruent with general cellular processes such as differentiation and proliferation. After stratifying the patients based on the subject-specific networks to study the patient-specific response, we observed different patient groups with different collagens, cytoskeleton and actin monomers in association with virulence factors, immunogenic proteins and housekeeping genes which we utilised to postulate differing modes of entry and immune evasion for different bacteria in relationship to the patients' phenotype.
Molecular interaction between methicillin-resistant Staphylococcus aureus (MRSA) and chicken breast reveals enhancement of pathogenesis and toxicity for food-borne outbreak.
Chung Han Young,Kim You-Tae,Kwon Joon-Gi,Im Han Hyeok,Ko Duhyun,Lee Ju-Hoon,Choi Sang Ho
To study pathogenesis and toxicity of Staphylococcus aureus in foods, FORC_062 was isolated from a human blood sample and complete genome sequence has a type II SCCmec gene cluster and a type II toxin-antitoxin system, indicating an MRSA strain. Its mobile gene elements has many pathogenic genes involved in host infection, biofilm formation, and various enterotoxin and hemolysin genes. Clinical MRSA is often found in animal foods and ingestion of MRSA-contaminated foods causes human infection. Therefore, it is very important to understand the role of contaminated foods. To elucidate the interaction between clinical MRSA FORC_062 and raw chicken breast, transcriptome analysis was conducted, showing that gene expressions of amino acid biosynthesis and metabolism were specifically down-regulated, suggesting that the strain may import and utilize amino acids from the chicken breast, but not able to synthesize them. However, toxin gene expressions were up-regulated, suggesting that human infection of S. aureus via contaminated food may be more fatal. In addition, the contaminated foods enhance multiple-antibiotic resistance activities and virulence factors in this clinical MRSA. Consequently, MRSA-contaminated food may play a role as a nutritional reservoir as well as in enhancing factor for pathogenesis and toxicity of clinical MRSA for severe food-borne outbreaks.
Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria.
Imdahl Fabian,Vafadarnejad Ehsan,Homberger Christina,Saliba Antoine-Emmanuel,Vogel Jörg
Bacteria respond to changes in their environment with specific transcriptional programmes, but even within genetically identical populations these programmes are not homogenously expressed. Such transcriptional heterogeneity between individual bacteria allows genetically clonal communities to develop a complex array of phenotypes, examples of which include persisters that resist antibiotic treatment and metabolically specialized cells that emerge under nutrient-limiting conditions. Fluorescent reporter constructs have played a pivotal role in deciphering heterogeneous gene expression within bacterial populations but have been limited to recording the activity of single genes in a few genetically tractable model species, whereas the vast majority of bacteria remain difficult to engineer and/or even to cultivate. Single-cell transcriptomics is revolutionizing the analysis of phenotypic cell-to-cell variation in eukaryotes, but technical hurdles have prevented its robust application to prokaryotes. Here, using an improved poly(A)-independent single-cell RNA-sequencing protocol, we report the faithful capture of growth-dependent gene expression patterns in individual Salmonella and Pseudomonas bacteria across all RNA classes and genomic regions. These transcriptomes provide important reference points for single-cell RNA-sequencing of other bacterial species, mixed microbial communities and host-pathogen interactions.
Analysis of the Transcriptome of Bordetella pertussis during Infection of Mice.
Wong Ting Y,Hall Jesse M,Nowak Evan S,Boehm Dylan T,Gonyar Laura A,Hewlett Erik L,Eby Joshua C,Barbier Mariette,Damron F Heath
causes the disease whooping cough through coordinated control of virulence factors by the virulence gene system. Microarrays and, more recently, RNA sequencing (RNA-seq) have been used to describe gene expression profiles of and other pathogens. In previous studies, we have analyzed the gene expression profiles of , and we hypothesize that the infection transcriptome profile is significantly different from that under laboratory growth conditions. To study the infection transcriptome of , we developed a simple filtration technique for isolation of bacteria from infected lungs. The work flow involves filtering the bacteria out of the lung homogenate using a 5-μm-pore-size syringe filter. The captured bacteria are then lysed to isolate RNA for Illumina library preparation and RNA-seq analysis. Upon comparing the and gene expression profiles, we identified 351 and 255 genes as activated and repressed, respectively, during murine lung infection. As expected, numerous genes associated with virulent-phase growth were activated in the murine host, including pertussis toxin (PT), the PT secretion apparatus, and the type III secretion system. A significant number of genes encoding iron acquisition and heme uptake proteins were highly expressed during infection, supporting iron acquisition as critical for survival Numerous metabolic genes were repressed during infection. Overall, these data shed light on the gene expression profile of during infection, and this method will facilitate efforts to understand how this pathogen causes infection. growth conditions for bacteria do not fully recapitulate the host environment. RNA sequencing transcriptome analysis allows for the characterization of the infection gene expression profiles of pathogens in complex environments. Isolation of the pathogen from infected tissues is critical because of the large amounts of host RNA present in crude lysates of infected organs. A filtration method was developed that enabled enrichment of the pathogen RNA for RNA-seq analysis. The resulting data describe the "infection transcriptome" of in the murine lung. This strategy can be utilized for pathogens in other hosts and, thus, expand our knowledge of what bacteria express during infection.
Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation.
Schmühl Carina,Beckstette Michael,Heroven Ann Kathrin,Bunk Boyke,Spröer Cathrin,McNally Alan,Overmann Jörg,Dersch Petra
Yersinia enterocolitica is a zoonotic pathogen and an important cause of bacterial gastrointestinal infections in humans. Large-scale population genomic analyses revealed genetic and phenotypic diversity of this bacterial species, but little is known about the differences in the transcriptome organization, small RNA (sRNA) repertoire, and transcriptional output. Here, we present the first comparative high-resolution transcriptome analysis of Y. enterocolitica strains representing highly pathogenic phylogroup 2 (serotype O:8) and moderately pathogenic phylogroup 3 (serotype O:3) grown under four infection-relevant conditions. Our transcriptome sequencing (RNA-seq) approach revealed 1,299 and 1,076 transcriptional start sites and identified strain-specific sRNAs that could contribute to differential regulation among the phylogroups. Comparative transcriptomics further uncovered major gene expression differences, in particular, in the temperature-responsive regulon. Multiple virulence-relevant genes are differentially regulated between the two strains, supporting an ecological separation of phylogroups with certain niche-adapted properties. Strong upregulation of the enterotoxin gene in combination with constitutive high expression of cell invasion factor InvA further showed that the toxicity of recent outbreak O:3 strains has increased. Overall, our report provides new insights into the specific transcriptome organization of phylogroups 2 and 3 and reveals gene expression differences contributing to the substantial phenotypic differences that exist between the lineages. Yersinia enterocolitica is a major diarrheal pathogen and is associated with a large range of gut-associated diseases. Members of this species have evolved into different phylogroups with genotypic variations. We performed the first characterization of the Y. enterocolitica transcriptional landscape and tracked the consequences of the genomic variations between two different pathogenic phylogroups by comparing their RNA repertoire, promoter usage, and expression profiles under four different virulence-relevant conditions. Our analysis revealed major differences in the transcriptional outputs of the closely related strains, pointing to an ecological separation in which one is more adapted to an environmental lifestyle and the other to a mostly mammal-associated lifestyle. Moreover, a variety of pathoadaptive alterations, including alterations in acid resistance genes, colonization factors, and toxins, were identified which affect virulence and host specificity. This illustrates that comparative transcriptomics is an excellent approach to discover differences in the functional output from closely related genomes affecting niche adaptation and virulence, which cannot be directly inferred from DNA sequences.
The Burkholderia pseudomallei intracellular 'TRANSITome'.
Heacock-Kang Yun,McMillan Ian A,Norris Michael H,Sun Zhenxin,Zarzycki-Siek Jan,Bluhm Andrew P,Cabanas Darlene,Norton Robert E,Ketheesan Natkunam,Miller Jeff F,Schweizer Herbert P,Hoang Tung T
Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.
Cross-species RNA-seq for deciphering host-microbe interactions.
Westermann Alexander J,Vogel Jörg
Nature reviews. Genetics
The human body is constantly exposed to microorganisms, which entails manifold interactions between human cells and diverse commensal or pathogenic bacteria. The cellular states of the interacting cells are decisive for the outcome of these encounters such as whether bacterial virulence programmes and host defence or tolerance mechanisms are induced. This Review summarizes how next-generation RNA sequencing (RNA-seq) has become a primary technology to study host-microbe interactions with high resolution, improving our understanding of the physiological consequences and the mechanisms at play. We illustrate how the discriminatory power and sensitivity of RNA-seq helps to dissect increasingly complex cellular interactions in time and space down to the single-cell level. We also outline how future transcriptomics may answer currently open questions in host-microbe interactions and inform treatment schemes for microbial disorders.
The ironclad truth: how in vivo transcriptomics and in vitro mechanistic studies shape our understanding of Neisseria gonorrhoeae gene regulation during mucosal infection.
Moreau Matthew R,Massari Paola,Genco Caroline A
Pathogens and disease
Neisseria gonorrhoeae is one of the most prevalent sexually transmitted infections worldwide. This obligate human pathogen has been extensively studied in vitro, where bacterial factors that are known to contribute to gonococcal disease and their regulation are relatively well defined. However, these in vitro experimental conditions only loosely replicate the host specific environment encountered by the bacteria in vivo. We recently reported on the complete gonococcal transcriptome expressed during natural human mucosal infection using RNA-seq analysis. Gene transcripts expressed in vivo (in vivo expressed factors) included genes encoding antibiotic resistance determinants, and a large number of hypothetical genes. A comparison of the gonococcal transcriptome expressed in vivo with the corresponding strain grown in vitro identified sets of genes regulated by infection, including those regulated by iron and the transcriptional regulatory protein Fur. We highlight here the role of Fur and gonococcal-specific regulatory processes important for infection and pathogenicity. We have determined that the genes controlled by Fur follow the same expression pattern in vivo as described previously in vitro, confirming Fur's regulatory role during infection. Collectively, these studies provide new insights into how bacterial fitness and pathogenicity are modulated during human mucosal infection.
Dual RNA-seq of pathogen and host.
Westermann Alexander J,Gorski Stanislaw A,Vogel Jörg
Nature reviews. Microbiology
A comprehensive understanding of host-pathogen interactions requires a knowledge of the associated gene expression changes in both the pathogen and the host. Traditional, probe-dependent approaches using microarrays or reverse transcription PCR typically require the pathogen and host cells to be physically separated before gene expression analysis. However, the development of the probe-independent RNA sequencing (RNA-seq) approach has begun to revolutionize transcriptomics. Here, we assess the feasibility of taking transcriptomics one step further by performing 'dual RNA-seq', in which gene expression changes in both the pathogen and the host are analysed simultaneously.
Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples.
Kumar Nikhil,Lin Mingqun,Zhao Xuechu,Ott Sandra,Santana-Cruz Ivette,Daugherty Sean,Rikihisa Yasuko,Sadzewicz Lisa,Tallon Luke J,Fraser Claire M,Dunning Hotopp Julie C
Despite numerous advances in genomics and bioinformatics, technological hurdles remain to examine host-microbe transcriptomics. Sometimes the transcriptome of either or both can be ascertained merely by generating more sequencing reads. However, many cases exist where bacterial mRNA needs to be enriched further to enable cost-effective sequencing of the pathogen or endosymbiont. While a suitable method is commercially available for mammalian samples of this type, development of such methods has languished for invertebrate samples. Furthermore, a common method across multiple taxa would facilitate comparisons between bacteria in invertebrate vectors and their vertebrate hosts. Here, a method is described to concurrently remove polyadenylated transcripts, prokaryotic rRNA, and eukaryotic rRNA, including those with low amounts of starting material (e.g. 100 ng). In a Wolbachia-Drosophila system, this bacterial mRNA enrichment yielded a 3-fold increase in Wolbachia mRNA abundance and a concomitant 3.3-fold increase in the percentage of transcripts detected. More specifically, 70% of the genome could be recovered by transcriptome sequencing compared to 21% in the total RNA. Sequencing of similar bacterial mRNA-enriched samples generated from Ehrlichia-infected canine cells covers 93% of the Ehrlichia genome, suggesting ubiquitous transcription across the entire Ehrlichia chaffeensis genome. This technique can potentially be used to enrich bacterial mRNA in many studies of host-microbe interactions.
Analysis of In Vivo Transcriptome of Intracellular Bacterial Pathogen Isolated from Mouse Spleen.
Sun Na,Song Yanying,Liu Cong,Liu Mengda,Yu Lanping,Wang Fangkun
Pathogens (Basel, Switzerland)
() is an important intracellular pathogen that poses a health threat to humans. This study tries to clarify the mechanism of survival and reproduction in the host. In this study, high-throughput sequencing analysis was performed on RNA extracted from the strains isolated from infected mouse spleens and an reference strain (ATCC 14028) based on the BGISEQ-500 platform. A total of 1340 significant differentially expressed genes (DEGs) were screened. Functional annotation revealed DEGs associated with regulation, metabolism, transport and binding, pathogenesis, and motility. Through data mining and literature retrieval, 26 of the 58 upregulated DEGs (FPKM > 10) were not reported to be related to the adaptation to intracellular survival and were classified as candidate key genes (CKGs) for survival and proliferation in vivo. Our data contribute to our understanding of the mechanisms used by to regulate virulence gene expression whilst replicating inside mammalian cells.
In vivo transcriptional profiling of Yersinia pestis reveals a novel bacterial mediator of pulmonary inflammation.
Pechous Roger D,Broberg Christopher A,Stasulli Nikolas M,Miller Virginia L,Goldman William E
UNLABELLED:Inhalation of Yersinia pestis results in primary pneumonic plague, a highly lethal and rapidly progressing necrotizing pneumonia. The disease begins with a period of extensive bacterial replication in the absence of disease symptoms, followed by the sudden onset of inflammatory responses that ultimately prove fatal. Very little is known about the bacterial and host factors that contribute to the rapid biphasic progression of pneumonic plague. In this work, we analyzed the in vivo transcription kinetics of 288 bacterial open reading frames previously shown by microarray analysis to be dynamically regulated in the lung. Using this approach combined with bacterial genetics, we were able to identify five Y. pestis genes that contribute to the development of pneumonic plague. Deletion of one of these genes, ybtX, did not alter bacterial survival but attenuated host inflammatory responses during late-stage disease. Deletion of ybtX in another lethal respiratory pathogen, Klebsiella pneumoniae, also resulted in diminished host inflammation during infection. Thus, our in vivo transcriptional screen has identified an important inflammatory mediator that is common to two Gram-negative bacterial pathogens that cause severe pneumonia. IMPORTANCE:Yersinia pestis is responsible for at least three major pandemics, most notably the Black Death of the Middle Ages. Due to its pandemic potential, ease of dissemination by aerosolization, and a history of its weaponization, Y. pestis is categorized by the Centers for Disease Control and Prevention as a tier 1 select agent most likely to be used as a biological weapon. To date, there is no licensed vaccine against Y. pestis. Importantly, an early "silent" phase followed by the rapid onset of nondescript influenza-like symptoms makes timely treatment of pneumonic plague difficult. A more detailed understanding of the bacterial and host factors that contribute to pathogenesis is essential to understanding the progression of pneumonic plague and developing or enhancing treatment options.
A Plasmid Confers Hypermucoviscosity-Like Phenotype and Alters Capsule Production and Virulence.
Rodríguez-Medina Nadia,Martínez-Romero Esperanza,De la Cruz Miguel Angel,Ares Miguel Angel,Valdovinos-Torres Humberto,Silva-Sánchez Jesús,Lozano-Aguirre Luis,Martínez-Barnetche Jesús,Andrade Veronica,Garza-Ramos Ulises
Frontiers in microbiology
Hypermucoviscosity (hmv) is a capsule-associated phenotype usually linked with hypervirulent strains. The key components of this phenotype are the RmpADC proteins contained in non-transmissible plasmids identified and studied in . is closely related to and recently has been identified as an emergent human pathogen. a normally contains plasmids, some of them carrying antibiotic resistance and virulence genes. Previously, we described a clinical isolate showing an hmv-like phenotype that harbors a 343-kb pKV8917 plasmid. Here, we investigated whether pKV8917 plasmid carried by 8917 is linked with the hmv-like phenotype and its contribution to virulence. We found that curing the 343-kb pKV8917 plasmid caused the loss of hmv, a reduction in capsular polysaccharide ( < 0.001) and virulence. In addition, pKV8917 was successfully transferred to and strains via conjugation. Notably, when pKV8917 was transferred to , the transconjugants displayed an hmv-like phenotype, and capsule production and virulence increased; these phenotypes were not observed in the transconjugants. These data suggest that the pKV8917 plasmid carries novel hmv and capsule determinants. Whole-plasmid sequencing and analysis revealed that pKV8917 does not contain / genes; thus, an alternative mechanism was searched. The 343-kb plasmid contains an IncFIB backbone and shares a region of ∼150 kb with a 99% identity and 49% coverage with a virulence plasmid from hypervirulent and multidrug-resistant . The pKV8917-unique region harbors a cellulose biosynthesis cluster (), fructose- and sucrose-specific () phosphotransferase systems, and the transcriptional regulators and , respectively, involved in membrane permeability. The hmv-like phenotype has been identified more frequently, and recent evidence supports the existence of /-independent hmv-like pathways in this bacterial genus.
Effect of multiple, compatible plasmids on the fitness of the bacterial host by inducing transcriptional changes.
Lee Haejeong,Ko Kwan Soo
The Journal of antimicrobial chemotherapy
OBJECTIVES:Bacteria that acquire plasmids incur a biological cost. Despite this fact, clinical Enterobacteriaceae isolates commonly contain multiple co-existing plasmids harbouring carbapenemase genes. METHODS:Six different plasmids carrying blaNDM-1, blaNDM-5, blaCTX-M-15, blaKPC-2, blaOXA-181 and blaOXA-232 genes were obtained from Klebsiella pneumoniae and Escherichia coli clinical isolates. Using the E. coli DH5α strain as recipient, 14 transconjugants with diverse plasmid combinations (single or double plasmids) were generated. For each of these, the effects of plasmid carriage on the bacterial host were investigated using in vitro and in vivo competition assays; additionally, the effects were investigated in the context of biofilm formation, serum resistance and survival inside macrophages. Transcriptomic changes in single- and double-plasmid recipients were also investigated. RESULTS:Increased in vitro and in vivo competitiveness was observed when two plasmids carrying blaNDM-1 and blaOXA-232 were co-introduced into the host bacteria. However, DH5α::pNDM5 + pOXA232 and other double-plasmid recipients did not show such competitiveness. DH5α::pNDM5 + pOXA181 did not show any fitness cost compared with a plasmid-free host and single-plasmid transconjugants, while both the double-plasmid recipients with pCTXM15 or pKPC2 exhibited a fitness burden. The double-plasmid recipient DH5α::pNDM1 + pOXA232 also exhibited increased biofilm formation, serum resistance and survival inside macrophages. Transcriptomic analysis revealed that the genes of DH5α::pNDM1 + pOXA232 involved in metabolic pathways, transport and stress response were up-regulated, while those involved in translation were down-regulated. CONCLUSIONS:Our study suggests that bacterial strains can gain fitness through the acquisition of multiple plasmids harbouring antibiotic resistance genes, which may be mediated by transcriptomic changes in the chromosomal genes of the bacterial host.
Reduced Virulence and Enhanced Host Adaption during Antibiotics Therapy: a Story of a Within-Host Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 Evolution in a Patient with a Serious Scrotal Abscess.
Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and threatened human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, but how this clone evolves then adapts to the host and facilitates dissemination remains largely unknown. Here, the genomic dynamics of 4 ST11-CRKP isolates, which were sequentially collected from the urine of a patient with initial serious scrotal abscess and finally recovered without effective medication, were analyzed. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4 to 24 mutations, among which 94% to 100% of them were synonymous or intergenic mutations. During 47 days of antibiotics therapy, CRKP underwent adaptive evolution, including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by a deletion within the ribosomal binding site, which has been described by us previously. On the other hand, mutations associated with two genes, acyltransferase () and , resulted in the attenuation phenotype of ST11-CRKP. deficiency reduced the capsular polysaccharide (CPS) production, enhanced biofilm formation, weakened capsular protection, and decreased induction of proinflammatory cytokines. Further RNA-Seq analysis revealed that influenced the expression of , , , and which likely participate in capsular synthesis and biofilm formation. affected the virulence by its overexpression caused by the deletion of the upstream repressor binding site. This study presents a within-host adaption of ST11-CRKP and suggests an important role of CPS in the adaptive evolution of virulence and persistence of CRKP. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated worldwide and can cause life-threatening infections, including pneumonia, bloodstream infections, urinary tract infections, intraabdominal infection, liver abscess, and meningitis. CRKP infection is the leading cause of high mortality in hospitals. The sequence type (ST) 11 CRKP is a dominant clone and accounts for 60% of CRKP infections in China. Recently, the ST11-CRKP with high transmissibility is increasingly identified. Understanding how this clone has evolved is crucial for developing strategies to control its further dissemination. The significance of our research is the identification of the genomic dynamics of ST11-CRKP and the genetic basis for ST11-CRKP that facilitate persistence and dissemination. Furthermore, our study also highlights the importance of monitoring the within-host evolution of pathogens during the treatment and developing interventions to minimize the potential impact of host adaptation on human health.
Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability.
Li Jia,Liu Fei,Wang Qi,Ge Pupu,Woo Patrick C Y,Yan Jinghua,Zhao Yanlin,Gao George F,Liu Cui Hua,Liu Changting
The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens.
Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance.
Wright Meredith S,Suzuki Yo,Jones Marcus B,Marshall Steven H,Rudin Susan D,van Duin David,Kaye Keith,Jacobs Michael R,Bonomo Robert A,Adams Mark D
Antimicrobial agents and chemotherapy
The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Col(r)) is increasingly reported from clinical settings. The genetic mechanisms that lead to Col(r) in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Col(r) clinical isolates. Col(r) was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-L-arabinose (Ara4N) modification of lipid A was found in all Col(r) strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Col(r) mechanisms may be dependent on the genetic background.
The Role of the Two-Component QseBC Signaling System in Biofilm Formation and Virulence of Hypervirulent ATCC43816.
Frontiers in microbiology
Hypervirulent (hvKP) is an evolving infectious pathogen associated with high mortality. The convergence of hypervirulence and multidrug resistance further challenges the clinical treatment options for infections. The QseBC two-component system (TCS) is a component of quorum-sensing regulatory cascade and functions as a global regulator of biofilm growth, bacterial motility, and virulence in . However, the functional mechanisms of QseBC in hvKP have not been reported, and we aim to examine the role of QseBC in regulating virulence in hvKP strain ATCC43816. The CRISPR-Cas9 system was used to construct , , and knockout in ATCC43816. No significant alterations in the growth and antibiotic susceptibility were detected between wild-type and mutants. The deletion of led to an increase of biofilm formation, resistance to serum killing, and high mortality in the model. RNAseq differential gene expression analysis exhibited that gene-associated biofilm formation (), bacterial type VI secretion system (), and biosynthesis of siderophore () were significantly upregulated in comparison with the wild-type control. In addition, , (encode OB-family protein), and AraC family transcriptional regulator IT767_23090 genes showed highest expressions in the absence of QseC, which might be related to increased virulence. The study provided new insights into the functional importance of QseBC in regulating the virulence of hvKP.
Phage resistance mutation triggered by OmpC deficiency in Klebsiella pneumoniae induced limited fitness costs.
Outer membrane proteins (OMPs) play an important role in bacterial fitness costs. Derived from the interaction between Klebsiella pneumoniae K7 and phage GH-K3, K7R is an outer membrane porin-deficient phage-resistant mutant strain triggered by ompC deletion, exhibits expression inhibition of OmpC, OmpN, KPN_02430 and OmpF, but its fitness costs and regulatory mechanism remains unknown. In this study, compared with K7, K7R showed almost unaffected growth rate, slightly decreased virulence, and increased resistance to some antibiotics. Transcriptome analysis showed that the pathways of glycerolipid metabolism and nitrogen metabolism in K7R were significantly inhibited, while the transcription of permeases belonging to ABC transporters tended to be active, nutrient uptakes such as citrate and phenylalanine were also enhanced. However, transcriptional up-regulation in K7R was inhibited by overexpression of OmpC, OmpN, KPN_02430 and OmpF in general. Overexpression of OmpN, KPN_02430 and OmpF, respectively, restoring the sensitivity of strains to antibiotics to varying degrees, while OmpC overexpression aggravated the bacterial drug-resistance especially to β-lactam antibiotics. Besides, unlike OmpC and OmpF, overexpression of OmpN and KPN_02430 reduced bacterial virulence. In brief, by revealing the limited fitness costs of phage-resistant mutant K. pneumoniae with porin-deficiency, our study providing a reference for the design and development of drugs to inhibit the ways of bacterial metabolic rewiring and to increase fitness costs.
Genome-Wide Identification of Klebsiella pneumoniae Fitness Genes during Lung Infection.
Bachman Michael A,Breen Paul,Deornellas Valerie,Mu Qiao,Zhao Lili,Wu Weisheng,Cavalcoli James D,Mobley Harry L T
UNLABELLED:Klebsiella pneumoniae is an urgent public health threat because of resistance to carbapenems, antibiotics of last resort against Gram-negative bacterial infections. Despite the fact that K. pneumoniae is a leading cause of pneumonia in hospitalized patients, the bacterial factors required to cause disease are poorly understood. Insertion site sequencing combines transposon mutagenesis with high-throughput sequencing to simultaneously screen thousands of insertion mutants for fitness defects during infection. Using the recently sequenced K. pneumoniae strain KPPR1 in a well-established mouse model of pneumonia, insertion site sequencing was performed on a pool of >25,000 transposon mutants. The relative fitness requirement of each gene was ranked based on the ratio of lung to inoculum read counts and concordance between insertions in the same gene. This analysis revealed over 300 mutants with at least a 2-fold fitness defect and 69 with defects ranging from 10- to >2,000-fold. Construction of 6 isogenic mutants for use in competitive infections with the wild type confirmed their requirement for lung fitness. Critical fitness genes included those for the synthesis of branched-chain and aromatic amino acids that are essential in mice and humans, the transcriptional elongation factor RfaH, and the copper efflux pump CopA. The majority of fitness genes were conserved among reference strains representative of diverse pathotypes. These results indicate that regulation of outer membrane components and synthesis of amino acids that are essential to its host are critical for K. pneumoniae fitness in the lung. IMPORTANCE:Klebsiella pneumoniae is a bacterium that commonly causes pneumonia in patients after they are admitted to the hospital. K. pneumoniae is becoming resistant to all available antibiotics, and when these infections spread to the bloodstream, over half of patients die. Since currently available antibiotics are failing, we must discover new ways to treat these infections. In this study, we asked what genes the bacterium needs to cause an infection, since the proteins encoded by these genes could be targets for new antibiotics. We identified over 300 genes that K. pneumoniae requires to grow in a mouse model of pneumonia. Many of the genes that we identified are found in K. pneumoniae isolates from throughout the world, including antibiotic-resistant forms. If new antibiotics could be made against the proteins that these genes encode, they may be broadly effective against K. pneumoniae.
Effects of Space Environment on Genome, Transcriptome, and Proteome of Klebsiella pneumoniae.
Guo Yinghua,Li Jia,Liu Jinwen,Wang Tong,Li Yinhu,Yuan Yanting,Zhao Jiao,Chang De,Fang Xiangqun,Li Tianzhi,Wang Junfeng,Dai Wenkui,Fang Chengxiang,Liu Changting
Archives of medical research
BACKGROUND AND AIMS:The aim of this study was to explore the effects of space flight on Klebsiella pneumoniae. METHODS:A strain of K. pneumoniae was sent to space for 398 h aboard the ShenZhou VIII spacecraft during November 1, 2011-November 17, 2011. At the same time, a ground simulation with similar temperature conditions during the space flight was performed as a control. After the space mission, the flight and control strains were analyzed using phenotypic, genomic, transcriptomic and proteomic techniques. RESULTS:The flight strains LCT-KP289 exhibited a higher cotrimoxazole resistance level and changes in metabolism relative to the ground control strain LCT-KP214. After the space flight, 73 SNPs and a plasmid copy number variation were identified in the flight strain. Based on the transcriptomic analysis, there are 232 upregulated and 1879 downregulated genes, of which almost all were for metabolism. Proteomic analysis revealed that there were 57 upregulated and 125 downregulated proteins. These differentially expressed proteins had several functions that included energy production and conversion, carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, posttranslational modification, protein turnover, and chaperone functions. At a systems biology level, the ytfG gene had a synonymous mutation that resulted in significantly downregulated expression at both transcriptomic and proteomic levels. CONCLUSIONS:The mutation of the ytfG gene may influence fructose and mannose metabolic processes of K. pneumoniae during space flight, which may be beneficial to the field of space microbiology, providing potential therapeutic strategies to combat or prevent infection in astronauts.
Klebsiella pneumoniae Type VI Secretion System Contributes to Bacterial Competition, Cell Invasion, Type-1 Fimbriae Expression, and In Vivo Colonization.
Hsieh Pei-Fang,Lu Yi-Rou,Lin Tzu-Lung,Lai Li-Yin,Wang Jin-Town
The Journal of infectious diseases
Background:We previously isolated a Klebsiella pneumoniae strain, NTUH-K2044, from a community-acquired pyogenic liver abscess (PLA) patient. Analysis of the NTUH-K2044 genome revealed that this strain harbors 2 putative type VI secretion system (T6SS)-encoding gene clusters. Methods:The distribution of T6SS genes in the PLA and intestinal-colonizing K pneumoniae clinical isolates was examined. icmF1-, icmF2-, icmF1/icmF2-, and hcp-deficient K pneumoniae strains were constructed using an unmarked deletion method. The roles of T6SSs in antibacterial activity, type-1 fimbriae expression, cell adhesion, and invasion and intestinal colonization were determined. Results:The prevalence of T6SSs is higher in the PLA strains than in the intestinal-colonizing strains (37 of 42 vs 54 of 130). Deletion of icmF1/icmF2 and hcp genes significantly reduced interbacterial and intrabacterial killing. Strain deleted for icmF1 and icmF2 exhibited decreased transcriptional expression of type-1 fimbriae and reduced adherence to and invasion of human colorectal epithelial cells and was attenuated for in vivo competition to enable colonization of the host gut. Finally, Hcp expression in K pneumoniae was silenced by the histone-like nucleoid structuring protein via direct binding. Conclusions:These results provide new insights into T6SS-mediated bacterial competition and attachment in K pneumoniae and could facilitate the prevention of K pneumoniae infection.
Toxin-antitoxin operon kacAT of Klebsiella pneumoniae is regulated by conditional cooperativity via a W-shaped KacA-KacT complex.
Qian Hongliang,Yu Hao,Li Peifei,Zhu E,Yao Qingqing,Tai Cui,Deng Zixin,Gerdes Kenn,He Xinyi,Gan Jianhua,Ou Hong-Yu
Nucleic acids research
Bacterial toxin-antitoxin pairs play important roles in bacterial multidrug tolerance. Gcn5-related N-acetyltransferase (GNAT) toxins inhibit translation by acetylation of aminoacyl-tRNAs and are counteracted by direct contacts with cognate ribbon-helix-helix (RHH) antitoxins. Our previous analysis showed that the GNAT toxin KacT and RHH antitoxin KacA of Klebsiella pneumoniae form a heterohexamer in solution and that the complex interacts with the cognate promoter DNA, resulting in negative autoregulation of kacAT transcription. Here, we present the crystal structure of DNA-bound KacAT complex at 2.2 Å resolution. The crystal structure revealed the formation of a unique heterohexamer, KacT-KacA2-KacA2-KacT. The direct interaction of KacA and KacT involves a unique W-shaped structure with the two KacT molecules at opposite ends. Inhibition of KacT is achieved by the binding of four KacA proteins that preclude the formation of an active KacT dimer. The kacAT operon is auto-regulated and we present an experimentally supported molecular model proposing that the KacT:KacA ratio controls kacAT transcription by conditional cooperativity. These results yield a profound understanding of how transcription GNAT-RHH pairs are regulated.
Reduced Fitness Costs of Compared to Mutated in Isogenic Colistin-Resistant KPC-3-Producing Klebsiella pneumoniae.
Giordano Cesira,Klak Adrian,Barnini Simona,Chlebowicz Monika A,Menconi Mariacristina,Rossen John W,Friedrich Alexander W,Bathoorn Erik
In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated and isogenic isolates of colR-KPC3-KP with isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in and of clonal variants with were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000-Δ, recovered in September 2014), a 17-nucleotide deletion in was detected. In subsequent isolates, the gene associated with the plasmid pIncX4-AOUP was found, while was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP by isogenic isolates with multiple resistance plasmids, including carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal , contributing to the success of this highly resistant hospital-adapted epidemiological lineage. Our study shows a successful prolonged human colonization by a colistin-resistant isolate harboring An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying showed fewer fitness costs than isogenic isolates with a mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment.
Sublethal concentrations of carbapenems alter cell morphology and genomic expression of Klebsiella pneumoniae biofilms.
Van Laar Tricia A,Chen Tsute,You Tao,Leung Kai P
Antimicrobial agents and chemotherapy
Klebsiella pneumoniae, a Gram-negative bacterium, is normally associated with pneumonia in patients with weakened immune systems. However, it is also a prevalent nosocomial infectious agent that can be found in infected surgical sites and combat wounds. Many of these clinical strains display multidrug resistance. We have worked with a clinical strain of K. pneumoniae that was initially isolated from a wound of an injured soldier. This strain demonstrated resistance to many commonly used antibiotics but sensitivity to carbapenems. This isolate was capable of forming biofilms in vitro, contributing to its increased antibiotic resistance and impaired clearance. We were interested in determining how sublethal concentrations of carbapenem treatment specifically affect K. pneumoniae biofilms both in morphology and in genomic expression. Scanning electron microscopy showed striking morphological differences between untreated and treated biofilms, including rounding, blebbing, and dimpling of treated cells. Comparative transcriptome analysis using RNA sequencing (RNA-Seq) technology identified a large number of open reading frames (ORFs) differentially regulated in response to carbapenem treatment at 2 and 24 h. ORFs upregulated with carbapenem treatment included genes involved in resistance, as well as those coding for antiporters and autoinducers. ORFs downregulated included those coding for metal transporters, membrane biosynthesis proteins, and motility proteins. Quantitative real-time PCR validated the general trend of some of these differentially regulated ORFs. Treatment of K. pneumoniae biofilms with sublethal concentrations of carbapenems induced a wide range of phenotypic and gene expression changes. This study reveals some of the mechanisms underlying how sublethal amounts of carbapenems could affect the overall fitness and pathogenic potential of K. pneumoniae biofilm cells.
Clinically Relevant Plasmid-Host Interactions Indicate that Transcriptional and Not Genomic Modifications Ameliorate Fitness Costs of Carbapenemase-Carrying Plasmids.
Buckner Michelle M C,Saw Howard T H,Osagie Rachael N,McNally Alan,Ricci Vito,Wand Matthew E,Woodford Neil,Ivens Alasdair,Webber Mark A,Piddock Laura J V
The rapid dissemination of antimicrobial resistance (AMR) around the globe is largely due to mobile genetic elements, such as plasmids. They confer resistance to critically important drugs, including extended-spectrum beta-lactams, carbapenems, and colistin. Large, complex resistance plasmids have evolved alongside their host bacteria. However, much of the research on plasmid-host evolution has focused on small, simple laboratory plasmids in laboratory-adapted bacterial hosts. These and other studies have documented mutations in both host and plasmid genes which occur after plasmid introduction to ameliorate fitness costs of plasmid carriage. We describe here the impact of two naturally occurring variants of a large AMR plasmid (pKpQIL) on a globally successful pathogen. In our study, after pKpQIL plasmid introduction, no changes in coding domain sequences were observed in their natural host, However, significant changes in chromosomal and plasmid gene expression may have allowed the bacterium to adapt to the acquisition of the AMR plasmid. We hypothesize that this was sufficient to ameliorate the associated fitness costs of plasmid carriage, as pKpQIL plasmids were maintained without selection pressure. The dogma that removal of selection pressure (e.g., antimicrobial exposure) results in plasmid loss due to bacterial fitness costs is not true for all plasmid/host combinations. We also show that pKpQIL impacted the ability of to form a biofilm, an important aspect of virulence. This study used highly relevant models to study the interaction between AMR plasmids and pathogens and revealed striking differences from results of studies done on laboratory-adapted plasmids and strains. Antimicrobial resistance is a serious problem facing society. Many of the genes that confer resistance can be shared between bacteria through mobile genetic elements, such as plasmids. Our work shows that when two clinically relevant AMR plasmids enter their natural host bacteria, there are changes in gene expression, rather than changes to gene coding sequences. These changes in gene expression ameliorate the potential fitness costs of carriage of these AMR plasmids. In line with this, the plasmids were stable within their natural host and were not lost in the absence of selective pressure. We also show that better understanding of the impact of resistance plasmids on fundamental pathogen biology, including biofilm formation, is crucial for fighting drug-resistant infections.
Transcriptional Landscape of a Plasmid and Response to Imipenem Exposure in TOP10.
Jousset Agnès B,Rosinski-Chupin Isabelle,Takissian Julie,Glaser Philippe,Bonnin Rémy A,Naas Thierry
Frontiers in microbiology
The diffusion of KPC-2 carbapenemase is closely related to the spread of of the clonal-group 258 and linked to IncFII plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using TOP10 strain harboring a multi-replicon IncFII-IncFIB -gene carrying plasmid pBIC1a from ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance ( , and (3')-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the oxydative stress regulons, the Fur regulon (for iron uptake machinery), and regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments.
The polymyxin B-induced transcriptomic response of a clinical, multidrug-resistant Klebsiella pneumoniae involves multiple regulatory elements and intracellular targets.
Ramos Pablo Ivan Pereira,Custódio Márlon Grégori Flores,Quispe Saji Guadalupe Del Rosario,Cardoso Thiago,da Silva Gisele Lucchetti,Braun Graziela,Martins Willames M B S,Girardello Raquel,de Vasconcelos Ana Tereza Ribeiro,Fernández Elmer,Gales Ana Cristina,Nicolás Marisa Fabiana
BACKGROUND:The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. RESULTS:We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13), magnesium deprivation (Kp13), high concentrations of calcium (Kp13) and iron (Kp13), and a control condition with PB (Kp13). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. CONCLUSIONS:We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.
Transcriptional profiling of Klebsiella pneumoniae defines signatures for planktonic, sessile and biofilm-dispersed cells.
Guilhen Cyril,Charbonnel Nicolas,Parisot Nicolas,Gueguen Nathalie,Iltis Agnès,Forestier Christiane,Balestrino Damien
BACKGROUND:Surface-associated communities of bacteria, known as biofilms, play a critical role in the persistence and dissemination of bacteria in various environments. Biofilm development is a sequential dynamic process from an initial bacterial adhesion to a three-dimensional structure formation, and a subsequent bacterial dispersion. Transitions between these different modes of growth are governed by complex and partially known molecular pathways. RESULTS:Using RNA-seq technology, our work provided an exhaustive overview of the transcriptomic behavior of the opportunistic pathogen Klebsiella pneumoniae derived from free-living, biofilm and biofilm-dispersed states. For each of these conditions, the combined use of Z-scores and principal component analysis provided a clear illustration of distinct expression profiles. In particular, biofilm-dispersed cells appeared as a unique stage in the bacteria lifecycle, different from both planktonic and sessile states. The K-means cluster analysis showed clusters of Coding DNA Sequences (CDS) and non-coding RNA (ncRNA) genes differentially transcribed between conditions. Most of them included dominant functional classes, emphasizing the transcriptional changes occurring in the course of K. pneumoniae lifestyle transitions. Furthermore, analysis of the whole transcriptome allowed the selection of an overall of 40 transcriptional signature genes for the five bacterial physiological states. CONCLUSIONS:This transcriptional study provides additional clues to understand the key molecular mechanisms involved in the transition between biofilm and the free-living lifestyles, which represents an important challenge to control both beneficial and harmful biofilm. Moreover, this exhaustive study identified physiological state specific transcriptomic reference dataset useful for the research community.
Continuous evaluation of changes in the serum proteome from early to late stages of sepsis caused by Klebsiella pneumoniae.
Raju M Swathi,V Jahnavi,Kamaraju Ratnakar S,Sritharan Venkataraman,Rajkumar Karthik,Natarajan Sumathi,Kumar Anil D,Burgula Sandeepta
Molecular medicine reports
Serum protein profiles of patients with bacterial sepsis from the day of diagnosis until recovery/mortality were compared from early to late stages in response to severe sepsis using two dimensional electrophoresis. The proteins exhibiting changes during the course of sepsis (20‑28 day mortality) were selected and identified by matrix‑assisted laser desorption ionization‑time of flight‑tandem mass spectrometry. Among the proteins identified, haptoglobin (Hp), transthyretin (TTR), orosomucoid 1/α1 acid glycoprotein (ORM1), α1 antitrypsin (A1AT), serum amyloid A (SAA) and S100A9 exhibited differential expression patterns between survivors (S; n=6) and non‑survivors (NS; n=6), particularly during the early stages of sepsis. Expression factors (EFs), taken as the ratio between the NS and S during early stages, showed ratios of Hp, 0.39 (P≤0.012); TTR, 3.96 (P≤0.03); ORM1, 0.69 (P≤0.79); A1AT, 0.92 (P≤0.87) and SAA, 0.69 (P≤0.01). S100A9, an acute phase protein, exhibited an EF ratio of 1.68 (P≤0.004) during the end stages of sepsis. A delayed rise in levels was observed in Hp, A1AT, ORM1, S100A9 and SAA, whereas TTR levels increased during the early stages of sepsis in NS. Analysis of inflammatory responses in the early stages of sepsis revealed increased mRNA expression in leukocytes of interleukin (IL)‑6 (EF, 2.50), IL‑10 (EF, 1.70) and prepronociceptin (EF, 1.6), which is a precursor for nociceptin in NS compared with S, and higher Toll‑like receptor‑4 (EF, 0.30) levels in S compared with NS. Therefore, a weaker acute phase response in the early stages of sepsis in NS, combined with an inefficient inflammatory response, may contribute to sepsis mortality.