Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae.
ABSTRACT: Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance.
Project description:The increasing global occurrence of recalcitrant multi-drug resistant <i>Klebsiella pneumoniae</i> infections warrants the investigation of alternative therapy options, such as the use of monoclonal antibodies (mAbs). We used a target-agnostic phage display approach to <i>K. pneumoniae</i> bacteria lacking bulky, highly variable surface polysaccharides in order to isolate antibodies targeting conserved epitopes among clinically relevant strains. One antibody population contained a high proportion of unique carbohydrate binders, and biolayer interferometry revealed these antibodies bound to lipopolysaccharide (LPS). Antibodies that bound to O1 and O1/O2 LPS were identified. Antibodies were found to promote opsonophagocytic killing by human monocyte-derived macrophages and clearance of macrophage-associated bacteria when assessed using high-content imaging. One antibody, B39, was found to protect mice in a lethal model of <i>K. pneumoniae</i> pneumonia against both O1 and O2 strains when dosed therapeutically. High-content imaging, western blotting and fluorescence-activated cell sorting were used to determine binding to a collection of clinical <i>K. pneumoniae</i> O1 and O2 strains. The data suggests B39 binds to D-galactan-I and D-galactan-II of the LPS of O1 and O2 strains. Thus, we have discovered an mAb with novel binding and functional activity properties that is a promising candidate for development as a novel biotherapeutic for the treatment and prevention of <i>K. pneumoniae</i> infections.
Project description:<i>Klebsiella pneumoniae</i> is a common cause of sepsis and is particularly associated with healthcare-associated infections. New strategies are needed to prevent or treat infections due to the emergence of multi-drug resistant <i>K. pneumoniae</i>. The goal of this study was to determine the diversity and distribution of O (lipopolysaccharide) and K (capsular polysaccharide) antigens on a large (>500) global collection of <i>K. pneumoniae</i> strains isolated from blood to inform vaccine development efforts. A total of 645 <i>K. pneumoniae</i> isolates were collected from the blood of patients in 13 countries during 2005-2017. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. O antigen types including the presence of modified O galactan types were determined by PCR. K types were determined by multiplex PCR and <i>wzi</i> capsular typing. Sequence types of isolates were determined by multilocus sequence typing (MLST) targeting seven housekeeping genes. Among 591 isolates tested for antimicrobial resistance, we observed that 19.3% of isolates were non-susceptible to carbapenems and 62.1% of isolates were multidrug resistant (from as low as 16% in Sweden to 94% in Pakistan). Among 645 isolates, four serotypes, O1, O2, O3, and O5, accounted for 90.1% of <i>K. pneumoniae</i> strains. Serotype O1 was associated with multidrug resistance. Fifty percent of 199 tested O1 and O2 strains were <i>gmlABC</i>-positive, indicating the presence of the modified polysaccharide subunit D-galactan III. The most common K type was K2 by both multiplex PCR and <i>wzi</i> capsular typing. Of 39 strains tested by MLST, 36 strains were assigned to 26 known sequence types of which ST14, ST25, and ST258 were the most common. Given the limited number of O antigen types, diverse K antigen types and the high multidrug resistance, we believe that an O antigen-based vaccine would offer an excellent prophylactic strategy to prevent <i>K. pneumoniae</i> invasive infection.
Project description:Klebsiella pneumoniae, Escherichia coli, and other members of the Enterobacteriaceae family are common human pathogens that have acquired broad antibiotic resistance, rendering infection by some strains virtually untreatable. Enterobacteriaceae are intestinal residents, but generally represent <1% of the adult colonic microbiota. Antibiotic-mediated destruction of the microbiota enables Enterobacteriaceae to expand to high densities in the colon, markedly increasing the risk of bloodstream invasion, sepsis, and death. Here, we demonstrate that an antibiotic-naive microbiota suppresses growth of antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis by acidifying the proximal colon and triggering short chain fatty acid (SCFA)-mediated intracellular acidification. High concentrations of SCFAs and the acidic environment counter the competitive edge that O2 and NO3 respiration confer upon Enterobacteriaceae during expansion. Reestablishment of a microbiota that produces SCFAs enhances clearance of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis from the intestinal lumen and represents a potential therapeutic approach to enhance clearance of antibiotic-resistant pathogens.
Project description:<h4>Objectives</h4>Urinary tract infections (UTIs) in children are rapidly increasing worldwide and are commonly caused by extensively drug-resistant bacteria. This study determines the prevalence of UTIs in paediatric patients and evaluates the pattern of extensively drug-resistance in <i>Escherichia coli</i> and <i>Klebsiella</i> <i>pneumoniae</i> strains isolated from paediatric UTI patients.<h4>Methods</h4>Uropathogenic bacterial strains were isolated from paediatric patients with UTIs admitted to the Institute of Child Health, Lahore, Pakistan. Strains of both <i>E. coli</i> and <i>K. pneumoniae</i> were identified using biochemical characterisation and subjected to antibiotic susceptibility assays for 21 common antimicrobial drugs in order to determine their extensively drug-resistant profile.<h4>Results</h4>We isolated 63 <i>E. coli</i> and 37 <i>K. pneumoniae</i> strains from 130 paediatric patients with UTIs over a period of six months. The antibiotic susceptibility assays showed that both the <i>E. coli</i> and <i>K. pneumoniae</i> strains exhibited a high degree of resistance against co-amoxiclav, cefuroxime, cefixime, cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, nalidixic acid, norfloxacin, pepedemic acid, and co-trimoxazole. However, several of the antimicrobial agents, including polymyxin B, colistin sulphate, chloramphenicol, nitrofurantoin, and fosfomycin, were found to retain their antimicrobial activities against both pathogens. The five highest antibiotic resistant strains were identified as <i>E. coli</i> strains ZK9, ZK40, and ZK60 and <i>K. pneumoniae</i> ZK32 and ZK89 using 16S rRNA gene sequencing.<h4>Conclusion</h4>Our study demonstrates that <i>E. coli</i> and <i>K. pneumonia</i> are the dominant extensively drug-resistant uropathogenic bacteria in community-acquired UTIs in our cohort. These uropathogens were found to be resistant to the majority of the routinely-used classes of β-lactams, pyridopyrimidines, quinolones, and fluoroquinolone antibiotics, and these findings may be useful for clinicians in their treatment of paediatric UTIs.
Project description:There is a global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. While the epidemiology of K. pneumoniae strains and occurrences of specific antibiotic resistance genes, such as plasmid-borne extended-spectrum ?-lactamases (ESBLs), have been extensively studied, only four complete genomes of K. pneumoniae are available. To better understand the multidrug resistance factors in K. pneumoniae, we determined by pyrosequencing the nearly complete genome DNA sequences of two strains with disparate antibiotic resistance profiles, broadly drug-susceptible strain JH1 and strain 1162281, which is resistant to multiple clinically used antibiotics, including extended-spectrum ?-lactams, fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles. Comparative genomic analysis of JH1, 1162281, and other published K. pneumoniae genomes revealed a core set of 3,631 conserved orthologous proteins, which were used for reconstruction of whole-genome phylogenetic trees. The close evolutionary relationship between JH1 and 1162281 relative to other K. pneumoniae strains suggests that a large component of the genetic and phenotypic diversity of clinical isolates is due to horizontal gene transfer. Using curated lists of over 400 antibiotic resistance genes, we identified all of the elements that differentiated the antibiotic profile of MDR strain 1162281 from that of susceptible strain JH1, such as the presence of additional efflux pumps, ESBLs, and multiple mechanisms of fluoroquinolone resistance. Our study adds new and significant DNA sequence data on K. pneumoniae strains and demonstrates the value of whole-genome sequencing in characterizing multidrug resistance in clinical isolates.
Project description:The last three decades have seen a dwindling number of novel antibiotic classes approved for clinical use and a concurrent increase in levels of antibiotic resistance, necessitating alternative methods to combat the rise of multi-drug resistant bacteria. A promising strategy employs antibiotic adjuvants, non-toxic molecules that disarm antibiotic resistance. When co-dosed with antibiotics, these compounds restore antibiotic efficacy in drug-resistant strains. Herein we identify derivatives of tryptamine, a ubiquitous biochemical scaffold containing an indole ring system, capable of disarming colistin resistance in the Gram-negative bacterial pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli while having no inherent bacterial toxicity. Resistance was overcome in strains carrying endogenous chromosomally-encoded colistin resistance machinery, as well as resistance conferred by the mobile colistin resistance-1 (mcr-1) plasmid-borne gene. These compounds restore a colistin minimum inhibitory concentration (MIC) below the Clinical & Laboratory Sciences Institute (CLSI) breakpoint in all resistant strains.
Project description:<h4>Background</h4>Klebsiella pneumoniae is one of the leading causes of hospital outbreaks worldwide. Also, antibiotic-resistant K. pneumoniae is progressively being involved in invasive infections with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the incidence of resistance genes (integron types and β-lactamase-encoded genes) among clinical isolates of K. pneumoniae.<h4>Methods</h4>In this cross-sectional study, a total of 100 clinical samples were obtained from hospitalized patients in three teaching hospitals in the north of Iran, from November 2018 and October 2019. Antimicrobial susceptibility testing was performed using disk agar diffusion test in line with CLSI recommendations. For colistin, minimum inhibitory concentration (MIC) was determined using broth microdilution. Based on antibiogram, multi-drug resistant (MDR) and extensive-drug resistant (XDR) strains were detected. Finally, integron types and β-lactamase resistance genes were identified using polymerase chain reaction technique.<h4>Results</h4>The most and least clinical samples were related to the urine and bronchoalveolar lavage, respectively. Based on the antibiogram results, amikacin and gentamicin exhibited good activity against K. pneumoniae strains in vitro. The high resistance rate (93%) to ampicillin/sulbactam predicts the limited efficacy of this antibiotic, in the hospitals studied. Among all the 100 isolates, the frequency of MDR and XDR phenotypes were 58% and 13%, respectively, while no pan-drug resistant (PDR) strains were found. In the MDR K. pneumoniae strains, the prevalence of bla<sub>SHV</sub>, bla<sub>TEM</sub>, bla<sub>CTX-M-15</sub>, bla<sub>KPC</sub>, bla<sub>OXA-48</sub>, bla<sub>NDM</sub> β-lactamase genes were 91.4%, 82.7%, 79.3%, 29.3%, 36.2% and 6.9%, respectively, however 91.4% of the isolates were carrying intI gene. Class II and III integrons were not detected in any isolates.<h4>Conclusion</h4>The MDR K. pneumoniae is becoming a serious problem in hospitals, with many strains developing resistance to most available antimicrobials. Our results indicate co-presence of a series of β-lactamase and integron types on the MDR strains recovered from hospitalized patients. The increasing rate of these isolates emphasizes the importance of choosing an appropriate antimicrobial regimen based on antibiotic susceptibility pattern.
Project description:The increasing prevalence of multidrug-resistant <i>Klebsiella pneumoniae</i> has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in <i>K. pneumoniae</i> is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the <i>in vitro</i> evolutionary trajectories toward colistin resistance in four clinical <i>K. pneumoniae</i> complex strains and their impact on fitness and virulence characteristics. Through population sequencing during <i>in vitro</i> evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one <i>K. pneumoniae</i> <i>sensu stricto</i> strain, but not those of the other three <i>K. pneumoniae</i> complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. <i>K. pneumoniae</i> <i>sensu stricto</i> strains exhibited cross-resistance to LL-37, in contrast to the <i>Klebsiella variicola</i> subsp. <i>variicola</i> strain. Virulence, as determined in a <i>Caenorhabditis elegans</i> survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial <i>K. pneumoniae</i> complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant <i>Klebsiella</i>.
Project description:<h4>Background</h4>The antimicrobial resistance of clinical, environmental and control strains of the WHO "Priority 1: Critical group" organisms, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa to various classes of antibiotics, colistin and surfactin (biosurfactant) was determined.<h4>Methods</h4>Acinetobacter baumannii was isolated from environmental samples and antibiotic resistance profiling was performed to classify the test organisms [A. baumannii (n =?6), P. aeruginosa (n =?5), E. coli (n =?7) and K. pneumoniae (n =?7)] as multidrug resistant (MDR) or extreme drug resistant (XDR). All the bacterial isolates (n =?25) were screened for colistin resistance and the mobilised colistin resistance (mcr) genes. Biosurfactants produced by Bacillus amyloliquefaciens ST34 were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS). The susceptibility of strains, exhibiting antibiotic and colistin resistance, to the crude surfactin extract (cell-free supernatant) was then determined.<h4>Results</h4>Antibiotic resistance profiling classified four A. baumannii (67%), one K. pneumoniae (15%) and one P. aeruginosa (20%) isolate as XDR, with one E. coli (15%) and three K. pneumoniae (43%) strains classified as MDR. Many of the isolates [A. baumannii (25%), E. coli (80%), K. pneumoniae (100%) and P. aeruginosa (100%)] exhibited colistin resistance [minimum inhibitory concentrations (MICs)???4?mg/L]; however, only one E. coli strain isolated from a clinical environment harboured the mcr-1 gene. UPLC-MS analysis then indicated that the B. amyloliquefaciens ST34 produced C<sub>13-16</sub> surfactin analogues, which were identified as Srf1 to Srf5. The crude surfactin extract (10.00?mg/mL) retained antimicrobial activity (100%) against the MDR, XDR and colistin resistant A. baumannii, P. aeruginosa, E. coli and K. pneumoniae strains.<h4>Conclusion</h4>Clinical, environmental and control strains of A. baumannii, P. aeruginosa, E. coli and K. pneumoniae exhibiting MDR and XDR profiles and colistin resistance, were susceptible to surfactin analogues, confirming that this lipopeptide shows promise for application in clinical settings.
Project description:BACKGROUND:The relationship between Klebsiella pneumoniae and nosocomial and community-acquired infections is well known, and K. pneumoniae resistance to most antibiotics is increasing worldwide. In contrast, tigecycline remains active against many bacterial strains, and serves as a last resort for treating multi-drug resistant bacterial infections. That tigecycline nonsusceptibility among K. pneumoniae isolates has been reported worldwide is worrying. However, the mechanisms of tigecycline resistance in K. pneumoniae are less well known. We report the genome sequence and genomic characterization of tigecycline-resistant K. pneumoniae strain 5422 isolated from the bile samples of a patient with cholangiocarcinoma. RESULTS:We sequenced the K. pneumoniae strain 5422 genome using next-generation sequencing technologies. Sequence data assembly revealed a 5,432,440-bp draft genome and 57.1% G?+?C content, which contained 5397 coding sequences. The genome has extensive similarity to other sequenced K. pneumoniae genomes, but also has several resistance-nodulation-cell division (RND) efflux pump genes that may be related to tigecycline resistance. CONCLUSIONS:K. pneumoniae strain 5422 is resistant to multiple antibiotics. The genome sequence of the isolate and comparative analysis with other K. pneumoniae strains presented in this paper are important for better understanding of K. pneumoniae multi-drug resistance. The RND efflux pump genes identified in the genome indicate the presence of an antibiotic resistance mechanism prior to antibiotics overuse. The availability of the genome sequence forms the basis for further comparative analyses and studies addressing the evolution of the K. pneumoniae drug resistance mechanism and the K. pneumoniae transcriptome.