Potential Pathogens in the Environment: Cultural Reactions and Nucleic Acid Studies on Klebsiella pneumoniae from Clinical and Environmental Sources

The phenotypic and nucleic acid properties of Klebsiella pneumoniae have been studied on cultures obtained from six different habitats (humans, vegetables, seeds, trees, rivers, and pulp mills). The 19 cultural reactions of 107 isolates varied significantly only in tryptophanase activity and dulcitol fermentation. The percentage of guanine plus cytosine base composition of 41 isolates varied from 53.9 to 59.2%. The range of percentage of guanine plus cytosine base composition for environmental klebsiellas was broader than that for the cultures of human origin. The range of deoxyribonucleic acid relative reassociation (homology) to the human K. pneumoniae reference strain extended from 5% to 100% and the chromosome molecular weights ranged from 2,200 × 10⁶ to 3,000 × 10⁶. The species of K. pneumoniae is thus molecularly more heterogeneous than previously thought and most isolates of human, pulp mill, and river origin are genetically indistinguishable. The presence of K. pneumoniae therefore represents a deterioration of the microbiological quality of the environment and should be considered of public health significance. At the present time the health significance of the molecularly more divergent strains, primarily of vegetable and seed origin, their relationship to klebsiellas of human origin, or to other genera of the Enterobacteriaceae is unclear.     

An optimized method for suicide vector-based allelic exchange in Klebsiella pneumoniae

Klebsiella pneumoniae is an important and versatile bacterium that can be found in diverse environments and is also a frequent cause of human infections. Limited data exists on the mechanisms of interaction between K. pneumoniae and the human host and of adaptations to other environments. Coupled with the high genetic diversity of this species, these factors highlight the necessity for substantial further K. pneumoniae-focused molecular genetics studies.In this report we describe a simple and efficient experimental protocol for suicide vector-based allelic exchange in K. pneumoniae. The protocol has been validated by mutating multiple loci in four distinct K. pneumoniae strains, including highly capsulated and/or multi-antibiotic resistant clinical isolates. Three key enhancements are reported:(1) Use of pDS132-derived conjugative plasmids carrying improved cloning sites, (2) Performance of sacB counterselection at 25 °C as opposed to higher temperatures, and (3) Exploitation of Flp-recombinase-mediated deletion of FRT (Flp recombinase target) flanked resistance cassettes to allow for reiterative manipulations with a single selectable marker. This study also highlights a problem that may be encountered when the aacC1 gentamicin resistance marker is used in K. pneumoniae and suggests alternative markers.The protocol developed in this study will help investigate the plethora of uncharacterized genes present in the K. pneumoniae pan-genome and shed further light upon clinically and industrially important phenotypes observed in this ubiquitous species.     

Klebsiella pneumoniae Produces No Histamine: Raoultella planticola and Raoultella ornithinolytica Strains Are Histamine Producers

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.     

Identification and characterization of Klebsiella pneumoniae aldehyde dehydrogenases increasing production of 3-hydroxypropionic acid from glycerol

Klebsiella pneumoniae produces 3-hydroxypropionic acid (3-HP) from glycerol with oxidation of 3-hydroxypropionaldehyde (3-HPA) to 3-HP in a reaction catalyzed by aldehyde dehydrogenase (ALDH). In the present study, two putative ALDHs of K. pneumoniae, YneI and YdcW were identified and characterized. Recombinant YneI was specifically active on 3-HPA and preferred NAD⁺ as a cofactor, whereas YdcW exhibited broad substrate specificity and preferred NADP⁺ as a cofactor. Overexpression of ALDHs in the glycerol oxidative pathway-deficient mutant K. pneumoniae AK resulted in a significant increase in 3-HP production upon shake-flask culture. The final titers of 3-HP were 2.4 and 1.8 g L^?1 by recombinants overexpressing YneI and YdcW, respectively. Deletion of the ALDH gene from K. pneumoniae did not affect the extent of 3-HP synthesis, implying non-specific activity of ALDHs on 3-HPA. The ALDHs might play major roles in detoxifying the aldehyde generated in glycerol metabolism.     

Immunolocalization of Dinitrogenase Reductase Produced by Klebsiella pneumoniae in Association with Zea mays L.

The endophytic lifestyle of Klebsiella pneumoniae is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by K. pneumoniae strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for K. pneumoniae and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.     

Phenotypic and Molecular Characterization of Multidrug Resistant Klebsiella pneumoniae Isolated from a University Teaching Hospital,China

The multidrug-resistant rate of Klebsiella pneumoniae has risen rapidly worldwide. To better understand the multidrug resistance situation and molecular characterization of Klebsiella pneumoniae, a total of 153 Klebsiella pneumoniae isolates were collected, and drug susceptibility test was performed to detect its susceptibility patterns to 13 kinds of antibiotics. Phenotypic tests for carbapenemases ESBLs and AmpC enzyme-producing strains were performed to detect the resistance phenotype of the isolates. Then PCR amplification and sequencing analysis were performed for the drug resistance determinants. The results showed that 63 strains harbored bla _CTX-M gene, and 14 strains harbored bla _DHA gene. Moreover, there were 5 strains carrying bla _KPC gene, among which 4 strains carried bla _CTX-M, bla _DHA and bla _KPC genes, and these 4 strains were also resistant to imipenem. Our data indicated that drug-resistant Klebsiella pneumoniae were highly prevalent in the hospital. Thus it is warranted that surveillance of epidemiology of those resistant isolates should be a cause for concern, and appropriate drugs should be chosen.     

Contemplating 3-Hydroxypropionic Acid Biosynthesis in Klebsiella pneumoniae

3-Hydroxypropionic acid (3-HP) is a commercially valuable platform chemical from which an array of C₃ compounds can be generated. Klebsiella pneumoniae has been considered a promising species for biological production of 3-HP. Despite a wealth of reports related to 3-HP biosynthesis in K. pneumoniae, its commercialization is still in infancy. The major hurdle hindering 3-HP overproduction lies in the poor understanding of glycerol dissimilation in K. pneumoniae. To surmount this problem, this review aims to portray a picture of 3-HP biosynthesis, involving 3-HP-synthesizing strains, biochemical attributes, metabolic pathways and key enzymes. Inspired by the state-of-the-art advances in metabolic engineering and synthetic biology, here we advocate protocols for overproducing 3-HP in K. pneumoniae. These protocols range from cofactor regeneration, alleviation of metabolite toxicity, genome editing, remodeling of transport system, to carbon flux partition via logic gate. The feasibility for these protocols was also discussed.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0513-0) contains supplementary material, which is available to authorized users.     

Pathway of galactitol catabolism in Klebsiella pneumoniae.

Cell extracts of galactitol-grown Klebsiella pneumoniae phosphorylate galactitol by means of a phosphoenolpyruvate:galactitol phosphotransferase system. Both the product and authentic L-galactitol-l-P are oxidized with NAD⁺ by a dehydrogenase to yield D-tagatose-6-P, which is phosphorylated with ATP by a kinase to form D-tagatose-1,6-P₂. This ketohexose diphosphate is cleaved by an aldolase to yield dihydroxyacetone-P and D-glyceraldehyde-3-P. Mutants deficient in either the dehydrogenase, kinase, or aldolase failed to grow on galactitol, indicating that the described pathway is of physiological significance in this organism.     

Metabolic Response to Klebsiella pneumoniae Infection in an Experimental Rat Model

Bacteremia, the presence of viable bacteria in the blood stream, is often associated with several clinical conditions. Bacteremia can lead to multiple organ failure if managed incorrectly, which makes providing suitable nutritional support vital for reducing bacteremia-associated mortality. In order to provide such information, we investigated the metabolic consequences of a Klebsiella pneumoniae (K. pneumoniae) infection in vivo by employing a combination of ¹H nuclear magnetic resonance spectroscopy and multivariate data analysis. K. pneumoniae was intravenously infused in rats; urine and plasma samples were collected at different time intervals. We found that K. pneumoniae-induced bacteremia stimulated glycolysis and the tricarboxylic acid cycle and also promoted oxidation of fatty acids and creatine phosphate to facilitate the energy-demanding host response. In addition, K. pneumoniae bacteremia also induced anti-endotoxin, anti-inflammatory and anti-oxidization responses in the host. Furthermore, bacteremia could cause a disturbance in the gut microbiotal functions as suggested by alterations in a range of amines and bacteria-host co-metabolites. Our results suggest that supplementation with glucose and a high-fat and choline-rich diet could ameliorate the burdens associated with bacteremia. Our research provides underlying pathological processes of bacteremia and a better understanding of the clinical and biochemical manifestations of bacteremia.     

Complete Genome Sequence of Klebsiella pneumoniae Phage JD001

Klebsiella pneumoniae is a member of the family Enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. The emergence of multidrug-resistant strains of K. pneumoniae has became a public health problem globally. To develop an effective antimicrobial agent, we isolated a bacteriophage, named JD001, from seawater and sequenced its genome. Comparative genome analysis of phage JD001 with other K. pneumoniae bacteriophages revealed that phage JD001 has little similarity to previously published K. pneumoniae phages KP15, KP32, KP34, and phiKO2. Here we announce the complete genome sequence of JD001 and report major findings from the genomic analysis.     

Expansion of the known Klebsiella pneumoniae species gene pool by characterization of novel alien DNA islands integrated into tmRNA gene sites

Klebsiella pneumoniae is an important bacterial pathogen of man that is commonly associated with opportunistic and hospital-associated infections. Increasing levels of multiple-antibiotic resistance associated with this species pose a major emerging clinical problem. This organism also occurs naturally in other diverse environments, including the soil. Consistent with its varied lifestyle and membership of the Enterobacteriaceae family, K. pneumoniae genomes exhibit highly plastic architecture comprising a core genome backbone interspersed with numerous and varied alien genomic islands. In this study the size of the presently known K. pneumoniae pan-genome gene pool was estimated through analysis of complete sequences of three chromosomes and 31 plasmids belonging to K. pneumoniae strains. In addition, using a PCR-based strategy the genomic content of eight tRNA/tmRNA gene sites that serve as DNA insertion hotspots were investigated in 28 diverse environmental and clinical strains of K. pneumoniae. Sequencing and characterization of five newly identified horizontally-acquired tmRNA-associated islands further expanded the archived K. pneumoniae gene pool to a total of 7648 unique gene members. Large-scale investigation of the content of tRNA/tmRNA hotspots will be useful to identify and/or survey accessory sequences dispersed amongst hundreds to thousands of members of many key bacterial species.     

Biodegradation of tributyl phosphate using Klebsiella pneumoniae sp. S3

Tributyl phosphate (TBP) has enormous applications in the field of extraction, fuel reprocessing, as defoamers and/or plasticizers. Excessive usage of this organophosphorus compound, poses an environmental threat. The present study deals with microbial degradation of TBP using Klebsiella pneumoniae S3 isolated from the soil. Diauxic growth curve pattern explains a preferential utilization of TBP. The strain S3 was able to biotransform TBP (1,000 mg L^?1) to dibutyl phosphate within 48 h and showed higher tolerance towards TBP up to 17.0 g L^?1. Toxicity of the parent as well as degraded product was assessed using comet assay. Generation of reactive oxygen species elaborates the oxidative stress imposed upon the bacterial strain by TBP. The antioxidant defense mechanism was studied using various biomarkers namely catalase, glutathione-S-transferase, and superoxide dismutase. The present study describes a faster and eco-friendly alternative for disposal of TBP.     

Complement mediated Klebsiella pneumoniae capsule changes

The Gram-negative bacterium Klebsiella pneumoniae is an opportunistic pathogen, which can cause life-threatening infections such as sepsis. Worldwide, emerging multidrug resistant K. pneumoniae infections are challenging to treat, hence leading to increased mortality. Therefore, understanding the interactions between K. pneumoniae and the immune system is important to develop new treatment options.We characterized ten clinical K. pneumoniae isolates obtained from blood of bacteremia patients. The interaction of the isolates with human serum was investigated to elucidate how K. pneumoniae escapes the host immune system, and how complement activation by K. pneumoniae changed the capsule structure. All K. pneumoniae isolates activated the alternative complement pathway despite serum resistance of seven isolates. One serum sensitive isolate activated two or all three pathways, and this isolate was lysed and had numerous membrane attack complexes in the outer membrane. However, we also found deposition of complement components in the capsule of serum resistant isolates resulting in morphological capsule changes and capsule shedding. These bacteria did not lyse, and no membrane attack complex was observed despite deposition of C5b-9 within the capsule, indicating that the capsule of serum resistant K. pneumoniae isolates is a defense mechanism against complement-mediated lysis.     

Enhanced aldehyde dehydrogenase activity by regenerating NAD+ in Klebsiella pneumoniae and implications for the glycerol dissimilation pathways

In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD⁺ as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD⁺ is exhausted. Here we show that NAD⁺ regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD⁺-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD⁺. When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD⁺ enhanced the activity of ALD4. More interestingly, the growth rates of all NAD⁺-regenerating strains were prolonged in comparison with the control, indicating that NAD⁺ regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD⁺ availability but also provides a method for regulating the dha regulon.     

Legionella pneumophila Persists within Biofilms Formed by Klebsiella pneumoniae,Flavobacterium sp., and Pseudomonas fluorescens under Dynamic Flow Conditions

Legionella pneumophila, the agent of Legionnaires'' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×10⁴ CFU per cm² of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species.     

Draft Genome Sequence of an Extremely Drug-Resistant KPC-Producing Klebsiella pneumoniae ST258 Epidemic Strain

Extremely drug-resistant (XDR) Klebsiella pneumoniae carbapenemase-producing clone ST258 has rapidly disseminated worldwide. We report here the draft genome sequence of the K. pneumoniae ST258 XDR clinical strain from Israel.     

Genome sequencing and functional characterization of the non-pathogenic Klebsiella pneumoniae KpGe bacteria

Klebsiella pneumoniae is an extensively studied human pathogen responsible for a wide variety of infections. Dictyostelium discoideum is a model host organism employed to study many facets of the complex interactions between phagocytic cells and bacteria. Historically, a non-pathogenic strain of K. pneumoniae has been used to feed Dictyostelium amoebae, and more recently to study cellular mechanisms involved in bacterial recognition, ingestion and killing. Here we provide the full genome sequence and functional characterization of this non-pathogenic KpGe strain.     

Erratum to: Production of isobutanol from crude glycerol by a genetically-engineered Klebsiella pneumoniae strain

Klebsiella pneumoniae was engineered to produce isobutanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ?ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced isobutanol (160 mg l^?1) from crude glycerol. To increase the yield of isobutanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of isobutanol from 160 to 320 mg l^?1. This represents the first successful attempt to produce isobutanol from crude glycerol.     

Production of 2-butanol from crude glycerol by a genetically-engineered Klebsiella pneumoniae strain

Klebsiella pneumoniae was engineered to produce 2-butanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ?ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced 2-butanol (160 mg l^?1) from crude glycerol. To increase the yield of 2-butanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of 2-butanol from 160 to 320 mg l^?1. This represents the first successful attempt to produce 2-butanol from crude glycerol.