Growth In Human Urine Research Articles

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum.

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Combined functional genomic and metabolomic approaches identify new genes required for growth in human urine by multidrug-resistant Escherichia coli ST131.

Uropathogenic Escherichia coli (UPEC) cause ~80% of all urinary tract infections (UTIs), with increasing rates of antibiotic resistance presenting an urgent threat to effective treatment. To cause infection, UPEC must grow efficiently in human urine (HU), necessitating a need to understand mechanisms that promote its adaptation and survival in this nutrient-limited environment. Here, we used a combination of functional genomic and metabolomic techniques and identified roles for the metabolism of small peptides, amino acids, nucleotides, and L-lactate, as well as the stringent response pathway, lipopolysaccharide biosynthesis, and fluoride resistance, for UPEC growth in HU. We further demonstrated that pathways involving nucleotide metabolism and the stringent response are required for UPEC colonization of the mouse bladder. The UPEC genes and metabolic pathways identified in this study represent targets for the development of innovative therapeutics to prevent UPEC growth during human UTI, an urgent need given the rapidly rising rates of global antibiotic resistance.

Defining the Roles of Pyruvate Oxidation, TCA Cycle, and Mannitol Metabolism in Methicillin-Resistant Staphylococcus aureus Catheter-Associated Urinary Tract Infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of complicated urinary tract infection (UTI) associated with the use of indwelling urinary catheters. Previous reports have revealed host and pathogen effectors critical for MRSA uropathogenesis. Here, we sought to determine the significance of specific metabolic pathways during MRSA UTI. First, we identified four mutants from the Nebraska transposon mutant library in the MRSA JE2 background that grew normally in rich medium but displayed significantly reduced growth in pooled human urine (HU). This prompted us to transduce the uropathogenic MRSA 1369 strain with the transposon mutants in sucD and fumC (tricarboxylic acid [TCA] cycle), mtlD (mannitol metabolism), and lpdA (pyruvate oxidation). Notably, sucD, fumC, and mtlD were also significantly upregulated in the MRSA 1369 strain upon exposure to HU. Compared to the WT, the MRSA 1369 lpdA mutant was significantly defective for (i) growth in HU, and (ii) colonization of the urinary tract and dissemination to the kidneys and the spleen in the mouse model of catheter-associated UTI (CAUTI), which may be attributed to its increased membrane hydrophobicity and higher susceptibility to killing by human blood. In contrast to their counterparts in the JE2 background, the sucD, fumC, and mtlD mutants in the MRSA 1369 background grew normally in HU; however, they displayed significant fitness defects in the CAUTI mouse model. Overall, identification of novel metabolic pathways important for the urinary fitness and survival of MRSA can be used for the development of novel therapeutics. IMPORTANCE While Staphylococcus aureus has historically not been considered a uropathogen, S. aureus urinary tract infection (UTI) is clinically significant in certain patient populations, including those with chronic indwelling urinary catheters. Moreover, most S. aureus strains causing catheter-associated UTI (CAUTI) are methicillin-resistant S. aureus (MRSA). MRSA is difficult to treat due to limited treatment options and the potential to deteriorate into life-threatening bacteremia, urosepsis, and shock. In this study, we found that pathways involved in pyruvate oxidation, TCA cycle, and mannitol metabolism are important for MRSA fitness and survival in the urinary tract. Improved understanding of the metabolic needs of MRSA in the urinary tract may help us develop novel inhibitors of MRSA metabolism that can be used to treat MRSA-CAUTI more effectively.

High resistance of fluoroquinolone and macrolide reported in avian pathogenic Escherichia coli isolates from the humid subtropical regions of Pakistan

ObjectivesThis study aimed to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of avian pathogenic Escherichia coli (APEC) that cause colibacillosis in poultry. MethodsAntibiotic susceptibility testing (AST) was measured via the Kirby-Bauer disc diffusion method against 27 commonly used antibiotics. Phylogrouping, virulence-associated gene detection, and hybrid strain detection via multiplex polymerase chain reaction (PCR) and genetic diversity were analysed via ERIC-PCR fingertyping method. ResultsAST analysis showed 100% of isolates were multidrug-resistant (MDR) and highest resistance was against penicillin, tetracycline, and macrolide classes of antibiotics. The mcr-1 gene was present in 40% of the isolates, though only 4% of isolates were showing phenotypic resistance. Despite the scarce use of fluoroquinolone, carbapenem, and cephalosporin in the poultry sector, resistance was evident because of the high prevalence of extended-spectrum β-lactamase (ESBL) (53.7%) and other β-lactamases in APEC isolates. β-lactamase genotyping of APEC isolates revealed that 85.7% of isolates contained either blaCTX or blaTEM and around 38% of isolates were complement resistant. Growth in human urine was evident in 67.3% of isolates. Phylogroup B1 (51%) was the most prevalent group followed by phylogroups A (30.6%), D (13.61%), and B2 (4.76%). The most prevalent virulence-associated genes were fimH, iss, and tatT. Results showed that 26 isolates (17.69%) can be termed hybrid strains and APEC/EHEC (enterohemorrhagic E. coli) was the most prevalent hybrid E. coli pathotype. ERIC-PCR fingerprinting genotype analysis clustered APEC isolates in 40 groups (E1-E40). This study provides insights into the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. ConclusionsThe findings of this study provide insights into that the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. This data can inform future studies designed to better estimate the severity of the colibacillosis in poultry farms.

High Occurrence of Multidrug-Resistant Escherichia coli Strains in Bovine Fecal Samples from Healthy Cows Serves as Rich Reservoir for AMR Transmission.

Antibiotics are valuable therapeutics. However, the unwarranted and excessive use of these antimicrobials in food animals and the consequent contamination of the environment have been associated with the emergence and spread of antimicrobial resistance. Continuous surveillance and monitoring of antimicrobial resistance among E. coli isolates is recommended, not only for bovine health but also for public health. This study aims to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of fecal E. coli isolates from healthy cows. The in vitro, phenotypic antibiotic resistance of isolates was measured via the Kirby-Bauer disc-diffusion method against twenty-seven antibiotics. The β-lactamase enzymatic activities of the strains were also investigated. For the assessment of virulence potential, fecal E. coli isolates were subjected to several in vitro pathogenicity assays, including biofilm formation ability, blood hemolysis, complement resistance, and growth in human urine. Phylogroup determination and virulence-associated genes were detected via multiplex PCR. In vitro antibiotic resistance profiling showed that 186/200 (93%) of the isolates were multidrug-resistant (MDR), with the highest resistance against penicillin, tetracycline, fluoroquinolone, and macrolide classes of antibiotics. Of particular concern was the phenotypic resistance to colistin in 52/200 isolates (26%), though 16% of the total isolates harbored mcr1, the genetic determinant of colistin. Despite the scarce use of fluoroquinolone, cephalosporin, and carbapenem in the agricultural sector, resistance to these classes was evident due to the presence of extended-spectrum β-lactamase (ESBL) in 41% of E. coli isolates. The β-lactamase genotyping of E. coli isolates showed that 47% of isolates harbored either blaCTX or blaTEM. Approximately 32% of isolates were resistant to serum complement, and their growth in human urine was evident in 18% of isolates, indicating a possible infection of these isolates in high nitrogenous condition. Phylogrouping showed that the most prevalent phylogenetic group among fecal E. coli isolates was phylogroup B1 (57%), followed by phylogroups A (33%), D (6%), and B2 (4%). The most prevalent virulence-associated genes in fecal E. coli were fimH, iss and tatT. Results showed that ten isolates (5%) harbored the stx1 gene, the genetic marker of enterohemorrhagic E. coli. This study provides insights into the antibiotic resistance and virulence profiling of the fecal E. coli isolates from healthy cows. These results emphasize the need for imposing regulations on the proper use of antibiotics and growth promoters in food-producing animals.

Genome-wide analysis of fitness factors in uropathogenic Escherichia coli in a pig urinary tract infection model

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs) in animals and humans. We applied Transposon-Directed Insertion Site sequencing (TraDIS) to determine the fitness genes in two well-characterized UPEC strains, UTI89 and CFT073, in order to identify fitness factors during UTI in a pig model. This novel animal model better reflects the course of UTI in humans than the commonly used mouse model, and facilitates the differentiation between sessile and planktonic UPEC populations. A total of 854 and 483 genes in UTI89 and CFT073, respectively, were predicted to contribute to growth in pig urine, and 1257 and 764, were scored as required for colonization of the bladder. The combined list of fitness genes for growth in urine and cystitis contained 741 (UTI89) and 439 (CFT073) genes. The essential genes for growth on LB agar media supplemented with kanamycin and the fitness factors during growth in human urine were also analyzed in CFT073. A total of 457 essential genes were identified and the pool of fitness genes for growth in human urine included 215 genes. The gene rfaG, which is involved in lipopolysaccharide biosynthesis, was included in all the fitness-gene-lists and was further confirmed to be relevant for all the conditions tested regardless of the host and the strain. Thus, this gene may represent a promising target for the development of new therapeutic strategies against UTI UPEC-associated. Besides this important observation, the study revealed strain-specific differences in gene-essentiality as well as in the fitness-gene-repertoire for growth in human urine and UTI of the pig model, and it identified novel factors required for UPEC-induced UTIs.

Preferential catabolism of l- vs d-serine by Proteus mirabilis contributes to pathogenesis and catheter-associated urinary tract infection.

Proteus mirabilis is a common cause of urinary tract infection, especially in catheterized individuals. Amino acids are the predominant nutrient for bacteria during growth in urine, and our prior studies identified several amino acid import and catabolism genes as fitness factors for P. mirabilis catheter-associated urinary tract infection (CAUTI), particularly those for d- and l-serine. In this study, we sought to determine the hierarchy of amino acid utilization by P. mirabilis and to examine the relative importance of d- vs l-serine catabolism for critical steps in CAUTI development and progression. Herein, we show that P. mirabilis preferentially catabolizes l-serine during growth in human urine, followed by d-serine, threonine, tyrosine, glutamine, tryptophan, and phenylalanine. Independently disrupting catabolism of either d- or l-serine has minimal impact on in vitro phenotypes while completely disrupting both pathways decreases motility, biofilm formation, and fitness due to perturbation of membrane potential and cell wall biosynthesis. In a mouse model of CAUTI, loss of either serine catabolism system decreased fitness, but disrupting l-serine catabolism caused a greater fitness defect than disrupting d-serine catabolism. We, therefore, conclude that the hierarchical utilization of amino acids may be a critical component of P. mirabilis colonization and pathogenesis within the urinary tract.

CsrA Coordinates Compatible Solute Synthesis in Acinetobacter baumannii and Facilitates Growth in Human Urine.

ABSTRACTCsrA is a global regulator widespread in bacteria and known to be involved in different physiological processes, including pathogenicity. Deletion of csrA of Acinetobacter baumannii strain ATCC 19606 resulted in a mutant that was unable to utilize a broad range of carbon and energy sources, including amino acids. This defect in amino acid metabolism was most likely responsible for the growth inhibition of the ΔcsrA mutant in human urine, where amino acids are the most abundant carbon source for A. baumannii. Recent studies revealed that deletion of csrA in the A. baumannii strains AB09-003 and ATCC 17961 resulted in an increase in hyperosmotic stress resistance. However, the molecular basis for this observation remained unknown. This study aimed to investigate the role of CsrA in compatible solute synthesis. We observed striking differences in the ability of different A. baumannii strains to cope with hyperosmotic stress. Strains AB09-003 and ATCC 17961 were strongly impaired in hyperosmotic stress resistance in comparison to strain ATCC 19606. These differences were abolished by deletion of csrA and are in line with the ability to synthesize compatible solutes. In the salt-sensitive strains AB09-003 and ATCC 17961, compatible solute synthesis was repressed by CsrA. This impairment is mediated via CsrA and could be overcome by deletion of csrA from the genome.IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii has become one of the leading causes of nosocomial infections around the world due to the increasing prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. Recently, it was found that CsrA, a global mRNA binding posttranscriptional regulator, plays a role in osmotic stress adaptation, virulence, and growth on amino acids of A. baumannii AB09-003 and ATCC 17961. Here, we report that this is also the case for A. baumannii ATCC 19606. However, we observed significant differences in the ΔcsrA mutants with respect to osmostress resistance, such as the AB09-003 and 17961 mutants being enhanced in osmostress resistance whereas the ATCC 19606 mutant was not. This suggests that the role of CsrA in osmotic stress adaptation is strain specific. Furthermore, we provide clear evidence that CsrA is essential for growth in human urine and at high temperatures.

Escherichia coli isolates from commercial chicken meat and eggs cause sepsis, meningitis and urinary tract infection in rodent models of human infections.

The zoonotic potential of Escherichia coli from chicken-source food products is important to define for public health purposes. Previously, genotypic and phenotypic screening of E.coli isolates from commercial chicken meat and shell eggs identified some E.coli strains that by molecular criteria resembled human-source extraintestinal pathogenic E.coli (ExPEC). Here, to clarify the zoonotic risk of such chicken-source E.coli, we compared selected E.coli isolates from chicken meat and eggs, stratified by molecularly defined ExPEC status, to human-source ExPEC and to laboratory E.coli for virulence in rodent models of sepsis, meningitis and UTI, and evaluated whether specific bacterial characteristics predict experimental virulence. Multiple chicken-source E.coli resembled human-source ExPEC in their ability to cause one or multiple different ExPEC-associated infections. Swimming ability corresponded with urovirulence, K1 capsule corresponded with ability to cause neonatal meningitis, and biofilm formation in urine corresponded with ability to cause sepsis. In contrast, molecularly defined ExPEC status and individual genotypic traits were uncorrelated with ability to cause sepsis, and neither complement sensitivity nor growth in human urine corresponded with virulence in any infection model. These findings establish that chicken-derived food products contain E.coli strains that, in rodent models of multiple human-associated ExPEC infections, are able to cause disease comparably to human-source E.coli clinical isolates, which suggests that they may pose a significant food safety threat. Further study is needed to define the level of risk they pose to human health, which if appreciable would justify efforts to monitor for and reduce or eliminate them.

DFI-seq identification of environment-specific gene expression in uropathogenic Escherichia coli

BackgroundDuring infection of the urinary tract, uropathogenic Escherichia coli (UPEC) are exposed to different environments, such as human urine and the intracellular environments of bladder epithelial cells. Each environment elicits a distinct bacterial environment-specific transcriptional response. We combined differential fluorescence induction (DFI) with next-generation sequencing, collectively termed DFI-seq, to identify differentially expressed genes in UPEC strain UTI89 during growth in human urine and bladder cells.ResultsDFI-seq eliminates the need for iterative cell sorting of the bacterial library and yields a genome-wide view of gene expression. By analysing the gene expression of UPEC in human urine we found that genes involved in amino acid biosynthesis were upregulated. Deletion mutants lacking genes involved in arginine biosynthesis were outcompeted by the wild type during growth in human urine and inhibited in their ability to invade or proliferate in the J82 bladder epithelial cell line. Furthermore, DFI-seq was used to identify genes involved in invasion of J82 bladder epithelial cells. 56 genes were identified to be differentially expressed of which almost 60% encoded hypothetical proteins. One such gene UTI89_C5139, displayed increased adhesion and invasion of J82 cells when deleted from UPEC strain UTI89.ConclusionsWe demonstrate the usefulness of DFI-seq for identification of genes required for optimal growth of UPEC in human urine, as well as potential virulence genes upregulated during infection of bladder cell culture. DFI-seq holds potential for the study of bacterial gene expression in live-animal infection systems. By linking fitness genes, such as those genes involved in amino acid biosynthesis, to virulence, this study contributes to our understanding of UPEC pathophysiology.

Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease–associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that enhances their persistence.

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae.

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU.

Human Urine Decreases Function and Expression of Type 1 Pili in Uropathogenic Escherichia coli.

ABSTRACTUropathogenic Escherichia coli (UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by the fim operon in nearly all E. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing the fimS promoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces the fimS phase OFF orientation, preventing fim expression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias in fimS orientation toward the phase OFF state. The dual effect of urine on fimS regulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such that fim expression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce the fimS phase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function.

1152 RENAL PROTEIN PHOSPHATASE 1A (PPM1A) REDUCTION ACCOMPANIES PATHOLOGIC HYPERACTIVATION OF TRANSFORMING GROWTH FACTOR-BETA (TGF-β1) / SMAD SIGNALING IN OBSTRUCTIVE UROPATHY

RNA extraction. A 3-way comparison approach was used to compare gene expression in human clinical samples to the cultures derived from either laboratory medium or human urine in vitro. Total RNA was extracted from urine samples and eukaryotic RNA was depleted before constructing cDNA libraries. Additionally, the genomes of these 5 clinical UPEC isolates were sequenced. RESULTS: Genes in the UPEC strain CFT073 that exhibit more than 2-fold change in expression in human urine compared to laboratory medium were identified; 1443 genes (26.8%) were up-regulated and 348 genes (6.5%) were down-regulated. The cDNA and DNA samples from human UTI samples have been sequenced successfully. At the time of submission of this abstract, bioinformatic analyses are being conducted to quantify gene expression in UPEC during human infection. CONCLUSIONS: Expression of the genes involved in amino acid uptake and biosynthetic pathways, capsule biosynthesis, fimbrial biogenesis, iron acquisition and peptide uptake are critical for growth in human urine. These genes and pathways represent novel targets that could be exploited to combat UPEC.

Escherichia coli Isolates That Carry vat , fyuA , chuA , and yfcV Efficiently Colonize the Urinary Tract

Extraintestinal Escherichia coli (ExPEC), a heterogeneous group of pathogens, encompasses avian, neonatal meningitis, and uropathogenic E. coli strains. While several virulence factors are associated with ExPEC, there is no core set of virulence factors that can be used to definitively differentiate these pathotypes. Here we describe a multiplex of four virulence factor-encoding genes, yfcV, vat, fyuA, and chuA, highly associated with uropathogenic E. coli strains that can distinguish three groups of E. coli: diarrheagenic and animal-associated E. coli strains, human commensal and avian pathogenic E. coli strains, and uropathogenic and neonatal meningitis E. coli strains. Furthermore, human intestinal isolates that encode all four predictor genes express them during exponential growth in human urine and colonize the bladder in the mouse model of ascending urinary tract infection in higher numbers than human commensal strains that do not encode the four predictor genes (P = 0.02), suggesting that the presence of the predictors correlates with uropathogenic potential.

Klebsiella pneumoniae Yersiniabactin Promotes Respiratory Tract Infection through Evasion of Lipocalin 2

Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent(+)) (81%), enterobactin and yersiniabactin positive (Ent(+) Ybt(+)) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent(+) gly-Ent(+)) with or without Ybt (2%). Ent(+) Ybt(+) strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and β-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent(+) strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2(-/-) mice. In contrast, an Ent(+) Ybt(+) strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

Identification of In Vivo -Induced Antigens Including an RTX Family Exoprotein Required for Uropathogenic Escherichia coli Virulence

Uncomplicated urinary tract infections (UTI) are caused most commonly by uropathogenic Escherichia coli (UPEC). Whole-genome screening approaches, including transcriptomic, proteomic, and signature-tagged mutagenesis, have shown that UPEC highly expresses or requires genes for translational machinery, capsule, lipopolysaccharide, type 1 fimbriae, and iron acquisition systems during UTI. To identify additional genes expressed by UPEC during UTI, an immunoscreening approach termed in vivo-induced antigen technology (IVIAT) was employed to identify antigens produced during experimental infection that are not produced during in vitro culture. An inducible protein expression library, constructed from genomic DNA isolated from UPEC strain CFT073, was screened using exhaustively adsorbed pooled sera from 20 chronically infected female CBA/J mice. Using this approach, we identified 93 antigens induced by UPEC in vivo. A representative subset of these genes was tested by quantitative PCR for expression by CFT073 in vivo and during growth in human urine or LB medium in vitro; proWX, narJI, lolA, lolD, tosA (upxA), c2432, katG, ydhX, kpsS, and yddQ were poorly expressed in vitro but highly expressed in vivo. Of these, tosA, a gene encoding a predicted repeat-in-toxin family member, was expressed exclusively during UTI. Deletion of tosA in UPEC strain CFT073 resulted in significant attenuation in bladder and kidney infections during ascending UTI. By screening for in vivo-induced antigens, we identified a novel UPEC virulence factor and additional proteins that could be useful as potential vaccine targets.

Comparative Genomic Analysis of Pathogenic and Probiotic Enterococcus faecalis Isolates, and Their Transcriptional Responses to Growth in Human Urine

Urinary tract infection (UTI) is the most common infection caused by enterococci, and Enterococcus faecalis accounts for the majority of enterococcal infections. Although a number of virulence related traits have been established, no comprehensive genomic or transcriptomic studies have been conducted to investigate how to distinguish pathogenic from non-pathogenic E. faecalis in their ability to cause UTI. In order to identify potential genetic traits or gene regulatory features that distinguish pathogenic from non-pathogenic E. faecalis with respect to UTI, we have performed comparative genomic analysis, and investigated growth capacity and transcriptome profiling in human urine in vitro. Six strains of different origins were cultivated and all grew readily in human urine. The three strains chosen for transcriptional analysis showed an overall similar response with respect to energy and nitrogen metabolism, stress mechanism, cell envelope modifications, and trace metal acquisition. Our results suggest that citrate and aspartate are significant for growth of E. faecalis in human urine, and manganese appear to be a limiting factor. The majority of virulence factors were either not differentially regulated or down-regulated. Notably, a significant up-regulation of genes involved in biofilm formation was observed. Strains from different origins have similar capacity to grow in human urine. The overall similar transcriptional responses between the two pathogenic and the probiotic strain suggest that the pathogenic potential of a certain E. faecalis strain may to a great extent be determined by presence of fitness and virulence factors, rather than the level of expression of such traits.

Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.