The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis CAUTI incidence and severity.
The Gram-negative bacterium Proteus mirabilis thrives in a wide variety of environments, including soil, water sources, sewage, and as a commensal in the intestinal tract of humans and animals [1,2]. P. mirabilis has also been identified as the causative agent of numerous human illnesses including cystitis, pyelonephritis, prostatitis, as well as intra-abdominal, wound, eye, and burn infections [2]. While it is capable of causing uncomplicated urinary tract infections (UTIs), this organism is a much more common cause of catheter-associated UTI (CAUTI) [3–5]. Indeed, we recently identified P. mirabilis as the most common cause of CAUTIs in twelve Michigan nursing homes [6]. CAUTIs are frequently polymicrobial [7,8], and P. mirabilis is one of the most common organisms present during polymicrobial urine colonization and infection [3,6]. UTIs and CAUTIs involving P. mirabilis are typically complicated by the formation of bladder and kidney stones (urolithiasis) and permanent renal damage [9–11], and may progress to bacteremia [12,13]. Despite these potentially severe complications of P. mirabilis infection, there are no currently licensed vaccines available for this organism and multidrug-resistant isolates are increasingly common [14,15]. No previous studies have explored P. mirabilis genes essential for growth in vitro, but numerous fitness and virulence factors have been examined in a murine model of ascending UTI, including but not limited to urease, fimbriae, and hemolysin (see [2,16] for review). Fitness factors for colonization of the urinary tract have also been explored in three non-saturating signature-tagged mutagenesis (STM) studies in the ascending UTI model using P. mirabilis strain HI4320, which achieved a combined 70% theoretical coverage of the genome and revealed traditional virulence factors, metabolic pathways important for infection, and fitness factors with no prior links to P. mirabilis pathogenicity [17–19]. For P. mirabilis infection studies, the murine model of ascending UTI is generally considered to represent complicated UTI due to the occurrence of urolithiasis in this infection model [20]. However, we recently adapted a murine model of CAUTI for investigation of P. mirabilis pathogenicity, and verified that maintenance of a catheter segment within the bladder dramatically increases inflammation and infection severity [21]. Thus, different genes and pathways are likely required for fitness in the catheterized bladder environment than those identified in the ascending UTI model. An additional consideration in identifying fitness and virulence factors of P. mirabilis is the frequent involvement of this organism in polymicrobial infections. We recently determined that other uropathogens, such as Escherichia coli, Enterococcus species, and Providencia stuartii, are capable of influencing P. mirabilis urease activity [21]. Co-culture with P. stuartii also enhanced P. mirabilis cytotoxicity independent of urease activity, altered the immune response to infection, and resulted in greater tissue damage, indicating that other virulence factors are affected by polymicrobial interactions [21]. All prior studies of P. mirabilis fitness factors have been conducted with pure cultures of P. mirabilis in isolation, yet the potential clearly exists for other organisms to influence expression of P. mirabilis virulence factors, metabolic requirements, and factors required for adaptation to changes in the bladder environment in response to the presence of a second pathogen. In this study, we generated a genome-saturating library of transposon mutants and utilized transposon insertion-site sequencing (Tn-Seq) to identify P. mirabilis genes essential for growth in rich medium and the full arsenal of fitness factors for single-species infection in a CAUTI model, while concurrently determining how fitness requirements changed during polymicrobial infection with P. stuartii, a common co-colonizing partner of P. mirabilis [3,4, 22–25].
Based on the P. mirabilis HI4320 genome size (4. 063 Mbp, with approximately 3747 genes [26]), it is estimated that 34,249 transposon mutants are required for 99. 99% probability of full genome coverage [27]. A similar strategy as detailed by Crimmins et al. [28] for maximum colonization density was used to determine the appropriate transposon library pool density. From our recent investigation of experimental CAUTI in CBA/J mice, the minimum colonization density achieved by P. mirabilis in the urine, bladder, and kidneys of mice 4-days post inoculation is expected to be ~1x104 CFU [21]. Preliminary experiments confirmed a minimum bladder colonization of 1x104 CFU/gram of tissue at 4-h and 4-days post-inoculation, and indicated lack of a significant bottleneck in the CAUTI model (S1 Fig). We therefore concluded that generation of transposon pools containing 1x104 transposon mutants would be ideal. Approximately 50,000 transposon mutants from three independent matings were collected and pooled in groups of 1x104 mutants to generate five transposon mutant libraries. Randomness of insertions was verified by Southern blot, and the majority of mutants harbored only one transposon insertion as expected (S2 Fig). Raw Illumina reads were filtered and transposon insertion-sites were uniquely mapped to genomic coordinates using a method adapted from the previously published work by Goodman et al. [29]. Saturation was achieved for the P. mirabilis HI4320 chromosome and 36 Kb plasmid (pHI4320), and the majority of insertion-sites were represented in three or more input pools (Fig 1). However, there were a few noticeable gaps in both the chromosome and plasmid maps. To determine if these gaps represented genes essential for growth of P. mirabilis in LB medium, a Bayesian mixture model was used to estimate essentiality based on absence or underrepresentation of transposon insertions in these genes within the input pools (see Materials and methods). The model identified 436 genes (11. 6% of the 3747 genes encoded by P. mirabilis HI4320) as potentially essential for P. mirabilis growth (S1 Table), 279 (64. 0%) of which were present in the bacterial section of the Database of Essential Genes (DEG, http: //www. essentialgene. org/). Three hundred thirty-three of the estimated essential genes had a Cluster of Orthologous Groups of proteins (COG) assignment (Fig 2). Among the list of P. mirabilis estimated essential genes are numerous genes listed as essential in the majority of bacterial species present in DEG, including genes pertaining to cell cycle control and division (such as the fts cell division proteins), cell wall biogenesis, replication (including DNA polymerase I, DNA polymerase III, and DNA gyrase), RNA polymerase σ70 (rpoD), σ32 (rpoH), and σ24 (rpoE), nucleoid protein H-NS (hns), numerous ribosomal proteins and tRNA synthetases, and ATP synthase. There are five ATP-dependent proteases in bacteria that comprise the majority of energy-dependent protein degradation: HflB (FtsH), the Clp proteases (ClpAP, ClpXP, and ClpYQ), and the Lon protease [30]. Insertions in three of these proteases (HflB, ClpAP, and ClpXP) were absent from the input pools and therefore identified as essential for growth of P. mirabilis in rich medium, underscoring the importance of degrading misfolded and unstable proteins for P. mirabilis growth in rich medium. The replication initiation protein PMIP01 on the P. mirabilis plasmid (pHI4320) was also identified as essential. Infection studies to screen for P. mirabilis fitness factors during single-species and polymicrobial CAUTI were conducted in parallel to utilize the same inocula as the input samples for each infection type. A conceptual model of the infection and sequencing scheme is shown in Fig 3. For each of the five transposon library pools, 5–10 CBA/J mice were transurethrally inoculated with 1x105 CFU (10X coverage of each mutant within the pool), and 5–10 CBA/J mice were inoculated with 1x105 CFU of a 1: 1 mixture of the transposon library and wild-type P. stuartii BE2467 (5x104 CFU of the P. mirabilis transposon mutant library for 5X coverage of each mutant within the pool). In all cases, a 4 mm segment of sterile silicone catheter tubing was carefully advanced into the bladder during inoculation and retained for the duration of the study as described previously [21]. Due to the level of encrustation in mice infected with urease-positive organisms, the catheter segments were generally embedded in the bladder tissue and therefore were not removed prior to homogenization of bladder samples. Thus, all CFUs recovered from bladder samples actually represent colonization of the catheter segment as well as the bladder. Single-species infections and coinfections with the P. mirabilis transposon mutant pools resulted in comparable bladder and kidney colonization in all mice (Fig 4A and 4B). Differential plating revealed that the majority of coinfected mice were highly colonized by both P. mirabilis and P. stuartii, as expected (Fig 4C). In cases where an individual mouse exhibited low colonization by P. mirabilis in either the bladder or kidneys (<1x104 CFU), the samples from that mouse were excluded from further study. Bladder and kidney output samples for sequencing were chosen from the most highly colonized mice as follows: four mice from the single-species infection group and four from the coinfection group, per transposon pool, resulting in 20 bladder samples and 20 kidney samples from each infection type. A fitness index was calculated for each gene by taking into account the number of unique insertion-sites within that gene and the depth of reads at each insertion-site for the combined output samples recovered from the organs of infected mice compared to the combined input pools (see Materials and methods). Tn-Seq from single-species CAUTI identified 629 genes (16. 8% of the total genes encoded by P. mirabilis HI4320) as candidate fitness factors. Due to the stringent cutoffs that were used for estimation of fitness factors (see Materials and methods section), the range of fitness defects was compressed, spanning a 2- to 40-fold reduction in recovery from output samples compared to the input samples. The top candidate fitness factors for colonization of the catheterized bladder (excluding tRNAs, pseudogenes, and genes not present in COG) are shown in Table 1. Four of these genes were previously identified by signature-tagged mutagenesis (STM) in the ascending UTI model [18,19], and eight were previously shown to be upregulated by P. mirabilis in the ascending UTI model [31]. Importantly, many of the top hits for bladder colonization represent multiple genes in an operon, including the phosphate ABC transporter (pstSCAB) and the cytochrome bo3 quinol oxidase (cyoABCDE). Top candidate fitness factors for kidney colonization (again excluding tRNAs, pseudogenes, and genes not present in COG) are shown in Table 2. These include the same four genes identified by STM that were important for bladder colonization, and fourteen genes that were upregulated by P. mirabilis in the ascending UTI model [31]. Upon further analysis, fitness factors for single-species CAUTI fell into three categories: 93 genes for colonization of the catheterized bladder that were not significant for kidney colonization (S2 Table), 209 genes for kidney colonization that were not significant for bladder colonization (S3 Table), and 286 genes that were important for colonization of both organs (S4 Table). Fitness factors for colonization of each organ were randomly distributed across the chromosome and plasmid (S3 Fig). Of the 286 genes important for both bladder and kidney colonization, 203 were present in COG: 24 pertained to transcription (10. 9% of functional category assignments); 21 (9. 5%) translation, ribosomal structure, and biogenesis; 19 (8. 6%) amino acid transport and metabolism; 19 (8. 6%) post-translational modification, protein turnover, and chaperones; 18 (8. 2%) energy production and conversion; and 16 (7. 3%) cell wall and envelope biogenesis (Fig 5A). Regarding fitness factors for bladder colonization alone, 64 of the 93 candidate factors were present in COG and revealed a greater proportion of genes pertaining to amino acid transport, carbohydrate transport and metabolism, and inorganic ion transport and metabolism than the genes important for colonization of both organs, and fewer candidate genes pertaining to translation and cell wall biogenesis (Fig 5B). Interestingly, the candidate fitness factors for kidney colonization alone followed a similar distribution as the genes important for colonization of both the bladder and kidneys with a few notable exceptions, including fewer genes pertaining to translation or carbohydrate transport and metabolism, and more pertaining to replication, recombination, and repair and extracellular structures. (Fig 5C). Thus, functional categories important for kidney colonization are likely to be involved in fitness during bladder colonization as well. Overall, of the fifty-four P. mirabilis fitness factors previously identified by signature-tagged mutagenesis (STM) in the ascending UTI model [17–19], 31 (57%) were significant for CAUTI (denoted with a “*” in the supplemental tables). These include the phosphate ABC transporter (pstSCAB), pyruvate dehydrogenase (aceE and aceF), an AsnC transcriptional regulator (PMI1431), a d-methionine ABC transporter protein (metN), inosine-5’-monophosphate dehydrogenase (guaB), a bifunctional polymyxin resistance protein (arnA), a polysaccharide deacetylase next to the Arn operon (PMI1046), a dihydrouridine synthase (dusB), and a hypothetical protein (PMI3700). Urease is another well-known fitness factor for P. mirabilis UTI and prior STM studies that we previously verified as important for colonization in the CAUTI model [21], and a urease accessory protein (ureG) required for the catalytically-active enzyme was also identified in the present CAUTI Tn-Seq. Tn-Seq from coinfection with P. stuartii revealed 1570 candidate P. mirabilis fitness factors, ranging from 2- to 30-fold reduction of transposon insertions in these genes from the combined output samples compared to the input samples. Candidate fitness factors represented 217/629 (34. 5%) of the genes identified above for single-species CAUTI, indicating substantial overlap between the two infection models, as well as 1353 additional factors that may specifically contribute to colonization during coinfection. Sixty-two genes were candidate fitness factors for bladder colonization alone (S5 Table), 10 of which were also important for bladder colonization during single-species infection. These bladder-specific fitness factors, regardless of infection type, include two members of a carbohydrate ABC transporter (ugpC and ugpE), a nucleoid-associated protein (ndpA), the anti σE protein (rseA), an HxlR-family transcriptional regulator, and a siderophore receptor (ireA). 213 genes were candidate fitness factors for kidney colonization alone (S6 Table), 61 of which were also important for kidney colonization during single-species infection. The majority of the kidney-specific fitness factors, regardless of infection type, pertained to amino acid transport and metabolism (including the d-serine ammonia-lyase dsdA and the l-serine ammonia lyase sdaA), cell wall and envelope biogenesis, energy production and conversion (including succinate dehydrogenase and formate acetyltransferase), posttranslational modification and protein turnover (sufD, an iron-regulated ABC transporter permease protein, and surA, a periplasmic chaperone for outer membrane proteins), transcriptional regulation (including the phoP two-component response regulator, and an RpiR-family transcriptional regulator encoded by PMI2974), and four plasmid-encoded genes (PMIP09, which encodes the pilX4 conjugal transfer protein, PMIP10, PMIP28, and PMIP30). The Sec-independent protein translocase TatC (twin arginine transporter) was also identified as important for kidney colonization but not bladder colonization in both infection types. 909 genes were important for both bladder and kidney colonization during coinfection (S7 Table), 717 of which were present in COG and primarily pertained to transport and metabolism of amino acids, inorganic ions, carbohydrates, as well as energy production and conversion (Fig 10A). The COG functional categories represented by P. mirabilis fitness factors during coinfection followed a similar trend as single-species infection: fitness factors for bladder colonization alone revealed a greater proportion of genes pertaining to translation and transcription compared to the genes important for colonization of both organs (Fig 10B), and fitness factors for kidney colonization alone followed a similar distribution as genes important for colonization of the bladder and kidneys, albeit with an increase in factors with unknown function and a decrease in factors pertaining to cell motility (Fig 10C). Fifteen of the genes important for colonization of both the bladder and kidneys during coinfection were also important for colonization of both organs during single-species infection. Seven of these genes have unknown functions or are not in COG; the other genes pertain to translation, ribosomal structure, and biogenesis (dusC, PMI3283, and rimI, an alanine acetyltransferase), cell wall/envelop biogenesis and defense (mdtA, a multidrug resistance efflux transporter), motility (PMI2642), and inorganic ion transport and metabolism (ppaA). The factors encoded by these genes appear to represent a putative core set of fitness requirements for P. mirabilis colonization of the murine urinary tract. Intriguingly, there were also 109 genes for which transposon insertions resulted in a fitness defect during single-species infection but provided a competitive advantage during polymicrobial CAUTI (S8 Table). Seventy-nine of these genes were present in COG, and included factors involved in energy production and conversion, transcription, carbohydrate transport and metabolism, inorganic ion transport (particularly iron and phosphate), amino acid transport and metabolism, and nucleotide transport and metabolism.
The majority of the large regions of the chromosome that lacked transposon insertions and were visible as gaps in Fig 1 did indeed correspond to genes identified as essential. For instance, the largest region that lacked transposon insertions was 12,423 bp encompassing 18 genes involved in cell division and cell wall biogenesis. The next largest regions that lacked transposon insertions were a 7,543 bp region encompassing genes necessary for translation, and a 6,650 bp region encompassing an intergenic region and murI, a glutamate racemase that is essential for cell wall biosynthesis. However, this was not always the case for gaps in transposon insertions on plasmid pHI4320. The largest region lacking insertions on the plasmid was 1,252 bp encompassing an intergenic region and a portion of PMIP30, which encodes a putative colicin. This gene was not identified as essential as there were 81 insertion sites after the gap which were well-represented in the input pools. Another gap on the plasmid of 826 bp corresponds to a portion of PMIP42, a putative RelB antitoxin. Similar to PMIP30, PMIP42 contained two insertion sites after the gap that were highly represented in the input pools, which likely caused it to fall below the threshold for being considered essential. Thus, the model used to identify putative essential genes provided very conservative estimates, and is likely an underrepresentation of the genes required for optimal growth of P. mirabilis in LB. It is therefore notable that the other ~500 bp gaps in transposon insertions on the plasmid were within a conjugal transfer protein (PMIP09), a colicin immunity protein (PMIP31), a plasmid stability/partitioning protein (PMIP34), a type IA DNA topoisomerase (PMIP25), and HN-S family protein (PMIP26). These findings are in agreement with the replication initiation protein (PMIP01) being essential, and underscore the importance of plasmid maintenance and replication to P. mirabilis growth. In addition to the numerous genes previously identified as essential for growth in other bacterial species, the list of P. mirabilis estimated essential genes contained a few unusual items. For instance, all but one of the genes in the non-oxidative pentose phosphate pathway were identified as putative essential genes (rpiA, rpe, tktA, and talB), indicating a central role for this pathway during growth of P. mirabilis in rich medium. Phosphoglycerate kinase (pgk), which is involved in glycolysis, gluconeogenesis, and glycerol degradation, was also identified as a possible essential gene. Factors involved in inorganic ion transport and metabolism were a surprising find, as these genes are not commonly identified as essential. These included genes involved in potassium uptake (trkA and trkH), intracellular sulfur oxidation (PMI2797 and PMI2798), tellurite resistance (terC), a sulfite reductase (PMI0794), and magnesium and cobalt efflux (corC). TrkA and TrkH comprise a high-rate low-affinity potassium-translocating system that requires ATP via the Sap system [37], and all members of the Sap dipeptide transport system transcriptional unit (sapBCDF) were also identified as putative essential genes. Six fimbrial genes were also identified as potential essentials, including three homologs of the mrpJ fimbrial operon regulator [38]. Little is known concerning the identified fimbrial genes (pmpB, fim3J, fim5G, fim7D, fim8J, fim10D, and fim10J), or why disruption of these genes would impact growth in rich medium. Three of these fimbrial genes encode homologs of mrpJ, a transcriptional regulator that represses motility but also influences expression of other adhesins, virulence factors, and metabolic pathways [39]. Homologs of mrpJ are also capable of regulating motility and adherence [38], so it is possible that these mrpJ homologs similarly play a role in a broad regulatory network that could contribute to growth, or that tight regulation of the expression of these operons is important during growth in rich medium. Two homologs of the type VI secretion system (T6SS) secreted protein Hcp (PMI0750 and PMI1117) were also identified as putative essential genes, as well as 17 transposases and 1 phage repressor protein. In each of these cases, the identified genes contained >15 TA sites for transposon insertion, but <3 total insertions were recovered from the five combined input pools. While these genes may be important for growth in rich medium, it is also possible that the mariner transposon wasn’t able to efficiently target these chromosomal locations. Thus, the importance of the unique genes estimated to be essential for P. mirabilis should be interpreted with caution. As mentioned above, the genes involved in putrescine uptake and biosynthesis were only identified as fitness requirements for P. mirabilis single-species CAUTI and not polymicrobial CAUTI, indicating that the presence of P. stuartii alleviates the polyamine requirement of P. mirabilis. The same appears to be true of the high-affinity zinc transport system encoded by znuABC. Stress responses also appear to be more important during single-species CAUTI than coinfection, as the majority of the stress-related genes identified as P. mirabilis fitness factors for single-species CAUTI were not significant during coinfection. Similarly, glutamine synthetase (glnA) was important for single-species fitness but not coinfection, further underscoring the differences in metabolic pathways favored by P. mirabilis during these two infection types. Further research is needed to determine which of these shifts in fitness requirements are specifically due to P. stuartii and which are due to the altered host environment and response to coinfection compared to P. mirabilis single-species infection. The combination of genome-saturating transposon mutant libraries and Tn-Seq has allowed for the first global estimation of P. mirabilis essential genes, validation of numerous P. mirabilis virulence factors and fitness determinants from decades of studies using the ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, proteobactin, a yersiniabactin-related siderophore (nrp), and heme receptors were promising targets for perturbing P. mirabilis ascending UTI [56], but these factors do not appear to be important for fitness during single-species infection in the CAUTI model. BCAA biosynthesis is an intriguing target for reducing bacterial colonization, but our results indicate that this pathway is only important for P. mirabilis during polymicrobial infection and therefore would not be a suitable target for single-species infection by P. mirabilis. In contrast, our data indicate that polyamine uptake and biosynthesis may be promising targets for perturbing P. mirabilis single-species CAUTI, but these pathways were not important during coinfection, which may limit their potential as therapeutic targets given the high frequency of P. mirabilis polymicrobial colonization and infection. Further research is needed concerning the numerous genes and pathways that provided a competitive advantage to P. mirabilis during both single-species and polymicrobial CAUTI, as the underlying mechanisms of these fitness requirements are the most likely to provide conserved targets for therapeutic intervention aimed at reducing P. mirabilis colonization or minimizing risk of progression to severe infection and urosepsis. We have clearly demonstrated that the CAUTI coinfection model can be used to examine the interplay between fitness requirements for both species during coinfection. It is therefore likely that investigation of the fitness requirements of other common CAUTI pathogens for single-species and polymicrobial CAUTI will further elucidate complex bacterial interactions that contribute to disease severity, and may even uncover conserved bacterial targets for therapeutic intervention.
Proteus mirabilis is a common cause of single-species and polymicrobial catheter-associated urinary tract infections (CAUTIs). Prior studies have uncovered P. mirabilis virulence factors for single-species ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. Using transposon insertion-site sequencing (Tn-Seq), we performed a global assessment of P. mirabilis fitness factors for CAUTI while simultaneously determining how coinfection with another CAUTI pathogen, Providencia stuartii, alters P. mirabilis fitness requirements. This approach provides six important contributions to the field: 1) the first global estimation of P. mirabilis genes essential for growth, 2) validation of a role for known P. mirabilis fitness factors during CAUTI, 3) identification of novel fitness factors, 4) identification of core fitness factors for both single-species and polymicrobial CAUTI, 5) identification of single-species fitness factors that are complemented during polymicrobial infection, and 6) identification of factors that only provide a competitive advantage during polymicrobial infection. We further demonstrate that the CAUTI model can be used to examine the interplay between fitness requirements of both species during coinfection. Investigation of fitness requirements for other pathogens during single-species and polymicrobial CAUTI will elucidate complex interactions that contribute to disease severity and uncover conserved targets for therapeutic intervention.
medicine and health sciences
pathology and laboratory medicine
evolutionary biology
pathogens
population genetics
bladder
microbiology
gene pool
genetic elements
population biology
kidneys
proteus mirabilis
bacterial pathogens
infectious diseases
medical microbiology
microbial pathogens
biochemistry
anatomy
polymicrobial infections
genetics
co-infections
biology and life sciences
transposable elements
renal system
genomics
mobile genetic elements
biosynthesis
Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements