Machine Learning with Alpha Toxin Phenotype to Predict Clinical Outcome in Patients with Staphylococcus aureus Bloodstream Infection.

Staphylococcus aureus bloodstream (SAB) infection remains a leading cause of sepsis-related mortality. Yet, current treatment does not account for variable virulence traits that mediate host dysregulated immune response, such as SA α-toxin (Hla)-mediated thrombocytopenia. Here, we applied machine learning (ML) to bacterial growth images combined with platelet count data to predict patient outcomes. We profiled Hla phenotypes of SA isolates collected from patients with bacteremia by taking smartphone images of beta-hemolytic growth on sheep blood agar (SBA). Electronic medical records were reviewed to extract relevant laboratory and clinical data. A convolutional neural network was applied to process the plate image data for input along with day 1 patient platelet count to generate ML-based models that predict thrombocytopenia on day 4 and mortality. A total of 229 patients infected with SA strains exhibiting varying zone sizes of beta-hemolysis on SBA were included. A total of 539 images of bacterial growth on SBA were generated as inputs for model development. One-third of patients developed thrombocytopenia at onset, with an overall mortality rate of 18.8%. The models developed from the ML algorithm showed strong performance (AUC 0.92) for predicting thrombocytopenia on day 4 of infection and modest performance (AUC 0.711) for mortality. Our findings support further development and validation of a proof-of-concept ML application in digital microbiology, with a measure of bacterial virulence factor production that carries prognostic significance and can help guide treatment selection.

Antimicrobial Activity of Tebipenem and Comparators against Enterobacterales from diverse Outpatient Centers and Nursing Homes in the United States.

INTRODUCTION: Tebipenem is a potential option for the treatment of a range of infections because of its oral dosing coupled with the safety profile of the ß-lactam antimicrobial class. OBJECTIVES: To evaluate tebipenem in vitro activity against a challenge set of clinical Enterobacterales collected from outpatient and community settings. METHODS: 618 Enterobacterales isolates were submitted by 11 geographically dispersed U.S medical centers that processed cultures from affiliated outpatient centers in 2022. Susceptibility tests for tebipenem and comparator agents were performed by broth microdilution. Extended-spectrum-ß-lactamase (ESBL)-like isolates were identified phenotypically. Multidrug-resistant isolates were non-susceptible to ≥1 agent in ≥3 antimicrobial classes. Genotypic testing (CarbaR) was conducted on select isolates. RESULTS: Isolates (59% Escherichia coli) were recovered from patients seen predominantly in urology/nephrology (24%), nursing home/long-term care (21%), and ambulatory/primary care (21%) clinics. Comparator agent susceptibility rates against all isolates were as follows: levofloxacin (67.5%), amoxicillin/clavulanate (73.6%), cefixime (70.4%), cefpodoxime (70%), cephalexin (61.7%), ceftriaxone (74.4%), cefazolin (63.8%), ertapenem (97.6%), meropenem (99.7%), nitrofurantoin (64.9%), and sulfamethoxazole/trimethoprim (70.9%). Overall, 90.3% (558/619) of isolates were inhibited at a tebipenem MIC of ≤0.125 mg/L (MIC50/90, 0.016/0.125 mg/L), including 85.7% inhibition of ESBL-phenotype isolates (n=161; MIC50/90, 0.03/0.25 mg/L), 86.3% of levofloxacin and sulfamethoxazole/trimethoprim co-resistant isolates (n=95; MIC50/90, 0.016/0.25 mg/L) and 84.3% of multidrug-resistant isolates (n = 172; MIC50/90, 0.03/0.25 mg/L). Carbapenemase genes were observed in 2 ESBL-phenotype isolates with a tebipenem MIC of ≥0.5 mg/L. CONCLUSION: Relative to common oral comparators, these data demonstrate excellent tebipenem in vitro activity against Enterobacterales isolated from patients receiving care in outpatient settings, including urology clinics and nursing homes.

CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.

Factors associated with prompt recovery among hospitalised patients with coronavirus disease 2019.

BACKGROUND: Patients who survived hospitalisation for COVID-19 experienced varying durations of illness but the factors associated with prompt recovery are unknown. This study identifies factors differentiating hospitalised patients who recovered promptly versus survived a prolonged course of illness because of COVID-19. METHODS: This was a retrospective study from March-August 2020 of hospitalised adults with COVID-19 which were grouped based on time to recovery: short (≤3 days), intermediate (4-10 days) and prolonged (>10 days). Recovery was defined as resolution of fever, tachypnea, hypotension, extubation and return of mental status at baseline. Multivariate analysis was used to evaluate factors associated with prompt recovery. RESULTS: Among 508 patients hospitalised for COVID-19, 401 (79%) survived. Of those, prompt recovery (within 3 days) was achieved in 43% (174/401), whereas 23% (92/401) recovered after a prolonged period of >10 days. Overall, median age was 64 years with 73% admitted from home and 25% from a skilled nursing facility. Predictors for prompt recovery upon admission included female sex (OR, 1.8; 95% CI, 1.1-2.7; P = .01), no fever (OR, 1.6; 95% CI, 1.1-2.6; P = .03), longer time from symptom onset to hospitalisation (OR, 1.1; 95% CI, 1.0-1.1; P = .001), no supplemental oxygen (OR, 1.9; 95% CI, 1.2-3.0; P = .004), no direct ICU admission (OR, 41.7; 95% CI, 2.4-740.4; P = .01) and absence of bacterial co-infections (OR, 2.5; 95% CI, 1.5-4.0, P = .0003). CONCLUSIONS: Our study provides relevant data that could help clinicians triage competing resources in health systems that are challenged by the ebb and flow of COVID-19 cases by identifying clinical features of COVID-19 patients who may require less intensive management including avoidance of unnecessary antibacterial therapy.

Evaluation of Antimicrobial Susceptibility Testing Methods for Burkholderia cenocepacia and Burkholderia multivorans Isolates from Cystic Fibrosis Patients.

The Burkholderia cepacia complex (BCC) is known for causing serious lung infections in people with cystic fibrosis (CF). These infections can require lung transplantation, eligibility for which may be guided by antimicrobial susceptibility testing (AST). While the Clinical and Laboratory Standards Institute recommends AST for BCC, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) does not, due to poor method performance and correlation with clinical outcomes. Furthermore, limited data exist on the performance of automated AST methods for BCC. To address these issues, reproducibility and accuracy were evaluated for disk diffusion (DD), broth microdilution (BMD), and MicroScan WalkAway using 50 B. cenocepacia and 50 B. multivorans isolates collected from people with CF. The following drugs were evaluated in triplicate: chloramphenicol (CAM), ceftazidime (CAZ), meropenem (MEM), trimethoprim-sulfamethoxazole (TMP-SMX), minocycline (MIN), levofloxacin (LVX), ciprofloxacin (CIP), and piperacillin-tazobactam (PIP-TAZ). BMD reproducibility was ≥ 95% for MEM and MIN only, and MicroScan WalkAway reproducibility was similar to BMD. DD reproducibility was < 90% for all drugs tested when a 3 mm cut-off was applied. When comparing the accuracy of DD to BMD, only MEM met all acceptance criteria. TMP-SMX and LVX had high minor errors, CAZ had unacceptable very major errors (VME), and MIN, PIP-TAZ, and CIP had both unacceptable minor errors and VMEs. For MicroScan WalkAway, no drugs met acceptance criteria. Analyses also showed that errors were not attributed to one species. In general, our data agree with EUCAST recommendations.

Risk factors and outcome associated with infection or colonization due to carbapenem-heteroresistant Escherichia coli.

BACKGROUND: Up to 32% of ESBL-producing Enterobacterales strains display a carbapenem-heteroresistant (cHR) phenotype but its clinical relevance is unknown. OBJECTIVES: To determine risk factors and clinical outcome associated with infection due to cHR ESBL-producing Escherichia coli (ESBL-EC). METHODS: A retrospective, case-control study was conducted on patients from whom a pair of clonally related E. coli strains were isolated during separate healthcare encounters with (case) or without (control) development of cHR phenotype in the latter strain. Study groups were compared for host and microbial characteristics and carbapenem exposure. Outcome measures included ICU admission, length of hospitalization, and mortality. RESULTS: Study patients (15 cases, 10 controls) were elderly (median age: 74 years) with half admitted from home (52%), most (80%) having ≥3 comorbid conditions and severe functional impairment. Case patients were more likely to have 'index' ESBL-EC isolating from blood (27% versus 0%; P = 0.125) and have greater cumulative amount and duration of carbapenem exposure than controls. All control 'subsequent' isolates were from urine whereas five cHR case isolates were from blood or respiratory sources. More hospitalized case patients required ICU admission (23% versus 0%; P = 0.257) and prolonged hospital stay (>7 days) than controls (62% versus 38%%; P = 0.387). CONCLUSIONS: Our findings deserve confirmation with a larger study population and call attention to the potential for increased morbidity with cHR ESBL-EC infections, which underscores the need to screen for cHR phenotype in patients with repeated growth of ESBL-EC, particularly from systemic sites and patients that have had extensive carbapenem exposure.

Contemporary analysis of ETEST for antibiotic susceptibility and minimum inhibitory concentration agreement against Pseudomonas aeruginosa from patients with cystic fibrosis.

OBJECTIVES: Cystic fibrosis (CF) acute pulmonary exacerbations are often caused by Pseudomonas aeruginosa, including multi-drug resistant strains. Optimal antibiotic therapy is required to return lung function and should be guided by in vitro susceptibility results. There are sparse data describing ETEST performance for CF isolates using contemporary isolates, methods and interpretation, as well as novel antibiotics, such as ceftazidime-avibactam and ceftolozane-tazobactam. METHODS: Pseudomonas aeruginosa (n = 105) isolated during pulmonary exacerbation from patients with CF were acquired from 3 US hospitals. Minimum inhibitory concentrations (MICs) were assessed by reference broth microdilution (BMD) and ETEST for aztreonam, cefepime, ceftazidime, ceftazidime-avibactam, ceftolozane-tazobactam, ciprofloxacin, levofloxacin, meropenem, piperacillin-tazobactam, and tobramycin. Broth microdilution was conducted in concordance with the Clinical and Laboratory Standards Institute M100. ETEST methodology reflected package insert recommendations. Performance of ETEST strips was evaluated using the Food and Drug Administration (FDA) and Susceptibility Testing Manufacturers Association (STMA) guidance. RESULTS: Of the 105 P. aeruginosa included, 46% had a mucoid phenotype. ETEST MICs typically read 0-1 dilution higher than BMD for all drugs. Categorical agreement and essential agreement ranged from 64 to 93% and 63 to 86%, respectively. The majority of observed errors were minor. A single very major error occurred with ceftazidime (4.2%). For ceftazidime-vibactam, 2 very major errors were observed and both were within essential agreement. Major errors occurred for aztreonam (3.3%), cefepime (9.4%), ceftazidime-avibactam (5.3%, adjusted 2.1%), ceftolozane-tazobactam (1%), meropenem (3.3%), piperacillin-tazobactam (2.9%), and tobramycin (1.5%). CONCLUSIONS: ETEST methods performed conservatively for most antibiotics against this challenging collection of P. aeruginosa from patients with CF.

Multicenter Evaluation of the BioFire FilmArray Pneumonia/Pneumonia Plus Panel for Detection and Quantification of Agents of Lower Respiratory Tract Infection.

The ability to provide timely identification of the causative agents of lower respiratory tract infections can promote better patient outcomes and support antimicrobial stewardship efforts. Current diagnostic testing options include culture, molecular testing, and antigen detection. These methods may require collection of various specimens, involve extensive sample treatment, and can suffer from low sensitivity and long turnaround times. This study assessed the performance of the BioFire FilmArray Pneumonia Panel (PN panel) and Pneumonia Plus Panel (PNplus panel), an FDA-cleared sample-to-answer assay that enables the detection of viruses, atypical bacteria, bacteria, and antimicrobial resistance marker genes from lower respiratory tract specimens (sputum and bronchoalveolar lavage [BAL] fluid). Semiquantitative results are also provided for the bacterial targets. This paper describes selected analytical and clinical studies that were conducted to evaluate performance of the panel for regulatory clearance. Prospectively collected respiratory specimens (846 BAL and 836 sputum specimens) evaluated with the PN panel were also tested by quantitative reference culture and molecular methods for comparison. The PN panel showed a sensitivity of 100% for 15/22 etiologic targets using BAL specimens and for 10/24 using sputum specimens. All other targets had sensitivities of ≥75% or were unable to be calculated due to low prevalence in the study population. Specificity for all targets was ≥87.2%, with many false-positive results compared to culture that were confirmed by alternative molecular methods. Appropriate adoption of this test could provide actionable diagnostic information that is anticipated to impact patient care and antimicrobial stewardship decisions.

Practical Comparison of the BioFire FilmArray Pneumonia Panel to Routine Diagnostic Methods and Potential Impact on Antimicrobial Stewardship in Adult Hospitalized Patients with Lower Respiratory Tract Infections.

Lower respiratory tract infections, including hospital-acquired and ventilator-associated pneumonia, are common in hospitalized patient populations. Standard methods frequently fail to identify the infectious etiology due to the polymicrobial nature of respiratory specimens and the necessity of ordering specific tests to identify viral agents. The potential severity of these infections combined with a failure to clearly identify the causative pathogen results in administration of empirical antibiotic agents based on clinical presentation and other risk factors. We examined the impact of the multiplexed, semiquantitative BioFire FilmArray Pneumonia panel (PN panel) test on laboratory reporting for 259 adult inpatients submitting bronchoalveolar lavage (BAL) specimens for laboratory analysis. The PN panel demonstrated a combined 96.2% positive percent agreement (PPA) and 98.1% negative percent agreement (NPA) for the qualitative identification of 15 bacterial targets compared to routine bacterial culture. Semiquantitative values reported by the PN panel were frequently higher than values reported by culture, resulting in semiquantitative agreement (within the same log10 value) of 43.6% between the PN panel and culture; however, all bacterial targets reported as >105 CFU/ml in culture were reported as ≥105 genomic copies/ml by the PN panel. Viral targets were identified by the PN panel in 17.7% of specimens tested, of which 39.1% were detected in conjunction with a bacterial target. A review of patient medical records, including clinically prescribed antibiotics, revealed the potential for antibiotic adjustment in 70.7% of patients based on the PN panel result, including discontinuation or de-escalation in 48.2% of patients, resulting in an average savings of 6.2 antibiotic days/patient.

Prevalence of the carbapenem-heteroresistant phenotype among ESBL-producing Escherichia coli and Klebsiella pneumoniae clinical isolates.

OBJECTIVES: Carbapenem-heteroresistant (cHR) Enterobacteriaceae strains have been reported worldwide; however, the prevalence among clinical ESBL-producing Enterobacteriaceae isolates obtained from patients with repeated hospital admissions remains largely unknown. METHODS: Heteroresistance was screened by disc diffusion and confirmed by a modified population analysis profiling (PAP) method against ertapenem, imipenem, meropenem and ceftolozane/tazobactam. MIC testing was performed by broth microdilution against carbapenems and a panel of agents with potential activity against ESBL-producing strains. RESULTS: One hundred and seventy-three ESBL-producing meropenem-susceptible Escherichia coli and Klebsiella pneumoniae isolates were selected for testing. A total of 519 bacteria/carbapenem combinations were screened by disc diffusion; 84 combinations were identified as cHR. Modified PAP confirmed 70 bacteria/carbapenem combinations as heteroresistant; most (63%, 44/70) confirmed cHR colonies grew within the ertapenem zone of inhibition, followed by imipenem (30%, 21/70), then meropenem (7%, 5/70). In total, one-third of the unique patient isolates (32%, 55/173) were identified as being heteroresistant to at least one carbapenem; of those patients, 16% (9/55) had a carbapenem-non-susceptible isolate on subsequent visits. Only two cHR isolates screened positive for ceftolozane/tazobactam heteroresistance (1%, 2/173), of which one was confirmed heteroresistant by modified PAP. cHR isolates were more likely to be collected from a non-urinary source (e.g. respiratory) compared with non-cHR isolates (31% versus 19%, P = 0.02). MIC distributions of all tested antibiotic agents did not differ between non-cHR and cHR isolates. CONCLUSIONS: Our findings raise concerns for the continued use of carbapenems as first-line therapy for ESBL infections and for the potential selection for strains with full carbapenem resistance.

Pseudomonas aeruginosa transcriptome during human infection.

Laboratory experiments have uncovered many basic aspects of bacterial physiology and behavior. After the past century of mostly in vitro experiments, we now have detailed knowledge of bacterial behavior in standard laboratory conditions, but only a superficial understanding of bacterial functions and behaviors during human infection. It is well-known that the growth and behavior of bacteria are largely dictated by their environment, but how bacterial physiology differs in laboratory models compared with human infections is not known. To address this question, we compared the transcriptome of Pseudomonas aeruginosa during human infection to that of P. aeruginosa in a variety of laboratory conditions. Several pathways, including the bacterium's primary quorum sensing system, had significantly lower expression in human infections than in many laboratory conditions. On the other hand, multiple genes known to confer antibiotic resistance had substantially higher expression in human infection than in laboratory conditions, potentially explaining why antibiotic resistance assays in the clinical laboratory frequently underestimate resistance in patients. Using a standard machine learning technique known as support vector machines, we identified a set of genes whose expression reliably distinguished in vitro conditions from human infections. Finally, we used these support vector machines with binary classification to force P. aeruginosa mouse infection transcriptomes to be classified as human or in vitro. Determining what differentiates our current models from clinical infections is important to better understand bacterial infections and will be necessary to create model systems that more accurately capture the biology of infection.

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii.

UNLABELLED: Acinetobacter baumannii is a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources for A. baumannii strain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that make A. baumannii a formidable public health threat. IMPORTANCE: Acinetobacter baumannii is one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate of A. baumannii that is highly virulent and representative of current outbreak strains.

Protein-to-mRNA ratios are conserved between Pseudomonas aeruginosa strains.

Recent studies have shown that the concentrations of proteins expressed from orthologous genes are often conserved across organisms and to a greater extent than the abundances of the corresponding mRNAs. However, such studies have not distinguished between evolutionary (e.g., sequence divergence) and environmental (e.g., growth condition) effects on the regulation of steady-state protein and mRNA abundances. Here, we systematically investigated the transcriptome and proteome of two closely related Pseudomonas aeruginosa strains, PAO1 and PA14, under identical experimental conditions, thus controlling for environmental effects. For 703 genes observed by both shotgun proteomics and microarray experiments, we found that the protein-to-mRNA ratios are highly correlated between orthologous genes in the two strains to an extent comparable to protein and mRNA abundances. In spite of this high molecular similarity between PAO1 and PA14, we found that several metabolic, virulence, and antibiotic resistance genes are differentially expressed between the two strains, mostly at the protein but not at the mRNA level. Our data demonstrate that the magnitude and direction of the effect of protein abundance regulation occurring after the setting of mRNA levels is conserved between bacterial strains and is important for explaining the discordance between mRNA and protein abundances.

Pseudomonas aeruginosa enhances production of a non-alginate exopolysaccharide during long-term colonization of the cystic fibrosis lung.

The gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.

Protein abundances are more conserved than mRNA abundances across diverse taxa.

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression data sets from seven different species: two bacteria, yeast, nematode, fly, human, and rice. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.

Parallel evolution in Pseudomonas aeruginosa over 39,000 generations in vivo.

The Gram-negative bacterium Pseudomonas aeruginosa is a common cause of chronic airway infections in individuals with the heritable disease cystic fibrosis (CF). After prolonged colonization of the CF lung, P. aeruginosa becomes highly resistant to host clearance and antibiotic treatment; therefore, understanding how this bacterium evolves during chronic infection is important for identifying beneficial adaptations that could be targeted therapeutically. To identify potential adaptive traits of P. aeruginosa during chronic infection, we carried out global transcriptomic profiling of chronological clonal isolates obtained from 3 individuals with CF. Isolates were collected sequentially over periods ranging from 3 months to 8 years, representing up to 39,000 in vivo generations. We identified 24 genes that were commonly regulated by all 3 P. aeruginosa lineages, including several genes encoding traits previously shown to be important for in vivo growth. Our results reveal that parallel evolution occurs in the CF lung and that at least a proportion of the traits identified are beneficial for P. aeruginosa chronic colonization of the CF lung.

Methanogens with pseudomurein use diaminopimelate aminotransferase in lysine biosynthesis.

Methanothermobacter thermautotrophicus uses lysine for both protein synthesis and cross-linking pseudomurein in its cell wall. A diaminopimelate aminotransferase enzyme from this methanogen (MTH0052) converts tetrahydrodipicolinate to l,l-diaminopimelate, a lysine precursor. This gene complemented an Escherichia coli diaminopimelate auxotrophy, and the purified protein catalyzed the transamination of diaminopimelate to tetrahydrodipicolinate. Phylogenetic analysis indicated this gene was recruited from anaerobic Gram-positive bacteria. These results expand the family of diaminopimelate aminotransferases to a diverse set of plant, bacterial and archaeal homologs. In contrast marine methanogens from the Methanococcales, which lack pseudomurein, appear to use a different diaminopimelate pathway for lysine biosynthesis.