Phenotypes of a Pseudomonas aeruginosa hypermutator lineage that emerged during prolonged mechanical ventilation in a patient without cystic fibrosis.

Hypermutator lineages of Pseudomonas aeruginosa arise frequently during the years of airway infection experienced by patients with cystic fibrosis and bronchiectasis but are rare in the absence of chronic infection and structural lung disease. Since the onset of the COVID-19 pandemic, large numbers of patients have remained mechanically ventilated for extended periods of time. These patients are prone to acquire bacterial pathogens that persist for many weeks and have the opportunity to evolve within the pulmonary environment. However, little is known about what types of adaptations occur in these bacteria and whether these adaptations mimic those observed in chronic infections. We describe a COVID-19 patient with a secondary P. aeruginosa lung infection in whom the causative bacterium persisted for >50 days. Over the course of this infection, a hypermutator lineage of P. aeruginosa emerged and co-existed with a non-hypermutator lineage. Compared to the parental lineage, the hypermutator lineage evolved to be less cytotoxic and less virulent. Genomic analyses of the hypermutator lineage identified numerous mutations, including in the mismatch repair gene mutL and other genes frequently mutated in individuals with cystic fibrosis. Together, these findings demonstrate that hypermutator lineages can emerge when P. aeruginosa persists following acute infections such as ventilator-associated pneumonia and that these lineages have the potential to affect patient outcomes.IMPORTANCEPseudomonas aeruginosa may evolve to accumulate large numbers of mutations in the context of chronic infections such as those that occur in individuals with cystic fibrosis. However, these "hypermutator" lineages are rare following acute infections. Here, we describe a non-cystic fibrosis patient with COVID-19 pneumonia who remained mechanically ventilated for months. The patient became infected with a strain of P. aeruginosa that evolved to become a hypermutator. We demonstrate that hypermutation led to changes in cytotoxicity and virulence. These findings are important because they demonstrate that P. aeruginosa hypermutators can emerge following acute infections and that they have the potential to affect patient outcomes in this setting.

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence.

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

Genome-wide screens reveal shared and strain-specific genes that facilitate enteric colonization by Klebsiella pneumoniae.

Gastrointestinal (GI) colonization by Klebsiella pneumoniae is a risk factor for subsequent infection as well as transmission to other patients. Additionally, colonization is achieved by many strain types that exhibit high diversity in genetic content. Thus, we aimed to study strain-specific requirements for K. pneumoniae GI colonization by applying transposon insertion sequencing to three classical clinical strains: a carbapenem-resistant strain, an extended-spectrum beta-lactamase-producing strain, and a non-epidemic antibiotic-susceptible strain. The transposon insertion libraries were screened in a murine model of GI colonization. At 3 days post-inoculation, 27 genes were required by all three strains for colonization. Isogenic deletion mutants for three genes/operons (acrA, carAB, and tatABCD) confirmed colonization defects in each of the three strains. Additionally, deletion of acrA reduced bile tolerance in vitro, while complementation restored both bile tolerance in vitro and colonization ability in vivo. Transposon insertion sequencing suggested that some genes were more important for the colonization of one strain than the others. For example, deletion of the sucrose porin-encoding gene scrY resulted in a colonization defect in the carbapenemase-producing strain but not in the extended-spectrum beta-lactamase producer or the antibiotic-susceptible strain. These findings demonstrate that classical K. pneumoniae strains use both shared and strain-specific strategies to colonize the mouse GI tract. IMPORTANCE Klebsiella pneumoniae is a common cause of difficult-to-treat infections due to its propensity to express resistance to many antibiotics. For example, carbapenem-resistant K. pneumoniae has been named an urgent threat by the United States Centers for Disease Control and Prevention. Gastrointestinal colonization in patients with K. pneumoniae has been linked to subsequent infection, making it a key process to control in the prevention of multidrug-resistant infections. However, the bacterial factors which contribute to K. pneumoniae colonization are not well understood. Additionally, individual strains exhibit large amounts of genetic diversity, begging the question of whether some colonization factors are strain dependent. This study identifies the enteric colonization factors of three classical strains using transposon mutant screens to define a core colonization program for K. pneumoniae as well as detecting strain-to-strain differences in colonization strategies.

Genome-wide screens reveal shared and strain-specific genes that facilitate enteric colonization by Klebsiella pneumoniae.

Gastrointestinal (GI) colonization by Klebsiella pneumoniae is a risk factor for subsequent infection as well as transmission to other patients. Additionally, colonization is achieved by many strain types that exhibit high diversity in genetic content. Thus, we aimed to study strain-specific requirements for K. pneumoniae GI colonization by applying transposon insertion sequencing to three classical clinical strains: a carbapenem-resistant strain, an extended-spectrum beta-lactamase producing strain, and a non-epidemic antibiotic-susceptible strain. The transposon insertion libraries were screened in a murine model of GI colonization. At three days post-inoculation, 27 genes were required by all three strains for colonization. Isogenic deletion mutants for three genes/operons (acrA, carAB, tatABCD) confirmed colonization defects in each of the three strains. Additionally, deletion of acrA reduced bile tolerance in vitro, while complementation restored both bile tolerance in vitro and colonization ability in vivo. Transposon insertion sequencing suggested that some genes were more important for colonization of one strain than the others. For example, deletion of the sucrose porin-encoding gene scrY resulted in a colonization defect in the carbapenemase-producing strain but not in the extended-spectrum beta-lactamase producer or the antibiotic-susceptible strain. These findings demonstrate that classical K. pneumoniae strains use both shared and strain-specific strategies to colonize the mouse GI tract. IMPORTANCE: Klebsiella pneumoniae is a common cause of difficult-to-treat infections due to its propensity to express resistance to many antibiotics. For example, carbapenem-resistant K. pneumoniae (CR-Kp) has been named an urgent threat by the United States Centers for Disease Control and Prevention. Gastrointestinal colonization of patients with K. pneumoniae has been linked to subsequent infection, making it a key process to control in prevention of multidrug-resistant infections. However, the bacterial factors which contribute to K. pneumoniae colonization are not well understood. Additionally, individual strains exhibit large amounts of genetic diversity, begging the question of whether some colonization factors are strain-dependent. This study identifies the enteric colonization factors of 3 classical strains using transposon mutant screens to define a core colonization program for K. pneumoniae as well as detecting strain-to-strain differences in colonization strategies.

Genomic surveillance for multidrug-resistant or hypervirulent Klebsiella pneumoniae among United States bloodstream isolates.

BACKGROUND: Klebsiella pneumoniae strains have been divided into two major categories: classical K. pneumoniae, which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulent K. pneumoniae, which cause severe community-acquired and disseminated infections in normal hosts. Both types of infections may lead to bacteremia and are associated with significant morbidity and mortality. The relative burden of these two types of K. pneumoniae among bloodstream isolates within the United States is not well understood. METHODS: We evaluated consecutive K. pneumoniae isolates cultured from the blood of hospitalized patients at Northwestern Memorial Hospital (NMH) in Chicago, Illinois between April 2015 and April 2017. Bloodstream isolates underwent whole genome sequencing, and sequence types (STs), capsule loci (KLs), virulence genes, and antimicrobial resistance genes were identified in the genomes using the bioinformatic tools Kleborate and Kaptive. Patient demographic, comorbidity, and infection information, as well as the phenotypic antimicrobial resistance of the isolates were extracted from the electronic health record. Candidate hypervirulent isolates were tested in a murine model of pneumonia, and their plasmids were characterized using long-read sequencing. We also extracted STs, KLs, and virulence and antimicrobial resistance genes from the genomes of bloodstream isolates submitted from 33 United States institutions between 2007 and 2021 to the National Center for Biotechnology Information (NCBI) database. RESULTS: Consecutive K. pneumoniae bloodstream isolates (n = 104, one per patient) from NMH consisted of 75 distinct STs and 51 unique capsule loci. The majority of these isolates (n = 58, 55.8%) were susceptible to all tested antibiotics except ampicillin, but 17 (16.3%) were multidrug-resistant. A total of 32 (30.8%) of these isolates were STs of known high-risk clones, including ST258 and ST45. In particular, 18 (17.3%) were resistant to ceftriaxone (of which 17 harbored extended-spectrum beta-lactamase genes) and 9 (8.7%) were resistant to meropenem (all of which harbored a carbapenemase genes). Four (3.8%) of the 104 isolates were hypervirulent K. pneumoniae, as evidenced by hypermucoviscous phenotypes, high levels of virulence in a murine model of pneumonia, and the presence of large plasmids similar to characterized hypervirulence plasmids. These isolates were cultured from patients who had not recently traveled to Asia. Two of these hypervirulent isolates belonged to the well characterized ST23 lineage and one to the re-emerging ST66 lineage. Of particular concern, two of these isolates contained plasmids with tra conjugation loci suggesting the potential for transmission. We also analyzed 963 publicly available genomes of K. pneumoniae bloodstream isolates from locations within the United States. Of these, 465 (48.3%) and 760 (78.9%) contained extended-spectrum beta-lactamase genes or carbapenemase genes, respectively, suggesting a bias towards submission of antibiotic-resistant isolates. The known multidrug-resistant high-risk clones ST258 and ST307 were the predominant sequence types. A total of 32 (3.3%) of these isolates contained aerobactin biosynthesis genes and 26 (2.7%) contained at least two genetic features of hvKP strains, suggesting elevated levels of virulence. We identified 6 (0.6%) isolates that were STs associated with hvKP: ST23 (n = 4), ST380 (n = 1), and ST65 (n = 1). CONCLUSIONS: Examination of consecutive isolates from a single center demonstrated that multidrug-resistant high-risk clones are indeed common, but a small number of hypervirulent K. pneumoniae isolates were also observed in patients with no recent travel history to Asia, suggesting that these isolates are undergoing community spread in the United States. A larger collection of publicly available bloodstream isolate genomes also suggested that hypervirulent K. pneumoniae strains are present but rare in the USA; however, this collection appears to be heavily biased towards highly antibiotic-resistant isolates (and correspondingly away from hypervirulent isolates).