Pseudomonas aeruginosa population dynamics in a vancomycin-induced murine model of gastrointestinal carriage.

Pseudomonas aeruginosa is a common nosocomial pathogen and a major cause of morbidity and mortality in hospitalized patients. Multiple reports highlight that P. aeruginosa gastrointestinal colonization may precede systemic infections by this pathogen. Gaining a deeper insight into the dynamics of P. aeruginosa gastrointestinal carriage is an essential step in managing gastrointestinal colonization and could contribute to preventing bacterial transmission and progression to systemic infection. Here, we present a clinically relevant mouse model relying on parenteral vancomycin pretreatment and a single orogastric gavage of a controlled dose of P. aeruginosa. Robust carriage was observed with multiple clinical isolates, and carriage persisted for up to 60 days. Histological and microbiological examination of mice indicated that this model indeed represented carriage and not infection. We then used a barcoded P. aeruginosa library along with the sequence tag-based analysis of microbial populations (STAMPR) analytic pipeline to quantify bacterial population dynamics and bottlenecks during the establishment of the gastrointestinal carriage. Analysis indicated that most of the P. aeruginosa population was rapidly eliminated in the stomach, but the few bacteria that moved to the small intestine and the caecum expanded significantly. Hence, the stomach constitutes a significant barrier against gastrointestinal carriage of P. aeruginosa, which may have clinical implications for hospitalized patients.

The global population stru cture of Lacticaseibacillus rhamnosus and its application to an investigation of a rare case of infective endocarditis.

BACKGROUND: Lacticaseibacillus (formerly Lactobacillus) rhamnosus is widely used in probiotics or food supplements to promote microbiome health and may also be part of the normal microbiota of the human gastrointestinal tract. However, it rarely also causes invasive or severe infections in patients. It has been postulated that these infections may originate from probiotics or from endogenous commensal reservoirs. In this report, we examine the population structure of Lacticaseibacillus rhamnosus and investigate the utility of using bacterial genomics to identify the source of invasive Lacticaseibacillus infections. METHODS: Core genome phylogenetic analysis was performed on 602 L. rhamnosus genome sequences from the National Center for Biotechnology public database. This information was then used along with newly generated sequences of L. rhamnosus isolates from yogurt to investigate a fatal case of L. rhamnosus endocarditis. RESULTS: Phylogenetic analysis demonstrated substantial genetic overlap of L. rhamnosus isolates cultured from food, probiotics, infected patients, and colonized individuals. This was applied to a patient who had both consumed yogurt and developed L. rhamnosus endocarditis to attempt to identify the source of his infection. The sequence of the isolate from the patient's bloodstream differed at only one nucleotide position from one of the yogurt isolates. Both isolates belonged to a clade, identified here as clade YC, composed of mostly gastrointestinal isolates from healthy individuals, some of which also differed by only a single nucleotide change from the patient's isolate. CONCLUSIONS: As illustrated by this case, whole genome sequencing may be insufficient to reliably determine the source of invasive infections caused by L. rhamnosus.

Activity and inhibition of the SARS-CoV-2 Omicron nsp13 R392C variant using RNA duplex unwinding assays.

SARS-CoV-2 nsp13 helicase is an essential enzyme for viral replication and a promising target for antiviral drug development. This study compares the double-stranded RNA (dsRNA) unwinding activity of nsp13 and the Omicron nsp13R392C variant, which is predominant in currently circulating lineages. Using in vitro gel- and fluorescence-based assays, we found that both nsp13 and nsp13R392C have dsRNA unwinding activity with equivalent kinetics. Furthermore, the R392C mutation had no effect on the efficiency of the nsp13-specific helicase inhibitor SSYA10-001. We additionally confirmed the activity of several other helicase inhibitors against nsp13, including punicalagin that inhibited dsRNA unwinding at nanomolar concentrations. Overall, this study reveals the utility of using dsRNA unwinding assays to screen small molecules for antiviral activity against nsp13 and the Omicron nsp13R392C variant. Continual monitoring of newly emergent variants will be essential for considering resistance profiles of lead compounds as they are advanced towards next-generation therapeutic development.

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.

Serodominant SARS-CoV-2 Nucleocapsid Peptides Map to Unstructured Protein Regions.

The SARS-CoV-2 nucleocapsid (N) protein is highly immunogenic, and anti-N antibodies are commonly used as markers for prior infection. While several studies have examined or predicted the antigenic regions of N, these have lacked consensus and structural context. Using COVID-19 patient sera to probe an overlapping peptide array, we identified six public and four private epitope regions across N, some of which are unique to this study. We further report the first deposited X-ray structure of the stable dimerization domain at 2.05 Å as similar to all other reported structures. Structural mapping revealed that most epitopes are derived from surface-exposed loops on the stable domains or from the unstructured linker regions. An antibody response to an epitope in the stable RNA binding domain was found more frequently in sera from patients requiring intensive care. Since emerging amino acid variations in N map to immunogenic peptides, N protein variation could impact detection of seroconversion for variants of concern. IMPORTANCE As SARS-CoV-2 continues to evolve, a structural and genetic understanding of key viral epitopes will be essential to the development of next-generation diagnostics and vaccines. This study uses structural biology and epitope mapping to define the antigenic regions of the viral nucleocapsid protein in sera from a cohort of COVID-19 patients with diverse clinical outcomes. These results are interpreted in the context of prior structural and epitope mapping studies as well as in the context of emergent viral variants. This report serves as a resource for synthesizing the current state of the field toward improving strategies for future diagnostic and therapeutic design.

Functional and Structural Characterization of OXA-935, a Novel OXA-10-Family ß-Lactamase from Pseudomonas aeruginosa.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic ß-lactamase AmpC. However, OXA-10-family ß-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA ß-lactamases have a propensity for mutation that leads to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk P. aeruginosa clonal complex CC446 with a resistance to ceftazidime. A genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of blaOXA-10, named blaOXA-935, which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of a glycine at position 157 and a serine instead of a phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with the resistance of P. aeruginosa to ceftazidime. Compared to OXA-14, OXA-935 showed increased catalytic efficiency for ceftazidime. The deletion of blaOXA-935 restored the sensitivity to ceftazidime, and susceptibility profiling of P. aeruginosa laboratory strains expressing blaOXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impacts of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. Compared to OXA-14, the F153S mutation in OXA-935 conferred increased flexibility in the omega (Ω) loop. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family ß-lactamases are concerning, given the rising reliance on novel ß-lactam/ß-lactamase inhibitor combinations, such as ceftolozane-tazobactam and ceftazidime-avibactam, to treat MDR P. aeruginosa infections.

Breakthrough daptomycin-, linezolid-, vancomycin-resistant Enterococcus faecium bacteremia during protracted daptomycin therapy: A case report.

Infections with multidrug resistant (MDR) Enterococcus faecium (Efm) are a growing problem. Vancomycin resistance in enterococci has long challenged treatment, necessitating the use of linezolid or daptomycin. Subsequently, daptomycin-, linezolid-, vancomycin-resistant Efm (DLVRE) infections have emerged. Case reports and guidelines for treating DLVRE infections are limited. Here, we describe the clinical and laboratory management of an MDR Efm protracted intraabdominal (IA) infection and breakthrough DLVRE bacteremia. Serial Efm resistance was evaluated using whole genome sequencing (WGS), susceptibility testing, and synergy analysis. Prior to in vitro synergy testing, combination antimicrobial therapy with daptomycin (DAP) and ceftaroline (CPT) was employed to treat the patient's central line-associated DLVRE bloodstream infection. In vitro antimicrobial testing revealed no synergy between daptomycin and ceftaroline; however, the patient's bacteremia cleared following initiation of both in conjunction with catheter removal. Sequencing of the DLVRE isolates revealed multiple genomic mutations which explained both linezolid and daptomycin resistance phenotypes and confirmed the presence of a plasmid containing the vanA operon. Sequential WGS of two additional bacterial isolates from the same patient revealed protracted colonization with a single DLVRE clone and suggested the development of bacterial subpopulations. Pairing clinical isolate susceptibilities with WGS and synergy testing should be encouraged in clinical practice to better inform antimicrobial management in cases of multidrug resistance.

Assessment of Virological Contributions to COVID-19 Outcomes in a Longitudinal Cohort of Hospitalized Adults.

BACKGROUND: While several demographic and clinical correlates of coronavirus disease 2019 (COVID-19) outcome have been identified, their relationship to virological and immunological parameters remains poorly defined. METHODS: To address this, we performed longitudinal collection of nasopharyngeal swabs and blood samples from a cohort of 58 hospitalized adults with COVID-19. Samples were assessed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load, viral genotype, viral diversity, and antibody titer. Demographic and clinical information, including patient blood tests and several composite measures of disease severity, was extracted from electronic health records. RESULTS: Several factors, including male sex, higher age, higher body mass index, higher 4C Mortality score, and elevated lactate dehydrogenase levels, were associated with intensive care unit admission. Of all measured parameters, only the retrospectively calculated median Deterioration Index score was significantly associated with death. While quantitative polymerase chain reaction cycle threshold (Ct) values and genotype of SARS-CoV-2 were not significantly associated with outcome, Ct value did correlate positively with C-reactive protein levels and negatively with D-dimer, lymphocyte count, and antibody titer. Intrahost viral genetic diversity remained constant through the disease course and resulted in changes in viral genotype in some participants. CONCLUSIONS: Ultimately, these results suggest that worse outcomes are driven by immune dysfunction rather than by viral load and that SARS-CoV-2 evolution in hospital settings is relatively constant over time.

Systemic infection facilitates transmission of Pseudomonas aeruginosa in mice.

Health care-associated infections such as Pseudomonas aeruginosa bacteremia pose a major clinical risk for hospitalized patients. However, these systemic infections are presumed to be a "dead-end" for P. aeruginosa and to have no impact on transmission. Here, we use a mouse infection model to show that P. aeruginosa can spread from the bloodstream to the gallbladder, where it replicates to extremely high numbers. Bacteria in the gallbladder can then seed the intestines and feces, leading to transmission to uninfected cage-mate mice. Our work shows that the gallbladder is crucial for spread of P. aeruginosa from the bloodstream to the feces during bacteremia, a process that promotes transmission in this experimental system. Further research is needed to test to what extent these findings are relevant to infections in patients.

Long-term Persistence of an Extensively Drug-Resistant Subclade of Globally Distributed Pseudomonas aeruginosa Clonal Complex 446 in an Academic Medical Center.

BACKGROUND: Antimicrobial resistance (AMR) is a major challenge in the treatment of infections caused by Pseudomonas aeruginosa. Highly drug-resistant infections are disproportionally caused by a small subset of globally distributed P. aeruginosa sequence types (STs), termed "high-risk clones." We noted that clonal complex (CC) 446 (which includes STs 298 and 446) isolates were repeatedly cultured at 1 medical center and asked whether this lineage might constitute an emerging high-risk clone. METHODS: We searched P. aeruginosa genomes from collections available from several institutions and from a public database for the presence of CC446 isolates. We determined antibacterial susceptibility using microbroth dilution and examined genome sequences to characterize the population structure of CC446 and investigate the genetic basis of AMR. RESULTS: CC446 was globally distributed over 5 continents. CC446 isolates demonstrated high rates of AMR, with 51.9% (28/54) being multidrug-resistant (MDR) and 53.6% of these (15/28) being extensively drug-resistant (XDR). Phylogenetic analysis revealed that most MDR/XDR isolates belonged to a subclade of ST298 (designated ST298*) of which 100% (21/21) were MDR and 61.9% (13/21) were XDR. XDR ST298* was identified repeatedly and consistently at a single academic medical center from 2001 through 2017. These isolates harbored a large plasmid that carries a novel antibiotic resistance integron. CONCLUSIONS: CC446 isolates are globally distributed with multiple occurrences of high AMR. The subclade ST298* is responsible for a prolonged epidemic (≥16 years) of XDR infections at an academic medical center. These findings indicate that CC446 is an emerging high-risk clone deserving further surveillance.

Draft Genome Sequence of Pseudomonas aeruginosa Strain BWH047, a Sequence Type 235 Multidrug-Resistant Clinical Isolate Expressing High Levels of Colistin Resistance.

The Gram-negative bacterium Pseudomonas aeruginosa is often multidrug resistant, associated with global epidemic outbreaks, and responsible for significant morbidity and mortality in hospitalized patients. Here, we present the draft genome sequence of BWH047, a multidrug-resistant P. aeruginosa clinical isolate belonging to the epidemic sequence type 235 and demonstrating high levels of colistin resistance.