COVID-19 outbreak and genomic investigation in an inpatient behavioral health unit.

Background: Inpatient behavioral health units (BHUs) had unique challenges in implementing interventions to mitigate coronavirus disease 2019 (COVID-19) transmission, in part due to socialization in BHU settings. The objective of this study was to identify the transmission routes and the efficacy of the mitigation strategies employed during a COVID-19 outbreak in an inpatient BHU during the Omicron surge from December 2021 to January 2022. Methods: An outbreak investigation was performed after identifying 2 COVID-19-positive BHU inpatients on December 16 and 20, 2021. Mitigation measures involved weekly point prevalence testing for all inpatients, healthcare workers (HCWs), and staff, followed by infection prevention mitigation measures and molecular surveillance. Whole-genome sequencing on a subset of COVID-19-positive individuals was performed to identify the outbreak source. Finally, an outbreak control sustainability plan was formulated for future BHU outbreak resurgences. Results: We identified 35 HCWs and 8 inpatients who tested positive in the BHU between December 16, 2021, and January 17, 2022. We generated severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomes from 15 HCWs and all inpatients. Phylogenetic analyses revealed 3 distinct but genetically related clusters: (1) an HCW and inpatient outbreak likely initiated by staff, (2) an HCW and inpatient outbreak likely initiated by an inpatient visitor, and (3) an HCW-only cluster initiated by staff. Conclusions: Distinct transmission clusters are consistent with multiple, independent SARS-CoV-2 introductions with further inpatient transmission occurring in communal settings. The implemented outbreak control plan comprised of enhanced personal protective equipment requirements, limited socialization, and molecular surveillance likely minimized disruptions to patient care as a model for future pandemics.

Updated Surveillance Metrics and History of the COVID-19 Pandemic (2020-2023) in the Middle East and North Africa: Longitudinal Trend Analysis.

BACKGROUND: This study updates the COVID-19 pandemic surveillance in the Middle East and North Africa (MENA) we first conducted in 2020 with 2 additional years of data for the region. OBJECTIVE: The objective of this study is to determine whether the MENA region meets the criteria for moving from a pandemic to endemic. In doing so, this study considers pandemic trends, dynamic and genomic surveillance methods, and region-specific historical context for the pandemic. These considerations continue through the World Health Organization (WHO) declaration of the end of the public health emergency for the COVID-19 pandemic on May 5, 2023. METHODS: In addition to updates to traditional surveillance data and dynamic panel estimates from the original study by Post et al, this study used data on sequenced SARS-CoV-2 variants from the Global Initiative on Sharing All Influenza Data (GISAID) to identify the appearance and duration of variants of concern. We used Nextclade nomenclature to collect clade designations from sequences and Pangolin nomenclature for lineage designations of SARS-CoV-2. Finally, we conducted a 1-sided t test to determine whether regional weekly speed of COVID-19 spread was greater than an outbreak threshold of 10. We ran the test iteratively with 6 months of data from September 4, 2020, to May 12, 2023. RESULTS: The speed of COVID-19 spread for the region had remained below the outbreak threshold for 7 continuous months by the time of the WHO declaration. Acceleration and jerk were also low and stable. Although the 1- and 7-day persistence coefficients remained statistically significant and positive, the weekly shift parameters suggested the coefficients had most recently turned negative, meaning the clustering effect of new COVID-19 cases became even smaller in the 2 weeks around the WHO declaration. From December 2021 onward, Omicron was the predominant variant of concern in sequenced viral samples. The rolling t test of the speed of spread equal to 10 became entirely insignificant from October 2022 onward. CONCLUSIONS: The COVID-19 pandemic had far-reaching effects on MENA, impacting health care systems, economies, and social well-being. Although COVID-19 continues to circulate in the MENA region, the rate of transmission remained well below the threshold of an outbreak for over 1 year ahead of the WHO declaration. COVID-19 is endemic in the region and no longer reaches the threshold of the pandemic definition. Both standard and enhanced surveillance metrics confirm that the pandemic had transitioned to endemic by the time of the WHO declaration.

Updated Surveillance Metrics and History of the COVID-19 Pandemic (2020-2023) in Latin America and the Caribbean: Longitudinal Trend Analysis.

BACKGROUND: In May 2020, the World Health Organization (WHO) declared Latin America and the Caribbean (LAC) the epicenter of the COVID-19 pandemic, with over 40% of worldwide COVID-19-related deaths at the time. This high disease burden was a result of the unique circumstances in LAC. OBJECTIVE: This study aimed to (1) measure whether the pandemic was expanding or contracting in LAC when the WHO declared the end of COVID-19 as a public health emergency of international concern on May 5, 2023; (2) use dynamic and genomic surveillance methods to describe the history of the pandemic in the region and situate the window of the WHO declaration within the broader history; and (3) provide, with a focus on prevention policies, a historical context for the course of the pandemic in the region. METHODS: In addition to updates of traditional surveillance data and dynamic panel estimates from the original study, we used data on sequenced SARS-CoV-2 variants from the Global Initiative on Sharing All Influenza Data (GISAID) to identify the appearance and duration of variants of concern (VOCs). We used Nextclade nomenclature to collect clade designations from sequences and Pangolin nomenclature for lineage designations of SARS-CoV-2. Additionally, we conducted a 1-sided t test for whether the regional weekly speed (rate of novel COVID-19 transmission) was greater than an outbreak threshold of 10. We ran the test iteratively with 6 months of data across the period from August 2020 to May 2023. RESULTS: The speed of pandemic spread for the region had remained below the outbreak threshold for 6 months by the time of the WHO declaration. Acceleration and jerk were also low and stable. Although the 1- and 7-day persistence coefficients remained statistically significant for the 120-day period ending on the week of May 5, 2023, the coefficients were relatively modest in magnitude (0.457 and 0.491, respectively). Furthermore, the shift parameters for either of the 2 most recent weeks around May 5, 2023, did not indicate any change in this clustering effect of cases on future cases. From December 2021 onward, Omicron was the predominant VOC in sequenced viral samples. The rolling t test of speed=10 became entirely insignificant from January 2023 onward. CONCLUSIONS: Although COVID-19 continues to circulate in LAC, surveillance data suggest COVID-19 is endemic in the region and no longer reaches the threshold of the pandemic definition. However, the region experienced a high COVID-19 burden in the early stages of the pandemic, and prevention policies should be an immediate focus in future pandemics. Ahead of vaccination development, these policies can include widespread testing of individuals and an epidemiological task force with a contact-tracing system.

Deviations in RSV epidemiological patterns and population structures in the United States following the COVID-19 pandemic.

Respiratory Syncytial Virus (RSV) is a leading cause of acute respiratory tract infection, with the greatest impact on infants, immunocompromised individuals, and older adults. RSV prevalence decreased substantially in the United States (US) following the implementation of COVID-19-related non-pharmaceutical interventions but later rebounded with abnormal seasonality. The biological and epidemiological factors underlying this altered behavior remain poorly defined. In this retrospective cohort study from 2009 to 2023 in Chicago, Illinois, US, we examined RSV epidemiology, clinical severity, and genetic diversity. We found that changes in RSV diagnostic platforms drove increased detections in outpatient settings post-2020 and that hospitalized adults infected with RSV-A were at higher risk of intensive care admission than those with RSV-B. While population structures of RSV-A remained unchanged, RSV-B exhibited a genetic shift into geographically distinct clusters. Mutations in the antigenic regions of the fusion protein suggest convergent evolution with potential implications for vaccine and therapeutic development.

Genomic characterization of SARS-CoV-2 in Guinea, West Africa.

SARS-CoV-2 has claimed several million lives since its emergence in late 2019. The ongoing evolution of the virus has resulted in the periodic emergence of new viral variants with distinct fitness advantages, including enhanced transmission and immune escape. While several SARS-CoV-2 variants of concern trace their origins back to the African continent-including Beta, Eta, and Omicron-most countries in Africa remain under-sampled in global genomic surveillance efforts. In an effort to begin filling these knowledge gaps, we conducted retrospective viral genomic surveillance in Guinea from October 2020 to August 2021. We found that SARS-CoV-2 clades 20A, 20B, and 20C dominated throughout 2020 until the coincident emergence of the Alpha and Eta variants of concern in January 2021. The Alpha variant remained dominant throughout early 2021 until the arrival of the Delta variant in July. Surprisingly, despite the small sample size of our study, we also found the persistence of the early SARS-CoV-2 clade 19B as late as April 2021. Together, these data help fill in our understanding of the SARS-CoV-2 population dynamics in West Africa early in the COVID-19 pandemic.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

The impact of an intervention to reduce dispersal from wastewater drain sites on carbapenem-resistant Pseudomonas aeruginosa colonization and bloodstream infection on a hematopoietic cell transplant and hematologic malignancy unit.

OBJECTIVE: To evaluate the impact of an intervention to limit dispersal from wastewater drain (WWD) sites on meropenem-nonsusceptible Pseudomonas aeruginosa patient and environmental colonization and bloodstream infection (BSI) on a hematopoietic cell transplant (HCT) and hematologic malignancy (HM) unit. DESIGN: This quasi-experimental study included pre/postintervention point-prevalence surveys in July 2019 and June 2020, respectively. The retrospective cohort included HCT/HM patients with P. aeruginosa BSI between 2012 and 2022. SETTING: Adult HCT/HM unit at an academic center. PARTICIPANTS: This study included consenting HCT/HM patients on the unit at the time of the point-prevalence surveys. HCT/HM patients with P. aeruginosa BSI between 2012 and 2022. METHODS: A quality improvement intervention targeting WWD sites was conceived and implemented on a HCT/HM unit. Pre and postintervention colonization samples were obtained from patients and environmental sites, cultivated on selective media, then characterized by susceptibility testing. Whole-genome sequencing and phylogenetic analysis were performed on select isolates. The impact of the intervention on colonization and BSI was evaluated, as was relatedness among isolates. RESULTS: Although colonization of WWD sites with meropenem-nonsusceptible P. aeruginosa was widespread before and after this intervention, we observed a substantial decline in patient colonization (prevalence rate ratio, 0.35; 95% confidence interval [CI], 0.04-3.12) and BSI (incidence rate ratio, 0.67; 95% CI, 0.31-1.42) after the intervention. Among 3 predominant sequence types (ST-111, ST-446, and ST-308), there was striking genetic conservation within groups and among environmental colonization, patient colonization, and BSI isolates. CONCLUSIONS: An intervention targeting WWD sites on a HCT/HM unit had a meaningful impact on meropenem-nonsusceptible P. aeruginosa patient colonization and BSI.

A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

Integration of individualized and population-level molecular epidemiology data to model COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune escape have emerged periodically throughout the coronavirus disease 2019 (COVID-19) pandemic, but the impact of these variants on disease severity has remained unclear. In this single-center, retrospective cohort study, we examined the association between SARS-CoV-2 clade and patient outcome over a two-year period in Chicago, Illinois. Between March 2020 and March 2022, 14,252 residual diagnostic specimens were collected from SARS-CoV-2-positive inpatients and outpatients alongside linked clinical and demographic metadata, of which 2,114 were processed for viral whole-genome sequencing. When controlling for patient demographics and vaccination status, several viral clades were associated with risk for hospitalization, but this association was negated by the inclusion of population-level confounders, including case count, sampling bias, and shifting standards of care. These data highlight the importance of integrating non-virological factors into disease severity and outcome models for the accurate assessment of patient risk.

Social/Sexual Networks of People Newly Diagnosed with HIV in Ibadan, Nigeria.

Young men who have sex with men (YMSM) in Nigeria are ten times more likely to be living with HIV-1 than other young men. Due to stigma and criminalization of same-sex sexual behavior, YMSM sexual networks are likely to overlap with those of the general population, leading to a generalized HIV-1 epidemic. Due to limited research on social/sexual network dynamics related to HIV-1 in Nigeria, our study focused on YMSM and sought to assess the feasibility and acceptability of collecting social and sexual network data in Network Canvas from individuals newly diagnosed with HIV-1 in Ibadan, Nigeria. The Network Canvas software was piloted at three sites in Ibadan, Nigeria to collect social/sexual network data from 151 individuals newly diagnosed with HIV-1. Our study sample included 37.7% YMSM; participants reported a mean of 2.6 social alters and 2.6 sexual alters. From the 151 egos and 634 alters, 85 potential unique individuals (194 total) were identified; 65 egos/alters were collapsed into 25 unique individuals. Our success collecting network data from individuals newly diagnosed with HIV-1 in Ibadan demonstrates clear feasibility and acceptability of the approach and the use of Network Canvas to capture and manage these data.

Evaluation of the analytical performance of the 15-minute point-of-care DASH™ SARS-CoV-2 RT-qPCR test.

Accurate and timely diagnosis for COVID-19 diagnosis allows highly effective antiviral medications to be prescribed. The DASH™ Rapid PCR System is a sample-to-answer point-of-care platform combining state-of-the-art PCR kinetics with sequence specific hybridization. The platform's first assay, the DASH™ SARS-CoV-2/S test for anterior nares direct swab specimens, received FDA Emergency Use Authorization in March 2022 for point-of-care use. Here we report the analytical characteristics of the assay including limit of detection, dynamic range, and robustness of SARS-CoV-2 variant detection. The limit of detection was determined by testing swabs contrived with one hundred copies of wild type or Omicron BA.5 virus and detecting 20/20 and 19/20, respectively. The dynamic range was assessed with contrived swabs containing 102-106 copies; the log-linear relationship between Cq and copy input was plotted, and the qPCR efficiency calculated from the slope of the line was 101.4%. Detection of seven SARS-CoV-2 variants was demonstrated.

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.

Individual meropenem epithelial lining fluid and plasma PK/PD target attainment.

It is unclear whether plasma is a reliable surrogate for target attainment in the epithelial lining fluid (ELF). The objective of this study was to characterize meropenem target attainment in plasma and ELF using prospective samples. The first 24-hour T>MIC was evaluated vs 1xMIC and 4xMIC targets at the patient (i.e., fixed MIC of 2 mg/L) and population [i.e., cumulative fraction of response (CFR) according to EUCAST MIC distributions] levels for both plasma and ELF. Among 65 patients receiving ≥24 hours of treatment, 40% of patients failed to achieve >50% T>4xMIC in plasma and ELF, and 30% of patients who achieved >50% T>4xMIC in plasma had <50% T>4xMIC in ELF. At 1xMIC and 4xMIC targets, 3% and 25% of patients with >95% T>MIC in plasma had <50% T>MIC in ELF, respectively. Those with a CRCL >115 mL/min were less likely to achieve >50%T>4xMIC in ELF (P < 0.025). In the population, CFR for Escherichia coli at 1xMIC and 4xMIC was >97%. For Pseudomonas aeruginosa, CFR was ≥90% in plasma and ranged 80%-85% in ELF at 1xMIC when a loading dose was applied. CFR was reduced in plasma (range: 75%-81%) and ELF (range: 44%-60%) in the absence of a loading dose at 1xMIC. At 4xMIC, CFR for P. aeruginosa was 60%-86% with a loading dose and 18%-62% without a loading dose. We found that plasma overestimated ELF target attainment inup to 30% of meropenem-treated patients, CRCL >115 mL/min decreased target attainment in ELF, and loading doses increased CFR in the population.

SARS-CoV-2 genomics and impact on clinical care for COVID-19.

The emergence and worldwide spread of SARS-CoV-2 during the COVID-19 pandemic necessitated the adaptation and rapid deployment of viral WGS and analysis techniques that had been previously applied on a more limited basis to other viral pathogens, such as HIV and influenza viruses. The need for WGS was driven in part by the low mutation rate of SARS-CoV-2, which necessitated measuring variation along the entire genome sequence to effectively differentiate lineages and characterize viral evolution. Several WGS approaches designed to maximize throughput and accuracy were quickly adopted by surveillance labs around the world. These broad-based SARS-CoV-2 genomic sequencing efforts revealed ongoing evolution of the virus, highlighted by the successive emergence of new viral variants throughout the course of the pandemic. These genomic insights were instrumental in characterizing the effects of viral mutations on transmissibility, immune escape and viral tropism, which in turn helped guide public health policy, the use of monoclonal antibody therapeutics and vaccine development strategies. As the use of direct-acting antivirals for the treatment of COVID-19 became more widespread, the potential for emergence of antiviral resistance has driven ongoing efforts to delineate resistance mutations and to monitor global sequence databases for their emergence. Given the critical role of viral genomics in the international effort to combat the COVID-19 pandemic, coordinated efforts should be made to expand global genomic surveillance capacity and infrastructure towards the anticipation and prevention of future pandemics.

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.

Resurgence of SARS-CoV-2 Delta after Omicron variant superinfection in an immunocompromised pediatric patient.

BACKGROUND: Persistent SARS-CoV-2 infection in immunocompromised hosts is thought to contribute to viral evolution by facilitating long-term natural selection and viral recombination in cases of viral co-infection or superinfection. However, there are limited data on the longitudinal intra-host population dynamics of SARS-CoV-2 co-infection/superinfection, especially in pediatric populations. Here, we report a case of Delta-Omicron superinfection in a hospitalized, immunocompromised pediatric patient. METHODS: We conducted Illumina whole genome sequencing (WGS) for longitudinal specimens to investigate intra-host dynamics of SARS-CoV-2 strains. Topoisomerase PCR cloning of Spike open-reading frame and Sanger sequencing of samples was performed for four specimens to validate the findings. Analysis of publicly available SARS-CoV-2 sequence data was performed to investigate the co-circulation and persistence of SARS-CoV-2 variants. RESULTS: Results of WGS indicate the patient was initially infected with the SARS-CoV-2 Delta variant before developing a SARS-CoV-2 Omicron variant superinfection, which became predominant. Shortly thereafter, viral loads decreased below the level of detection before resurgence of the original Delta variant with no residual trace of Omicron. After 54 days of persistent infection, the patient tested negative for SARS-CoV-2 but ultimately succumbed to a COVID-19-related death. Despite protracted treatment with remdesivir, no antiviral resistance mutations emerged. These results indicate a unique case of persistent SARS-CoV-2 infection with the Delta variant interposed by a transient superinfection with the Omicron variant. Analysis of publicly available sequence data suggests the persistence and ongoing evolution of Delta subvariants despite the global predominance of Omicron, potentially indicative of continued transmission in an unknown population or niche. CONCLUSION: A better understanding of SARS-CoV-2 intra-host population dynamics, persistence, and evolution during co-infections and/or superinfections will be required to continue optimizing patient care and to better predict the emergence of new variants of concern.

A cluster of six respiratory cultures positive for Mycobacterium xenopi -Clinical characteristics and genomic characterization.

Mycobacterium xenopi is a slow growing non-tuberculous mycobacterium (NTM) isolated from water systems and has been associated with pseudo-outbreaks and pulmonary infections in humans. We observed a cluster of six respiratory cultures positive for M. xenopi within a six-month period at our institution, approximately double our normal isolation rate of this organism. Only three of the six cases met clinical, radiographic, and microbiologic criteria for NTM infection. An investigation led by our hospital's Healthcare Epidemiology and Infection Program found no epidemiologic link between the six patients. Three isolates underwent whole-genome sequencing (WGS) and phylogenetic analysis confirmed they were non-clonal. In vitro susceptibility data found the isolates were sensitive to macrolides, moxifloxacin, and rifabutin. Our findings suggest that isolation of M. xenopi from pulmonary specimens may be increasing, further defines the genomic population structure of this potentially emerging infection, and establishes WGS as a useful tool for outbreak investigation strain typing.

Quantity of SARS-CoV-2 RNA copies exhaled per minute during natural breathing over the course of COVID-19 infection.

SARS-CoV-2 is spread through exhaled breath of infected individuals. A fundamental question in understanding transmission of SARS-CoV-2 is how much virus an individual is exhaling into the environment while they breathe, over the course of their infection. Research on viral load dynamics during COVID-19 infection has focused on internal swab specimens, which provide a measure of viral loads inside the respiratory tract, but not on breath. Therefore, the dynamics of viral shedding on exhaled breath over the course of infection are poorly understood. Here, we collected exhaled breath specimens from COVID-19 patients and used RTq-PCR to show that numbers of exhaled SARS-CoV-2 RNA copies during COVID-19 infection do not decrease significantly until day 8 from symptom-onset. COVID-19-positive participants exhaled an average of 80 SARS-CoV-2 viral RNA copies per minute during the first 8 days of infection, with significant variability both between and within individuals, including spikes over 800 copies a minute in some patients. After day 8, there was a steep drop to levels nearing the limit of detection, persisting for up to 20 days. We further found that levels of exhaled viral RNA increased with self-rated symptom-severity, though individual variation was high. Levels of exhaled viral RNA did not differ across age, sex, time of day, vaccination status or viral variant. Our data provide a fine-grained, direct measure of the number of SARS-CoV-2 viral copies exhaled per minute during natural breathing-including 312 breath specimens collected multiple times daily over the course of infection-in order to fill an important gap in our understanding of the time course of exhaled viral loads in COVID-19.

Taxonomic characterization of Pseudomonas hygromyciniae sp. nov., a novel species discovered from a commercially purchased antibiotic.

In an attempt to identify novel bacterial species, microbiologists have examined a wide range of environmental niches. We describe the serendipitous discovery of a novel gram-negative bacterial species from a different type of extreme niche: a purchased vial of antibiotic. The vial of antibiotic hygromycin B was found to be factory contaminated with a bacterial species, which we designate Pseudomonas hygromyciniae sp. nov. The proposed novel species belongs to the P. fluorescens complex and is most closely related to P. brenneri, P. proteolytica, and P. fluorescens. The type strain Pseudomonas hygromyciniae sp. nov. strain SDM007T (SDM007T) harbors a novel 250 kb megaplasmid which confers resistance to hygromycin B and contains numerous other genes predicted to encode replication and conjugation machinery. SDM007T grows in hygromycin concentrations of up to 5 mg/mL but does not use the antibiotic as a carbon or nitrogen source. While unable to grow at 37°C ruling out its ability to infect humans, it grows and survives at temperatures between 4 and 30°C. SDM007T can infect plants, as demonstrated by the lettuce leaf model, and is highly virulent in the Galleria mellonella infection model but is unable to infect mammalian A549 cells. These findings indicate that commercially manufactured antibiotics represent another extreme environment that may support the growth of novel bacterial species. IMPORTANCE Physical and biological stresses in extreme environments may select for bacteria not found in conventional environments providing researchers with the opportunity to not only discover novel species but to uncover new enzymes, biomolecules, and biochemical pathways. This strategy has been successful in harsh niches such as hot springs, deep ocean trenches, and hypersaline brine pools. Bacteria belonging to the Pseudomonas species are often found to survive in these unusual environments, making them relevant to healthcare, food, and manufacturing industries. Their ability to survive in a variety of environments is mainly due to the high genotypic and phenotypic diversity displayed by this genus. In this study, we discovered a novel Pseudomonas sp. from a desiccated environment of a sealed antibiotic bottle that was considered sterile. A close genetic relationship with its phylogenetic neighbors reiterated the need to use not just DNA-based tools but also biochemical characteristics to accurately classify this organism.