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Background A recent study from Taiwan suggested that Clostridium innocuum may be an unrecognized cause of antibiotic-associated diarrhea (AAD) and clinically indistinguishable from Clostridioides difficile infection. Our objective was to compare C. innocuum prevalence and strain between those with AAD and asymptomatic controls. Methods In this cross-sectional study, we collected stool from 200 individuals with AAD and 100 asymptomatic controls. We evaluated the association between AAD and C. innocuum in stool using anaerobic culture and quantitative polymerase chain reaction (qPCR). To identify strain-specific associations with AAD, we performed whole-genome sequencing of C. innocuum isolates using Illumina MiSeq and constructed comparative genomics analyses. Results C. innocuum was isolated from stool of 126/300 (42%) subjects and more frequently from asymptomatic controls than AAD subjects (50/100 [50%] vs 76/200 [38%], respectively; P = .047). C. innocuum isolation frequency was not associated with AAD in either the adult or pediatric subgroups. C. innocuum and C. difficile were frequently co-prevalent in individuals with and without diarrhea. There were no phylogenetic differences or accessory genome associations between C. innocuum isolates from AAD subjects and asymptomatic controls. Conclusions C. innocuum was frequently isolated and at a greater frequency in asymptomatic controls than those with AAD. We did not identify strain lineages or accessory genomic elements associated with AAD. These data highlight that differentiating C. innocuum–associated diarrhea from asymptomatic colonization, and differentiating diarrhea caused by C. difficile from C. innocuum, are clinical microbiology challenges that require additional investigation to identify host-specific factors and/or biomarkers that distinguish these conditions.

New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.

Objectives. The goal of this study is to describe placental pathology after infection with SARS-CoV-2 before the predominance of variants of concern (pre-VOC) and during eras of predominant transmission of the Alpha & Gamma (co-circulating), Delta, and Omicron variants. Methods. We used county-level variant data to establish population-level variant proportions, SARS-CoV-2 PCR to identify cases, and IgG serology to exclude latent infections from controls and histopathologic examination to identify placental pathology. Results. We report findings in 870 placentas from pregnancies complicated by SARS-CoV-2 including 90 with infection in the Alpha/Gamma era, 60 from the Delta era and 56 from the Omicron era. Features of maternal vascular malperfusion (MVM), including decidual arteriopathy, were significantly more frequent after SARS-CoV-2 infection. The risk of these findings varied over time, with the highest rates in the Delta era. Increased COVID-19 severity and the presence of comorbidities strengthened these associations. Conclusion. MVM is a feature of SARS-CoV-2 infection in pregnancy. Lesion frequency changed with the predominant circulating virus and should be considered with new variants.

Pseudomonas aeruginosa is frequently resistant to multiple antibiotics, including aminoglycosides. The rates of resistance to aminoglycosides in bloodstream isolates collected over 2 decades at a United States hospital remained constant, suggesting that antibiotic stewardship programs may be effective in countering an increase in resistance.

Disparities in SARS-CoV-2 genomic surveillance have limited our understanding of the viral population dynamics and may delay identification of globally important variants. Despite being the most populated country in Africa, Nigeria has remained critically under sampled. Here, we report sequences from 378 SARS-CoV-2 isolates collected in Oyo State, Nigeria between July 2020 and August 2021. In early 2021, most isolates belonged to the Alpha “variant of concern” (VOC) or the Eta lineage. Eta outcompeted Alpha in Nigeria and across West Africa, persisting in the region even after expansion of an otherwise rare Delta sub-lineage. Spike protein from the Eta variant conferred increased infectivity and decreased neutralization by convalescent sera in vitro. Phylodynamic reconstructions suggest that Eta originated in West Africa before spreading globally and represented a VOC in early 2021. These results demonstrate a distinct distribution of SARS-CoV-2 lineages in Nigeria, and emphasize the need for improved genomic surveillance worldwide.

Background P. aeruginosa is a cause of hospital-acquired and ventilator-associated pneumonia. Hypermutator P. aeruginosa strains have been described in patients with cystic fibrosis and chronic respiratory infections but are rare in patients with acute P. aeruginosa infection. This case describes a hypermutator strain of P. aeruginosa found in a patient with COVID-19-associated acute respiratory distress syndrome (ARDS). Methods Serial respiratory and blood cultures were collected. Short-read sequencing libraries were prepared using the Illumina Nextera XT kit, and whole-genome sequencing was performed using the Illumina NextSeq platform. Long-read sequencing libraries were prepared from unsheared genomic DNA using ligation sequencing kit SQK-LSK109 and sequenced on the Oxford MinION platform. Single nucleotide variants were identified by aligning reads from each isolate to the complete genome of the first available clinical isolate. Hypermutator assays were performed by measuring the mutation frequency rate for rifampin resistance. Antibiotic minimal inhibitory concentrations (MICs) were performed. Growth curves were performed with a starting OD600 of 0.1 with measurements taken every 30 minutes for 24 hours. Results Seventeen respiratory and five blood isolates were obtained throughout 62 days of hospitalization. Fourteen of the 22 isolates exhibited hypermutator phenotypes by rifampin resistance assays, which demonstrated rapid accumulation of mutations. All five bloodstream isolates were hypermutators. MIC testing noted increased resistance to aminoglycosides, fluoroquinolones, and aztreonam in the hypermutator isolates. All bloodstream isolates descended from a single progenitor noted on whole-genome sequencing. Each hypermutator strain contained a mutation in the mismatch repair gene mutL, previously associated with the hypermutator phenotype. Genetic Tree of Patient Isolates The genetic tree highlights hypermutator versus non-hypermutator single nucleotide variants Conclusion This case was notable for multiple isolates of hypermutator P. aeruginosa that persisted over weeks. The patient’s COVID-19 infection and acute respiratory distress syndrome may have facilitated persistence of the P. aeruginosa lineage, allowing a hypermutator lineage to emerge. Disclosures All Authors: No reported disclosures.

Background Cefiderocol (FDC) is a novel antimicrobial agent used for multi-drug resistant Gram-negative pathogens. To date, reports of mutations in β-lactamase and siderophore complex genes have been described and may contribute to FDC resistance. This case describes a New Dehli metallo-β-lactamase (NDM)-producing strain of Enterobacter hormaechei that developed FDC resistance following antibiotic exposure. Methods Serial respiratory and blood cultures were collected from a lung transplant recipient throughout 72 days of hospitalization. Confirmatory susceptibility and combination minimal inhibitory concentration (MIC) testing were performed using broth dilution and E-test assays. Short-read sequencing libraries were prepared using a seqWell plexWell 96 kit, and whole-genome sequencing was performed using the Illumina NovaSeq platform. Reads from the sample genomes were aligned to the chromosome and three plasmid sequences of reference genome ENCL48880. Results Four serial respiratory E. hormaechei isolates and one blood isolate were evaluated. Although initial isolates were susceptible to FDC (MICs 1-2 µg/mL), two respiratory isolates cultured after 41 days of FDC therapy had MICs of 128 µg/mL. The blood isolate remained FDC susceptible despite respiratory resistance. The combination of ceftazidime/avibactam and aztreonam was determined to be active via synergy MIC testing in all isolates, and aztreonam therapeutic drug monitoring confirmed an adequate dosing strategy. Whole-genome sequencing revealed no nonsynonymous single nucleotide variants (SNVs) within the chromosomes but identified a deletion of a large urease island in the resistant isolates. In four of the five isolates, a plasmid (p48880_mcr) was identified and analyzed for possible contributions to FDC resistance. Enterobacter Isolate Assemblies This figure demonstrates genomic assemblies from the five Enterobacter clinical isolates, noting an absence of sequence from ECResp2. Conclusion This case demonstrates development of FDC resistance in E. hormaechei isolates during a 41 day course of FDC therapy. Possible causes of resistance include a large chromosomal deletion and plasmid alleles, demonstrating a potential novel mechanism for FDC resistance. Partnering molecular testing and enhanced antimicrobial stewardship should be encouraged to optimize selection of regimens and durations to prevent resistance to FDC. Disclosures Michael G. Ison, MD MS, GlaxoSmithKlein: Grant/Research Support|Pulmocide: Grant/Research Support|Viracor Eurfins: Advisor/Consultant Nathaniel J. Rhodes, PharmD, MSc, American Academy of Colleges of Pharmacy: Grant/Research Support|Paratek: Grant/Research Support|Third Pole Therapeutics: Advisor/Consultant.

Background Streptococcus pyogenes (GAS) is the major cause of bacterial pharyngitis in children. GAS can cause conditions ranging from asymptomatic pharyngeal carriage to invasive infection. We compared the genomic characteristics of GAS pharyngeal and invasive isolates from U.S. children. Methods We cultured GAS from throat swabs collected from children aged 3–18 years with acute GAS pharyngitis, diagnosed by rapid antigen or molecular test, and from a convenience sample of asymptomatic children during 1/2020 to 3/2022 in Chicago, IL; Atlanta, GA; Portland, OR; Phoenix, AZ. We collected invasive isolates in Chicago. Whole-genome sequencing (WGS) was performed on the Illumina MiSeq for species confirmation, isolate typing, and virulence and antimicrobial gene identification. Reference-based read alignment was used for whole genome phylogenetic analysis. Results Of 1144 collected throat swabs, 684 were from children with pharyngitis and 460 from asymptomatic children. 13 invasive GAS isolates were collected. GAS were cultured from 359 pharyngitis and 20 asymptomatic swabs. Of these, 371 GAS isolates could be recovered and underwent WGS. Thirty isolates (8%) were either low-quality sequences and/or identified as species other than S. pyogenes and were excluded. Whole-genome phylogenetic analysis of 341 GAS (215 pharyngitis, 13 asymptomatic, 13 invasive) identified 25 related clusters and 8 singletons (Figure 1). Virulence gene complements within given emm types were generally concordant with recently published CDC strain data. The most frequently identified resistance genes were tetM and ermB. 281 isolates (82%) carried no antibiotic resistance genes. Some clusters were significantly more represented at certain sites within apparent geographically defined clusters of nearly indistinguishable isolates (Figure 1): cluster 1 in Chicago, emm type 3; cluster 14 in Phoenix and Portland, emm 28, 12; and cluster 15 in Atlanta, emm 89. No major genomic factors were significantly associated with asymptomatic vs pharyngitis vs invasive presentation. Figure 1:Whole genome phylogenetic tree of pediatric GAS isolates Maximum likelihood phylogenetic analysis based on whole-genome alignment of 371 Group A Streptococcus isolates. Tip colors represent cluster assignments based on genetic similarity. Inner ring represents geographic location of GAS isolation. Outer ring represents patient presentation: asymptomatic colonization, symptomatic pharyngitis, or invasive infection. Conclusion Pharyngeal and invasive GAS in children within individual clonal complexes are co-included in closely related clusters, with evidence of close transmission within individual sites Disclosures All Authors: No reported disclosures.

Background COVID-19 affected the epidemiology of many respiratory pathogens including GAS. Assessing genetic heterogeneity (emm type, antimicrobial resistance, virulence factors) can inform treatment recommendations and targets for potential GAS vaccines. We assessed GAS clinical antibiotic susceptibility and performed whole genome sequencing (WGS) among pediatric pharyngeal isolates from 2020–2022. Methods From 1/2020–3/2022 we collected throat swabs in pediatric clinics and EDs from children aged 3-18 years in Chicago, IL; Atlanta, GA; Portland, OR; and Phoenix, AZ: 1) with acute GAS pharyngitis and 2) among a convenience sample of asymptomatic children to assess for GAS colonization. Swabs were plated on blood agar. E-tests were used to assess clinical susceptibility to erythromycin (ERY) and ciprofloxacin (CIP). emm type and antimicrobial resistance genes (ERY, Clindamycin (CLI), and fluoroquinolones) were assessed by WGS. Results 1144 pharyngeal swabs were collected: 359/684 (52%) from children with GAS pharyngitis by rapid test and 20/460 (4.3%) from asymptomatic children yielded GAS on culture. Phenotypic resistance: 55/364 (15%) tested isolates were ERY resistant and 5/364 (1.4%) CIP resistant. The proportion of isolates with ERY resistance increased significantly from 2020 (6%) to 2021–2022 (25%) (χ2 = 23.70, p< .00001) (Figure 1). MICs were high among ERY resistant GAS (Table 1). Genotypic resistance: Of 304 sequenced GAS isolates 40/304 (13%) were ERY resistant, 35/304 (11.5%) were both ERY resistant and CLI (inducible or constitutive) resistant, and 4/304 (1.3%) fluoroquinolone resistant. ermB (62%) was the most common gene for ERY resistance and constitutive CLI resistance, followed by ermTR (23%) and ermT (11%) both conferring inducible CLI resistance. Among the 20 isolates from asymptomatic children no ERY, CLI, or CIP resistance occurred, and no resistance genes were identified. emm types 11, 9, 77, 58 and 94 were associated with ERY and CLI resistance. Conclusion ERY resistance increased from 2020–2022. The high rate of CLI resistance among ERY resistant GAS was associated with erm genes. These results are important to inform treatment recommendations for GAS pharyngitis and targets for vaccine development that can reduce antimicrobial-resistant GAS disease. Disclosures All Authors: No reported disclosures.

Background Pseudomonas aeruginosa (PA) readily acquires genomic mutations and exogenous genetic elements that confer antimicrobial resistance (AMR). With the rise in AMR, there are limited antibiotics available to treat multidrug-resistant (MDR) PA. As such, clinicians have returned to previously used antibiotics. Colistin, sidelined for neurotoxicity and nephrotoxicity, has returned to clinical practice as a viable but suboptimal option for MDR-PA treatment. The most common mechanism of resistance to colistin involves modifications of the lipid A moiety within the bacterial lipopolysaccharide (LPS). Following the identification of a MDR PA isolate, BWH047, we experimentally determined its colistin MIC to be > 1,280 µg/mL and used genomic approaches to identify novel genetic mechanisms of extreme colistin resistance. Methods We created a random, saturated transposon (Tn) insertion library in PA BWH047 using the Himar1 mariner system. After exposure of the library to 640 µg/mL colistin for 10 hours, genomic DNA was harvested, and the Tn insertion sites were sequenced. Insertion sequencing (INSeq) analysis was performed. We identified 27 genes conditionally important for BWH047 growth in the presence of colistin. We selected five initial targets arnC, dedA, wapH, speE2, and bchE and tested their impact on colistin resistance using standard microbroth dilution methods. Results Of our deletion mutants, three showed loss of resistance to colistin. ArnC was chosen as a positive control as its role in colistin resistance in PA is well described. Colistin MICs of BWH047 ΔarnC, ΔdedA, ΔwapH, ΔspeE2, and ΔbchE were determined to be 0.5, 0.5, 1, > 1,280 and > 1280 μg/mL, respectively. Conclusion Here, we used INSeq to identify novel genes involved in extreme colistin resistance. Thus far, we have identified two new candidate genes dedA and wapH, critical for colistin resistance in PA BWH047. Neither gene has been associated with colistin resistance in PA; However, dedA orthologs in Burkholderia thailandenesis and Klebsiella pneumoniae have been shown to be important for colistin resistance. The gene wapH is part of the LPS core oligosaccharide biosynthetic pathway and its discovery hints that additional alterations in the bacterial outer membrane may impact colistin resistance. Disclosures All Authors: No reported disclosures.

SARS-CoV-2 infection in pregnancy and COVID placentitis are associated with an increased risk of stillbirth. We sought to investigate the presence of maternal viremia in people with SARS-CoV-2 infection during pregnancy who had histologic placentitis versus those without placentitis. SARS-CoV-2 qRT-PCR was performed on plasma from 6 patients with COVID placentitis and 12 matched controls without placentitis. SARS-CoV-2 infection occurred between 4/2020-1/2021; the latency between SARS-CoV-2 diagnosis and delivery was 0-76 days. Two placentitis cases demonstrated viremia (1 stillbirth and 1 well infant), while 12/12 controls were negative. Future research may consider viremia as a possible marker of COVID placentitis.

The unprecedented growth of publicly available SARS-CoV-2 genome sequence data has increased demand for effective and accessible SARS-CoV-2 data analysis and visualization tools. A majority of the currently available tools either require computational expertise to deploy or limit user input to pre-selected subsets of SARS-CoV-2 genomes. To address these limitations, we developed ViralVar, a publicly available, point-and-click webtool that gives users the freedom to investigate and visualize user-selected subsets of SARS-CoV-2 genomes obtained from the GISAID public database. ViralVar has two primary features that enable: 1) visualization of spatiotemporal dynamics of SARS-CoV-2 lineages, and 2) structural/functional analysis of genomic mutations. As proof-of-principle, ViralVar was used to explore the evolution of the SARS-CoV-2 pandemic in the USA in the pediatric, adult, and elderly population (n > 1.7 million genomes). While the spatiotemporal dynamics of variants did not differ between these age groups, several USA-specific sublineages arose relative to the rest of the world. Our development and utilization of ViralVar to provide insights on the evolution of SARS-CoV-2 in the USA demonstrates the importance of developing accessible tools to facilitate and accelerate large-scale surveillance of circulating pathogens. The ViralVar webserver is freely available at http://viralvar.org/.

Vibrio vulnificus is an aquatic pathogen that is capable of causing severe disease in humans. Previous studies have suggested that pathogenic isolates were restricted to certain phylogenetic lineages and possibly toxinotype.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic β-lactamase AmpC. However, OXA-10-family β-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA β-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

The continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies is of clinical importance. Several spike mutations associated with immune escape have evolved independently in association with different variants of concern (VOCs). How and when these mutations arise is still unclear. We hypothesized that such mutations might arise in the context of persistent viral replication in immunosuppressed hosts.Nasopharyngeal specimens were collected longitudinally from two immunosuppressed patients with persistent SARS-CoV-2 infection. Plasma was collected from these same patients late in disease course. SARS-CoV-2 whole genome sequencing was performed to assess the emergence and frequency of mutations over time. Select Spike mutations were assessed for their impact on viral entry and antibody neutralization in vitro.Our sequencing results revealed the intrahost emergence of spike mutations that are associated with circulating VOCs in both immunosuppressed patients (del241-243 and E484Q in one patient, and E484K in the other). These mutations decreased antibody-mediated neutralization of pseudotyped virus particles in cell culture, but also decreased efficiency of spike-mediated cell entry.These observations demonstrate the de novo emergence of SARS-CoV-2 spike mutations with enhanced immune evasion in immunosuppressed patients with persistent infection. These data suggest one potential mechanism for the evolution of VOCs and emphasize the importance of continued efforts to develop antiviral drugs for suppression of viral replication in hospitalized settings.

BackgroundKlebsiella pneumoniaestrains have been divided into two major categories: classicalK. pneumoniae,which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulentK. 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 ofK. pneumoniaeamong bloodstream isolates within the United States is not well understood.MethodsWe evaluated consecutiveK. pneumoniaeisolates 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 toolsKleborateandKaptive.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.ResultsConsecutiveK. pneumoniaebloodstream 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 hypervirulentK. 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 withtraconjugation loci suggesting the potential for transmission. We also analyzed 963 publicly available genomes ofK. pneumoniaebloodstream 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).ConclusionsExamination of consecutive isolates from a single center demonstrated that multidrug-resistant high-risk clones are indeed common, but a small number of hypervirulentK. pneumoniaeisolates 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 hypervirulentK. pneumoniaestrains 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).

Emerging evidence suggests the Pseudomonas aeruginosa accessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. Here, we utilized a mouse bacteremia model to quantify the virulence of 100 individual P. aeruginosa bloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulent P. aeruginosa isolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that this P. aeruginosa CDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems in P. aeruginosa pathogenesis.

Hypermutator lineages of Pseudomonas aeruginosa arise frequently during the years of lung infection seen in patients with cystic fibrosis and bronchiectasis but are rare in the absence of 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 described in chronic infections. We describe a COVID-19 patient with a secondary Pseudomonas aeruginosa lung infection in which the causative bacterium persisted for >90 days. During 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 more extensively resistant to antibiotics, to change its type III secretion profile, and to grow more slowly. 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 phenotypes can emerge when clearance of P. aeruginosa fails to occur in typically acute infections such as ventilator-associated pneumonia and suggest that hypermutator lineages can affect patient treatments and outcomes.

BACKGROUND The Omicron variant of SARS-CoV-2 is more transmissible than prior variants of concern (VOCs). It has caused the largest outbreaks in the pandemic, with increases in mortality and hospitalizations. Early data on the spread of Omicron were captured in countries with relatively low case counts, so it was unclear how the arrival of Omicron would impact the trajectory of the pandemic in countries already experiencing high levels of community transmission of Delta. OBJECTIVE The objective of this study is to quantify and explain the impact of Omicron on pandemic trajectories and how they differ between countries that were or were not in a Delta outbreak at the time Omicron occurred. METHODS We used SARS-CoV-2 surveillance and genetic sequence data to classify countries into 2 groups: those that were in a Delta outbreak (defined by at least 10 novel daily transmissions per 100,000 population) when Omicron was first sequenced in the country and those that were not. We used trend analysis, survival curves, and dynamic panel regression models to compare outbreaks in the 2 groups over the period from November 1, 2021, to February 11, 2022. We summarized the outbreaks in terms of their peak rate of SARS-CoV-2 infections and the duration of time the outbreaks took to reach the peak rate. RESULTS Countries that were already in an outbreak with predominantly Delta lineages when Omicron arrived took longer to reach their peak rate and saw greater than a twofold increase (2.04) in the average apex of the Omicron outbreak compared to countries that were not yet in an outbreak. CONCLUSIONS These results suggest that high community transmission of Delta at the time of the first detection of Omicron was not protective, but rather preluded larger outbreaks in those countries. Outbreak status may reflect a generally susceptible population, due to overlapping factors, including climate, policy, and individual behavior. In the absence of strong mitigation measures, arrival of a new, more transmissible variant in these countries is therefore more likely to lead to larger outbreaks. Alternately, countries with enhanced surveillance programs and incentives may be more likely to both exist in an outbreak status and detect more cases during an outbreak, resulting in a spurious relationship. Either way, these data argue against herd immunity mitigating future outbreaks with variants that have undergone significant antigenic shifts.

As public health guidelines throughout the world have relaxed in response to vaccination campaigns against SARS-CoV-2, it is likely that SARS-CoV-2 will remain endemic, fueled by the rise of more infectious SARS-CoV-2 variants. Moreover, in the setting of waning natural and vaccine immunity, reinfections have emerged across the globe, even among previously infected and vaccinated individuals. As such, the ability to detect reexposure to and reinfection by SARS-CoV-2 is a key component for global protection against this virus and, more importantly, against the potential emergence of vaccine escape mutations. Accordingly, there is a strong and continued need for the development and deployment of simple methods to detect emerging hot spots of reinfection to inform targeted pandemic response and containment, including targeted and specific deployment of vaccine booster campaigns. In this study, we identify simple, rapid immune biomarkers of reinfection in rhesus macaques, including IgG3 antibody levels against nucleocapsid and FcgR2A receptor binding activity of anti-RBD antibodies, that are recapitulated in human reinfection cases. As such, this cross-species analysis underscores the potential utility of simple antibody titers and function as price-effective and scalable markers of reinfection to provide increased resolution and resilience against new outbreaks. IMPORTANCE As public health and social distancing guidelines loosen in the setting of waning global natural and vaccine immunity, a deeper understanding of the immunological response to reexposure and reinfection to this highly contagious pathogen is necessary to maintain public health. Viral sequencing analysis provides a robust but unrealistic means to monitor reinfection globally. The identification of scalable pathogen-specific biomarkers of reexposure and reinfection, however, could significantly accelerate our capacity to monitor the spread of the virus through naive and experienced hosts, providing key insights into mechanisms of disease attenuation. Using a nonhuman primate model of controlled SARS-CoV-2 reexposure, we deeply probed the humoral immune response following rechallenge with various doses of viral inocula. We identified virus-specific humoral biomarkers of reinfection, with significant increases in antibody titer and function upon rechallenge across a range of humoral features, including IgG1 to the receptor binding domain of the spike protein of SARS-CoV-2 (RBD), IgG3 to the nucleocapsid protein (N), and FcgR2A receptor binding to anti-RBD antibodies. These features not only differentiated primary infection from reexposure and reinfection in monkeys but also were recapitulated in a sequencing-confirmed reinfection patient and in a cohort of putatively reinfected humans that evolved a PCR-positive test in spite of preexisting seropositivity. As such, this cross-species analysis using a controlled primate model and human cohorts reveals increases in antibody titers as promising cross-validated serological markers of reinfection and reexposure. CA extern

Background Aminoglycoside-containing regimens may be an effective treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CR-Kp), but aminoglycoside-resistance genes are common in these strains. The relationship between the aminoglycoside-resistance genes and aminoglycoside MICs remains poorly defined. Objectives To identify genotypic signatures capable of predicting aminoglycoside MICs for CR-Kp. Methods Clinical CR-Kp isolates (n = 158) underwent WGS to detect aminoglycoside-resistance genes. MICs of amikacin, gentamicin, plazomicin and tobramycin were determined by broth microdilution (BMD). Principal component analysis was used to initially separate isolates based on genotype. Multiple linear regression was then used to generate models that predict aminoglycoside MICs based on the aminoglycoside-resistance genes. Last, the performance of the predictive models was tested against a validation cohort of 29 CR-Kp isolates. Results Among the original 158 CR-Kp isolates, 91.77% (145/158) had at least one clinically relevant aminoglycoside-resistance gene. As a group, 99.37%, 84.81%, 82.28% and 10.76% of the CR-Kp isolates were susceptible to plazomicin, amikacin, gentamicin and tobramycin, respectively. The first two principal components explained 72.23% of the total variance in aminoglycoside MICs and separated isolates into four groups with aac(6′)-Ib, aac(6′)-Ib′, aac(6′)-Ib+aac(6′)-Ib′ or no clinically relevant aminoglycoside-resistance genes. Regression models predicted aminoglycoside MICs with adjusted R2 values of 56%–99%. Within the validation cohort, the categorical agreement when comparing the observed BMD MICs with the predicated MICs was 96.55%, 89.66%, 86.21% and 82.76% for plazomicin, gentamicin, amikacin and tobramycin, respectively. Conclusions Susceptibility to each aminoglycoside varies in CR-Kp. Detection of aminoglycoside-resistance genes may be useful to predict aminoglycoside MICs for CR-Kp.

BACKGROUND Variants of the SARS-CoV-2 virus carry differential risks to public health. The Omicron (B.1.1.529) variant, first identified in Botswana on November 11, 2021, has spread globally faster than any previous variant of concern. Understanding the transmissibility of Omicron is vital in the development of public health policy. OBJECTIVE The aim of this study is to compare SARS-CoV-2 outbreaks driven by Omicron to those driven by prior variants of concern in terms of both the speed and magnitude of an outbreak. METHODS We analyzed trends in outbreaks by variant of concern with validated surveillance metrics in several southern African countries. The region offers an ideal setting for a natural experiment given that most outbreaks thus far have been driven primarily by a single variant at a time. With a daily longitudinal data set of new infections, total vaccinations, and cumulative infections in countries in sub-Saharan Africa, we estimated how the emergence of Omicron has altered the trajectory of SARS-CoV-2 outbreaks. We used the Arellano-Bond method to estimate regression coefficients from a dynamic panel model, in which new infections are a function of infections yesterday and last week. We controlled for vaccinations and prior infections in the population. To test whether Omicron has changed the average trajectory of a SARS-CoV-2 outbreak, we included an interaction between an indicator variable for the emergence of Omicron and lagged infections. RESULTS The observed Omicron outbreaks in this study reach the outbreak threshold within 5-10 days after first detection, whereas other variants of concern have taken at least 14 days and up to as many as 35 days. The Omicron outbreaks also reach peak rates of new cases that are roughly 1.5-2 times those of prior variants of concern. Dynamic panel regression estimates confirm Omicron has created a statistically significant shift in viral spread. CONCLUSIONS The transmissibility of Omicron is markedly higher than prior variants of concern. At the population level, the Omicron outbreaks occurred more quickly and with larger magnitude, despite substantial increases in vaccinations and prior infections, which should have otherwise reduced susceptibility to new infections. Unless public health policies are substantially altered, Omicron outbreaks in other countries are likely to occur with little warning.

Disparities in SARS-CoV-2 genomic surveillance have limited our understanding of the viral population dynamics and may delay identification of globally important variants. Despite being the most populated country in Africa, Nigeria has remained critically under sampled. Here, we report sequences from 378 SARS-CoV-2 isolates collected in Oyo State, Nigeria between July 2020 and August 2021. In early 2021, most isolates belonged to the Alpha "variant of concern" (VOC) or the Eta lineage. Eta outcompeted Alpha in Nigeria and across West Africa, persisting in the region even after expansion of an otherwise rare Delta sub-lineage. Spike protein from the Eta variant conferred increased infectivity and decreased neutralization by convalescent sera in vitro. Phylodynamic reconstructions suggest that Eta originated in West Africa before spreading globally and represented a VOC in early 2021. These results demonstrate a distinct distribution of SARS-CoV-2 lineages in Nigeria, and emphasize the need for improved genomic surveillance worldwide.

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.

BackgroundRecent surges in coronavirus 2019 disease (COVID-19) is attributed to the emergence of more transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs). However, the relative severity of SARS-CoV-2 VOCs in children is unknown.MethodsThis retrospective single-center cohort study was performed at the Ann & Robert H. Lurie Children’s Hospital of Chicago, academic free-standing children’s hospital. We included all children ≤ 18 years-old diagnosed with COVID-19 between October 15th, 2020 and August 31st, 2021 and whose SARS-CoV-2 isolate was sequenced using the Illumina platform. For each patient sample, we identified the SARS-CoV-2 lineage, which was assigned to one of the following groups: Non-VOC, alpha VOC, beta VOC, gamma VOC, or delta VOC. We measured frequency of 5 markers of COVID-19 severity: hospitalization; COVID-19 pharmacologic treatment; respiratory support; intensive care unit admission; and severe disease as classified by the COVID-19 World Health Organization (WHO) Clinical Progression Scale (severe disease; score ≥ 6). A series of logistic regression models were fitted to estimate odds of each severity marker with each VOC (in comparison to non-VOCs), adjusting for COVID-19 community incidence and demographic and clinical co-variates.ResultsDuring the study period, 2,025 patients tested positive for SARS-CoV-2; 1,422 (70.2%) had sufficient viral load to permit sequencing. Among the 499 (35.1%) patients whose isolate was sequenced, median (inter-quartile range) age was 7 (1,12) years; 256 (51.3%) isolates were a VOC: 96 (37.5%) alpha, 38 (14.8%) gamma, and 119 (46.5%) delta. After adjusting for age, Black race, Hispanic ethnicity, high-risk medical conditions, and COVID-19 community incidence, neither alpha nor delta was associated with severe COVID-19. Gamma was independently associated with hospitalization (OR 5.9, 95% CI 1.6-21.5, p=0.007), respiratory support (OR 8.3, 95% CI 1.5-56.3, p=0.02), and severe disease as classified by the WHO Clinical Progression Scale (OR 7.7, 95% CI 1.0-78.1, p=0.05).ConclusionsCompared to non-VOC COVID-19 infections, the gamma VOC, but not the alpha or delta VOCs, was associated with increased severity. These data suggest that recent increased in pediatric COVID-19 hospitalizations are related to increased delta COVID-19 incidence rather than increased delta virulence in children.

A purchased lot of the antibiotic hygromycin B was found to be contaminated with a novel bacterial species, which we designate Pseudomonas hygromyciniae. Characteristics of P. hygromyciniae include its ability to use a variety of compounds as carbon sources, its pathogenicity towards lettuce and Galleria mellonella, and its ability to inhibit the growth of an E. coli strain. P. hygromyciniae is unlikely to be a human pathogen, as it did not survive at 37 °C and was not cytotoxic towards a mammalian cell line. The P. hygromyciniae strain harbors a novel 250 kb megaplasmid which confers resistance to hygromycin B and contains numerous other genes predicted to encode replication and conjugation machinery. These findings indicate that commercially manufactured antibiotics represent another extreme environment that may support the growth of novel bacterial species.IMPORTANCEMicrobial ecologists have surveyed numerous natural and manmade environments in search of new microbial species. In some instances, these microbes are discovered in harsh conditions, such as deep-sea vents, and their discovery leads to better understanding of how microbes adapt to their environment. Here, we have discovered a new species of bacteria from an extreme manmade environment: a lyophilized, commercially available bottle of the antibiotic hygromycin B.

Objective:To identify the impact of universal masking on COVID-19 incidence and putative SARS-CoV-2 transmissions events among children’s hospital healthcare workers (HCWs).Design:Quasi-experimental study.Setting:Single academic free-standing children’s hospital.Methods:We performed whole-genome sequencing of SARS-CoV-2- PCR-positive samples collected from HCWs 3 weeks before and 6 weeks after implementing a universal masking policy. Phylogenetic analyses were performed to identify clusters of clonally related SARS-CoV-2 indicative of putative transmission events. We measured COVID-19 incidence, SARS-CoV-2 test positivity rates, and frequency of putative transmission events before and after the masking policy was implemented.Results:HCW COVID-19 incidence and test positivity declined from 14.3 to 4.3 cases per week, and from 18.4% to 9.0%, respectively. Putative transmission events were only identified prior to universal masking.Conclusions:A universal masking policy was associated with reductions in HCW COVID-19 infections and occupational acquisition of SARS-CoV-2.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic β-lactamase AmpC. However, OXA-10-family β-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA β-lactamases have a propensity for mutation leading to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk clonal complex CC446 with resistance to ceftazidime. 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 glycine at position 157 and a serine instead of phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with resistance to ceftazidime. Deletion of blaOXA-935 restored 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 impact of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. In OXA-14, one of two monomers contained the canonical carbamylated lysine-70 (K70). In contrast, both monomers of OXA-935 were decarbamylated at K70, and the F153S mutation conferred increased flexibility to the omega (Ω) loop. Compared to OXA-14, the catalytic efficiency of OXA-935 for nitrocefin was significantly reduced. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family β-lactamases are concerning given rising reliance on novel β-lactam/β-lactamase inhibitor combinations such as ceftolozane-tazobactam and ceftazidime-avibactam to treat MDR P. aeruginosa infections.

Background The global effort to vaccinate people against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during an ongoing pandemic has raised questions about how vaccine breakthrough infections compare with infections in immunologically naive individuals and the potential for vaccinated individuals to transmit the virus. Methods We examined viral dynamics and infectious virus shedding through daily longitudinal sampling in 23 adults infected with SARS-CoV-2 at varying stages of vaccination, including 6 fully vaccinated individuals. Results The durations of both infectious virus shedding and symptoms were significantly reduced in vaccinated individuals compared with unvaccinated individuals. We also observed that breakthrough infections are associated with strong tissue compartmentalization and are only detectable in saliva in some cases. Conclusions Vaccination shortens the duration of time of high transmission potential, minimizes symptom duration, and may restrict tissue dissemination.

The rapid spread of COVID-19 including recent emergence of new variants with its extreme range of pathologies create an urgent need to develop a versatile sensor for a rapid, precise, and highly sensitive detection of SARS-CoV-2. Herein, we report a microcantilever-based optical detection of SARS-CoV-2 antigenic proteins in just few minutes with high specificity by employing fluidic-atomic force microscopy (f-AFM) mediated nanomechanical deflection method. The corresponding antibodies against the target antigens were first grafted on the gold-coated microcantilever surface pre-functionalized with EDC-NHS chemistry for a suitable antibody-antigen interaction. Rapid detection of SARS-CoV-2 nucleocapsid (N) and spike (S1) receptor binding domain (RBD) proteins was first demonstrated at a clinically relevant concentration down to 1 ng/mL (33 pM) by real-time monitoring of nanomechanical signal induced by antibody-antigen interaction. More importantly, we further show high specific detection of antigens with nasopharyngeal swab specimens from patients pre-determined with qRT-PCR. The results take less than 5 min (swab to signal ≤5 min) and exhibit high selectivity and analytical sensitivity (LoD: 100 copies/ ml; 0.71 ng/ml of N protein). These findings demonstrate potential for nanomechanical signal transduction towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.

Carbapenem-resistant Klebsiella pneumoniae strains cause severe infections that are difficult to treat. The production of carbapenemases such as the K. pneumoniae carbapenemase (KPC) is a common mechanism by which these strains resist killing by the carbapenems. However, the degree of phenotypic carbapenem resistance (MIC) may differ markedly between isolates with similar carbapenemase genes, suggesting that our understanding of the underlying mechanisms of carbapenem resistance remains incomplete. To address this problem, we determined the whole-genome sequences of 166 K. pneumoniae clinical isolates resistant to meropenem, imipenem, or ertapenem. Multiple linear regression analysis of this collection of largely blaKPC-3-containing sequence type 258 (ST258) isolates indicated that blaKPC copy number and some outer membrane porin gene mutations were associated with higher MICs to carbapenems. A trend toward higher MICs was also observed with those blaKPC genes carried by the d isoform of Tn4401. In contrast, ompK37 mutations were associated with lower carbapenem MICs, and extended spectrum β-lactamase genes were not associated with higher or lower MICs in carbapenem-resistant K. pneumoniae. A machine learning approach based on the whole-genome sequences of these isolates did not result in a substantial improvement in prediction of isolates with high or low MICs. These results build upon previous findings suggesting that multiple factors influence the overall carbapenem resistance levels in carbapenem-resistant K. pneumoniae isolates. IMPORTANCE Klebsiella pneumoniae can cause severe infections in the blood, urinary tract, and lungs. Resistance to carbapenems in K. pneumoniae is an urgent public health threat, since it can make these isolates difficult to treat. While individual contributors to carbapenem resistance in K. pneumoniae have been studied, few reports explore their combined effects in clinical isolates. We sequenced 166 clinical carbapenem-resistant K. pneumoniae isolates to evaluate the contribution of known genes to carbapenem MICs and to try to identify novel genes associated with higher carbapenem MICs. The blaKPC copy number and some outer membrane porin gene mutations were associated with higher carbapenem MICs. In contrast, mutations in one specific porin, ompK37, were associated with lower carbapenem MICs. Machine learning did not result in a substantial improvement in the prediction of carbapenem resistance nor did it identify novel genes associated with carbapenem resistance. These findings enhance our understanding of the many contributors to carbapenem resistance in K. pneumoniae.

BackgroundAntimicrobial 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.MethodsWe 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.ResultsCC446 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.ConclusionsCC446 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.

Background Clostridioides (Clostridium) difficile colonization is common among infants. Serological sequelae of infant C. difficile colonization are poorly understood. Methods In this prospective cohort study of healthy infants, stools serially collected between ages 1-2 and 9-12 months were tested for non-toxigenic and toxigenic C. difficile (TCD). Cultured isolates underwent whole-genome sequencing. Serum collected at 9–12 months underwent measurement of IgA, IgG, and IgM against TCD toxins A and B and neutralizing antibody (NAb) titers against toxin B. For comparison, antitoxin IgG and NAb were measured in cord blood from 50 mothers unrelated to study infants. Results Among 32 infants, 16 (50%) were colonized with TCD; 12 were first colonized >1 month before serology measurements. A variety of sequence types were identified, and there was evidence of putative in-home (enrolled siblings) and outpatient clinic transmission. Infants first colonized with TCD >1 month prior had significantly greater serum antitoxin IgA and IgG against toxins A (P = .02 for both) and B (P = .009 and .008, respectively) compared with non–TCD-colonized infants, and greater IgG compared with unrelated cord blood (P = .005). Five of 12 (42%) colonized infants had detectable NAb titers compared with zero non–TCD-colonized infants (P = .02). Breastfeeding was not associated with differences in serological measurements. Conclusions TCD colonization is associated with a humoral immune response against toxins A and B, with evidence of toxin B neutralization in vitro. The extent and duration of protection against CDI later in life afforded by natural C. difficile immunization events require further investigation.

Global SARS-CoV-2 genomic surveillance efforts have provided critical data on the ongoing evolution of the virus to inform best practices in clinical care and public health throughout the pandemic. Impactful genomic surveillance strategies generally follow a multi-disciplinary pipeline involving clinical sample collection, viral genotyping, metadata linkage, data reporting, and public health responses. Unfortunately, current limitations in each of these steps have compromised the overall effectiveness of these strategies. Biases from convenience-based sampling methods can obfuscate the true distribution of circulating variants. The lack of standardization in genotyping strategies and bioinformatic expertise can create bottlenecks in data processing and complicate interpretation. Limitations and inconsistencies in clinical and demographic data collection and sharing can slow the compilation and limit the utility of comprehensive datasets. This likewise can complicate data reporting, restricting the availability of timely data. Finally, gaps and delays in the implementation of genomic surveillance data in the public health sphere can prevent officials from formulating effective mitigation strategies to prevent outbreaks. In this review, we outline current SARS-CoV-2 global genomic surveillance methods and assess roadblocks at each step of the pipeline to identify potential solutions. Evaluating the current obstacles that impede effective surveillance can improve both global coordination efforts and pandemic preparedness for future outbreaks.

Effector proteins translocated into host cells by the Pseudomonas aeruginosa type III secretion system (T3SS) are critical for phagocytic avoidance and systemic spread of the microorganism. The T3SS genes are present in virtually all P. aeruginosa strains. When examined in environmental isolates and clinical specimens, expression of the T3SS genes is the rule. Isolates from the airways of cystic fibrosis (CF) patients are one exception, and these isolates usually carry mutations that disable T3SS gene expression. In this study, we describe two P. aeruginosa isolates, one pigmented brown and one green, from a keratitis-ichthyosis-deafness (KID) syndrome patient with a chronic cutaneous ankle wound. Similar to most isolates from CF, both of the KID isolates were defective for T3SS gene expression. Providing the primary activator of T3SS transcription (exsA) in trans restored T3SS function. Since the exsA sequences were identical to that of a reference strain with active T3SS gene expression, we examined the cAMP-Vfr system, a critical regulator of T3SS gene expression. Vfr is a cAMP-dependent transcription factor that activates exsA expression. Whereas T3SS activity was corrected in the brown isolate by restoring cAMP synthesis, the same was not observed for the green isolate. These findings suggest that distinct mechanisms resulted in loss of T3SS gene expression in the KID isolates. The mutations responsible for the T3SS defects were not clearly evident by comparison of the whole-genome sequences to a reference strain. Our findings suggest that loss of T3SS gene expression may be a trait common to both CF and non-CF chronic infections. IMPORTANCE A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation. In this study, we report loss of T3SS gene expression in non-CF chronic isolates. This finding suggests that loss of the T3SS may be a common and important trait that contributes to persistence and may open avenues to explore the significance further using non-CF chronic infection models.

BackgroundWhile several demographic and clinical correlates of Coronavirus Disease 2019 (COVID-19) outcome have been identified, they remain imprecise tools for clinical management of disease. Furthermore, there are limited data on how these factors are associated with virological and immunological parameters over time.Methods and FindingsNasopharyngeal swabs and blood samples were longitudinally collected from a cohort of 58 hospitalized adults with COVID-19 in Chicago, Illinois between March 27 and June 9, 2020. Samples were assessed for 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, were extracted from electronic health records. All parameters were assessed for association with three patient outcome groups: discharge without intensive care unit (ICU) admission (n = 23), discharge with ICU admission (n = 29), and COVID-19 related death (n = 6). Higher age, male sex, and higher body mass index (BMI) were significantly associated with ICU admission. At hospital admission, higher 4C Mortality scores and lactate dehydrogenase (LDH) levels were likewise associated with ICU admission. Longitudinal trends in Deterioration Index (DI) score, Modified Early Warning Score (MEWS), and serum neutrophil count were also associated with ICU admission, though only the retrospectively calculated median DI score was predictive of death. While viral load and genotype were not significantly associated with outcome in this study, viral load did correlate positively with C-reactive protein levels and negatively with D-dimer, lymphocyte count, and antibody titer. Intra-host viral genetic diversity resulted in changes in viral genotype in some participants over time, though intra-host evolution was not associated with outcome. A stepwise-generated multivariable model including BMI, lymphocyte count at admission, and neutrophil count at admission was sufficient to predict outcome with a 0.82 accuracy rate in this cohort.ConclusionsThese studies suggest that COVID-19 disease severity and poor outcomes among hospitalized patients are likely driven by dysfunctional host responses to infection and underlying co-morbid conditions rather than SARS-CoV-2 viral loads. Several parameters, including 4C mortality score, LDH levels, and DI score, were ultimately predictive of participant outcome and warrant further exploration in larger cohort studies for use in clinical management and risk assessment. Finally, the prevalence of intra-host diversity and viral evolution in hospitalized patients suggests a mechanism for population-level change, further emphasizing the need for effective antivirals to suppress viral replication and to avoid the emergence of new variants.