Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice.

Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes, and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial, and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a preclinical model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC-infected SLPI-deficient (Slpi-/-) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi+/+) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi-/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with a history of recent or recurrent UTI, suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI promotes clearance of UPEC in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.IMPORTANCEAnnually, millions of people suffer from urinary tract infections (UTIs) and more than $3 billion are spent on work absences and treatment of these patients. While the early response to UTI is known to be important in combating urinary pathogens, knowledge of host factors that help curb infection is still limited. Here, we use a preclinical model of UTI to study secretory leukocyte protease inhibitor (SLPI), an antimicrobial protein, to determine how it protects the bladder against infection. We find that SLPI is increased during UTI, accelerates the clearance of bacteriuria, and upregulates genes and pathways needed to fight an infection while preventing prolonged bladder inflammation. In a small clinical study, we show SLPI is readily detectable in human urine and is associated with the presence of a uropathogen in patients without a previous history of UTI, suggesting SLPI may play an important role in protecting from bacterial cystitis.

Diagnostic and commensal Staphylococcus pseudintermedius genomes reveal niche adaptation through parallel selection of defense mechanisms.

Staphylococcus pseudintermedius is historically understood as a prevalent commensal and pathogen of dogs, though modern clinical diagnostics reveal an expanded host-range that includes humans. It remains unclear whether differentiation across S. pseudintermedius populations is driven primarily by niche-type or host-species. We sequenced 501 diagnostic and commensal isolates from a hospital, veterinary diagnostic laboratory, and within households in the American Midwest, and performed a comparative genomics investigation contrasting human diagnostic, animal diagnostic, human colonizing, pet colonizing, and household-surface S. pseudintermedius isolates. Though indistinguishable by core and accessory gene architecture, diagnostic isolates harbor more encoded and phenotypic resistance, whereas colonizing and surface isolates harbor similar CRISPR defense systems likely reflective of common household phage exposures. Furthermore, household isolates that persist through anti-staphylococcal decolonization report elevated rates of base-changing mutations in - and parallel evolution of - defense genes, as well as reductions in oxacillin and trimethoprim-sulfamethoxazole susceptibility. Together we report parallel niche-specific bolstering of S. pseudintermedius defense mechanisms through gene acquisition or mutation.

Secretory Leukocyte Protease Inhibitor Protects Against Severe Urinary Tract Infection in Mice.

Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a mouse model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC infected SLPI-deficient (Slpi-/-) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi+/+) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi-/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with history of recent or recurrent UTI (rUTI), suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI protects against acute UTI in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.

Seroprevalence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) antibodies among healthcare personnel in the Midwestern United States, September 2020-April 2021.

Objective: To determine the prevalence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) IgG nucleocapsid (N) antibodies among healthcare personnel (HCP) with no prior history of COVID-19 and to identify factors associated with seropositivity. Design: Prospective cohort study. Setting: An academic, tertiary-care hospital in St. Louis, Missouri. Participants: The study included 400 HCP aged ≥18 years who potentially worked with coronavirus disease 2019 (COVID-19) patients and had no known history of COVID-19; 309 of these HCP also completed a follow-up visit 70-160 days after enrollment. Enrollment visits took place between September and December 2020. Follow-up visits took place between December 2020 and April 2021. Methods: At each study visit, participants underwent SARS-CoV-2 IgG N-antibody testing using the Abbott SARS-CoV-2 IgG assay and completed a survey providing information about demographics, job characteristics, comorbidities, symptoms, and potential SARS-CoV-2 exposures. Results: Participants were predominately women (64%) and white (79%), with median age of 34.5 years (interquartile range [IQR], 30-45). Among the 400 HCP, 18 (4.5%) were seropositive for IgG N-antibodies at enrollment. Also, 34 (11.0%) of 309 were seropositive at follow-up. HCP who reported having a household contact with COVID-19 had greater likelihood of seropositivity at both enrollment and at follow-up. Conclusions: In this cohort of HCP during the first wave of the COVID-19 pandemic, ∼1 in 20 had serological evidence of prior, undocumented SARS-CoV-2 infection at enrollment. Having a household contact with COVID-19 was associated with seropositivity.

Molecular epidemiology of Clostridioides difficile isolates in a non-outbreak setting at a comprehensive cancer center.

Ribotyping was performed on Clostridioides difficile isolates from patients with malignancies. Thirty-one (27.9%) isolates from 111 episodes of colitis were recovered representing 14 ribotypes with 25 (80.6%) belonging to 6 ribotypes (014/020, 1/VPI/077/087, 05/015, 015/046, 05/053, 106/174). We identified three novel ribotypes with 1 carrying gene encoding for binary toxin.

Intensive care unit sinks are persistently colonized with multidrug resistant bacteria and mobilizable, resistance-conferring plasmids.

Contamination of hospital sinks with microbial pathogens presents a serious potential threat to patients, but our understanding of sink colonization dynamics is largely based on infection outbreaks. Here, we investigate the colonization patterns of multidrug-resistant organisms (MDROs) in intensive care unit sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. Using culture-based methods, we recovered 822 bacterial isolates representing 104 unique species and genomospecies. Genomic analyses revealed long-term colonization by Pseudomonas spp. and Serratia marcescens strains across multiple rooms. Nanopore sequencing uncovered examples of long-term persistence of resistance-conferring plasmids in unrelated hosts. These data indicate that antibiotic resistance (AR) in Pseudomonas spp. is maintained both by strain colonization and horizontal gene transfer (HGT), while HGT maintains AR within Acinetobacter spp. and Enterobacterales, independent of colonization. These results emphasize the importance of proactive, genomic-focused surveillance of built environments to mitigate MDRO spread. IMPORTANCE Hospital sinks are frequently linked to outbreaks of antibiotic-resistant bacteria. Here, we used whole-genome sequencing to track the long-term colonization patterns in intensive care unit (ICU) sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. We analyzed 822 bacterial genomes, representing over 100 different species. We identified long-term contamination by opportunistic pathogens, as well as transient appearance of other common pathogens. We found that bacteria recovered from the ICU had more antibiotic resistance genes (ARGs) in their genomes compared to matched community spaces. We also found that many of these ARGs are harbored on mobilizable plasmids, which were found shared in the genomes of unrelated bacteria. Overall, this study provides an in-depth view of contamination patterns for common nosocomial pathogens and identifies specific targets for surveillance.

Association between SARS-CoV-2 Symptoms, Ct Values, and Serological Response in Vaccinated and Unvaccinated Healthcare Personnel.

BACKGROUND: SARS-CoV-2 vaccines are effective at reducing symptomatic and asymptomatic COVID-19. Limited studies have compared symptoms, threshold cycle (Ct) values from reverse transcription (RT)-PCR testing, and serological testing results between previously vaccinated vs unvaccinated populations with SARS-CoV-2 infection. METHODS: Healthcare personnel (HCP) with a positive SARS-CoV-2 RT-PCR test within the previous 14 to 28 days completed surveys including questions about demographics, medical conditions, social factors, and symptoms of COVID-19. Ct values were observed, and serological testing was performed for anti-nucleocapsid (anti-N) and anti-Spike (anti-S) antibodies at enrollment and 40 to 90 days later. Serological results were compared to HCP with no known SARS-CoV-2 infection and negative anti-N testing. RESULTS: There were 104 unvaccinated/not fully vaccinated and 77 vaccinated HCP with 2 doses of an mRNA vaccine at time of infection. No differences in type or duration of symptoms were reported (P = 0.45). The median (interquartile range [IQR]) Ct was 21.4 (17.6-24.6) and 21.5 (18.1-24.6) for the unvaccinated and vaccinated HCP, respectively. Higher anti-N IgG was observed in unvaccinated HCP (5.08 S/CO, 3.08-6.92) than vaccinated (3.61 signal to cutoff ratio [S/CO], 2.16-5.05). Anti-S IgG was highest among vaccinated HCP with infection (34 285 aribitrary units [AU]/mL, 17 672-61 775), followed by vaccinated HCP with no prior infection (1452 AU/mL, 791-2943), then unvaccinated HCP with infection (829 AU/mL, 290-1555). Anti-S IgG decreased 1.56% (0.9%-1.79%) per day in unvaccinated and 0.38% (0.03%-0.94%) in vaccinated HCP. CONCLUSIONS: Vaccinated HCP infected with SARS-CoV-2 reported comparable symptoms and had similar Ct values relative to unvaccinated. However, vaccinated HCP had increased and prolonged anti-S and decreased anti-N response relative to unvaccinated.

Uncharacterized and lineage-specific accessory genes within the Proteus mirabilis pan-genome landscape.

Proteus mirabilis is a Gram-negative bacterium recognized for its unique swarming motility and urease activity. A previous proteomic report on four strains hypothesized that, unlike other Gram-negative bacteria, P. mirabilis may not exhibit significant intraspecies variation in gene content. However, there has not been a comprehensive analysis of large numbers of P. mirabilis genomes from various sources to support or refute this hypothesis. We performed comparative genomic analysis on 2,060 Proteus genomes. We sequenced the genomes of 893 isolates recovered from clinical specimens from three large US academic medical centers, combined with 1,006 genomes from NCBI Assembly and 161 genomes assembled from Illumina reads in the public domain. We used average nucleotide identity (ANI) to delineate species and subspecies, core genome phylogenetic analysis to identify clusters of highly related P. mirabilis genomes, and pan-genome annotation to identify genes of interest not present in the model P. mirabilis strain HI4320. Within our cohort, Proteus is composed of 10 named species and 5 uncharacterized genomospecies. P. mirabilis can be subdivided into three subspecies; subspecies 1 represented 96.7% (1,822/1,883) of all genomes. The P. mirabilis pan-genome includes 15,399 genes outside of HI4320, and 34.3% (5,282/15,399) of these genes have no putative assigned function. Subspecies 1 is composed of several highly related clonal groups. Prophages and gene clusters encoding putatively extracellular-facing proteins are associated with clonal groups. Uncharacterized genes not present in the model strain P. mirabilis HI4320 but with homology to known virulence-associated operons can be identified within the pan-genome. IMPORTANCE Gram-negative bacteria use a variety of extracellular facing factors to interact with eukaryotic hosts. Due to intraspecies genetic variability, these factors may not be present in the model strain for a given organism, potentially providing incomplete understanding of host-microbial interactions. In contrast to previous reports on P. mirabilis, but similar to other Gram-negative bacteria, P. mirabilis has a mosaic genome with a linkage between phylogenetic position and accessory genome content. P. mirabilis encodes a variety of genes that may impact host-microbe dynamics beyond what is represented in the model strain HI4320. The diverse, whole-genome characterized strain bank from this work can be used in conjunction with reverse genetic and infection models to better understand the impact of accessory genome content on bacterial physiology and pathogenesis of infection.

Gut pathogen colonization precedes bloodstream infection in the neonatal intensive care unit.

Bacterial bloodstream infections (BSIs) resulting in late-onset sepsis affect up to half of extremely preterm infants and have substantial morbidity and mortality. Bacterial species associated with BSIs in neonatal intensive care units (NICUs) commonly colonize the preterm infant gut microbiome. Accordingly, we hypothesized that the gut microbiome is a reservoir of BSI-causing pathogenic strains that increase in abundance before BSI onset. We analyzed 550 previously published fecal metagenomes from 115 hospitalized neonates and found that recent ampicillin, gentamicin, or vancomycin exposure was associated with increased abundance of Enterobacteriaceae and Enterococcaceae in infant guts. We then performed shotgun metagenomic sequencing on 462 longitudinal fecal samples from 19 preterm infants (cases) with BSI and 37 non-BSI controls, along with whole-genome sequencing of the BSI isolates. Infants with BSI caused by Enterobacteriaceae were more likely than infants with BSI caused by other organisms to have had ampicillin, gentamicin, or vancomycin exposure in the 10 days before BSI. Relative to controls, gut microbiomes of cases had increased relative abundance of the BSI-causing species and clustered by Bray-Curtis dissimilarity according to BSI pathogen. We demonstrated that 11 of 19 (58%) of gut microbiomes before BSI, and 15 of 19 (79%) of gut microbiomes at any time, harbored the BSI isolate with fewer than 20 genomic substitutions. Last, BSI strains from the Enterobacteriaceae and Enterococcaceae families were detected in multiple infants, indicating BSI-strain transmission. Our findings support future studies to evaluate BSI risk prediction strategies based on gut microbiome abundance in hospitalized preterm infants.

Genomic Analyses of Longitudinal Mycobacterium abscessus Isolates in a Multicenter Cohort Reveal Parallel Signatures of In-Host Adaptation.

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and an increasingly frequent cause of opportunistic infections. Mycobacterium abscessus complex (MABC) is one of the major NTM lung pathogens that disproportionately colonize and infect the lungs of individuals with cystic fibrosis (CF). MABC infection can persist for years, and antimicrobial treatment is frequently ineffective. METHODS: We sequenced the genomes of 175 isolates longitudinally collected from 30 patients with MABC lung infection. We contextualized our cohort amidst the broader MABC phylogeny and investigated genes undergoing parallel adaptation across patients. Finally, we tested the phenotypic consequences of parallel mutations by conducting antimicrobial resistance and mercury-resistance assays. RESULTS: We identified highly related isolate pairs across hospital centers with low likelihood of transmission. We further annotated nonrandom parallel mutations in 22 genes and demonstrated altered macrolide susceptibility co-occurring with a nonsynonymous whiB1 mutation. Finally, we highlighted a 23-kb mercury-resistance plasmid whose loss during chronic infection conferred phenotypic susceptibility to organic and nonorganic mercury compounds. CONCLUSIONS: We characterized parallel genomic processes through which MABC is adapting to promote survival within the host. The within-lineage polymorphisms we observed have phenotypic effects, potentially benefiting fitness in the host at the putative detriment of environmental survival.

Evaluation of the NG-Test CARBA 5 Lateral Flow Assay with an IMP-27-Producing Morganella morganii and Other Morganellaceae.

An isolate of Morganella morganii (MMOR1) that tested susceptible to 3rd/4th-generation cephalosporins and intermediate to meropenem was characterized as positive for NDM and IMP carbapenemases by NG-Test CARBA 5. Our objective was to further investigate this result, given the inconsistent susceptibility profile and unusual epidemiological profile for our region. The MMOR1 isolate was retested for antimicrobial susceptibilities and characterized for carbapenemase production. MMOR1 tested susceptible to ceftazidime, ceftriaxone, cefepime, aztreonam, and ertapenem, and intermediate to meropenem and imipenem. The isolate tested positive by carbapenem inactivation method (CIM) and CIM+EDTA (eCIM) testing, indicating metallo-ß-lactamase production. The isolate tested negative for all carbapenemase genes on Xpert Carba-R, but positive for IMP on repeat testing of NG-Test CARBA 5. Whole-genome sequencing revealed MMOR1 contained blaIMP-27, but no other carbapenemase genes. Additional testing with NG-Test CARBA 5 revealed a false-positive NDM band when the assay was overloaded with test inoculum. Supplementary isolates were tested with an overloaded inoculum (n = 6 M. morganii; n = 1 P. mirabilis; n = 1 IMP-27-producing P. rettgeri; n = 1 IMP-1-producing E. coli; n = 1 K. pneumoniae), and two non-carbapenemase-producing carbapenem non-susceptible M. morganii also generated a false-positive NDM band; though, this was not universal among this species. A dual IMP+/NDM+ M. morganii is an unusual result that should prompt additional investigation, especially in nonendemic regions and when the susceptibility profile is incompatible. IMP-27 is not detected by Xpert Carba-R but is variably detected by NG-Test CARBA 5. The microorganism inoculum used for NG-Test CARBA 5 must be carefully controlled for accurate results. IMPORTANCE The detection of carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) is an important function of the clinical microbiology laboratory, where positive identifications have immediate implications for infection control and surveillance strategies in the inpatient setting and can inform appropriate selection of therapy among the various novel anti-CP-CRE agents. NG-Test CARBA 5 is a relatively new lateral flow assay used for detection of carbapenemases in CP-CRE. Here, we describe the characterization of a Morganella morganii isolate that generated a false-positive NDM carbapenemase detection by this assay, and perform bacterial test inoculum experiments with additional isolates to further investigate a cause of false-positive results using the NG-Test CARBA 5. While a lateral flow assay like the NG-Test CARBA 5 is a very desirable test format for clinical laboratories, there are pitfalls to avoid when performing this test and interpreting results, including recognizing an overloaded test assay, which could lead to false-positive results.

Comparative Evaluation of Current Biochemical-, Sequencing-, and Proteomic-Based Identification Methods for the Streptococcus bovis Group.

The Streptococcus bovis group (previously group D streptococci) consists of seven distinct species and subspecies. Definitive identification within the group is important, as certain organisms have been associated with gastrointestinal carcinoma, bacteremia, infective endocarditis, meningitis, biliary tract disease, and carcinoma, among others. Definitive identification, however, remains elusive due to limitations and inconsistencies across commonly used identification platforms in the United States. Here, we compared the performance of standard biochemical (Trek Gram-positive identification [GPID] plate, Vitek 2 GPID), sequencing (16S rDNA, sodA) databases (NCBI, RDP, CDC MicrobeNet), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) platforms (Vitek MS, Bruker Biotyper MS) using a set of eight type strains representing all seven strains within the S. bovis group. Despite the evaluation of contemporary methods, no single platform was able to definitively identify all type strains within the S. bovis group. Vitek MS (85.7%, 7/8) provided the most accurate definitive identifications, followed by sodA sequencing (75%, 6/8). Vitek 2 and Bruker Biotyper RUO platforms performed the next best (62.5%, 5/8). All remaining platforms failed to adequately differentiate type strains within the S. bovis group (range, 0 to 37.5%). Laboratorians and clinicians should be aware of the identification limitations of routine testing algorithms and incorporate reflex testing, when appropriate, to platforms such as Vitek MS and/or sodA sequencing that are more able to definitively identify S. bovis group organisms. Further clinical evaluation was conducted using 65 clinical isolates from three geographically distinct U.S. institutions. Future improvements in identification platforms may reveal new clinical and epidemiological trends for members of the S. bovis group.

Evaluation of Variability in Interpretation of Disk Diffusion Testing for Cefiderocol Using Different Brands of Mueller-Hinton Agar.

BACKGROUND: Cefiderocol is a new antibiotic used to treat infections with antibiotic resistant Gram-negative bacilli. The impact of differences between Mueller-Hinton agar (MHA) brands on susceptibility testing is underexplored. Compounding the implementation of cefiderocol susceptibility testing is a lack of harmonization between different regulatory body breakpoint criteria. METHODS: We performed Kirby-Bauer disk diffusion using BD, Hardy, and Remel MHA, in addition to broth microdilution for Acinetobacter baumannii (n = 25), Enterobacterales (n = 25), Stenotrophomonas maltophilia (n = 24), and Pseudomonas aeruginosa (n = 23). We analyzed disk diffusion diameters and minimum inhibitory concentrations using interpretive criteria from the Clinical and Laboratory Standards Institute (CLSI), US Food and Drug Administration (FDA), and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). RESULTS: Breakpoint criteria impacted interpretation of susceptibly testing results, for example with the broth microdilution we found 8% (2/25) of A. baumannii isolates change interpretation between CLSI and EUCAST and 32% (8/25) change between CLSI and FDA, 12% (3/25) of Enterobacterales change between CLSI and EUCAST, 13% (3/23) of P. aeruginosa interpretations change between CLSI and FDA, and 4% (1/25) S. maltophilia change between CLSI and FDA. There was a significant difference between the zone disk diffusion diameters for P. aeruginosa and S. maltophilia between Hardy and BD; which changed interpretation (using CLSI criteria) for 8.7% (2/23) for P. aeruginosa but 0% (0/24) for S. maltophilia. CONCLUSIONS: Breakpoint criteria impact cefiderocol susceptibility testing interpretation for broth microdilution and disk diffusion. Choice of MHA brand can also affect result interpretation.

Outpatient Clonal Propagation and Rapid Regional Establishment of an Emergent Carbapenem-Resistant Acinetobacter baumannnii Lineage Sequence Type 499Pas.

Eliminating carbapenem-resistant Acinetobacter baumannii (CRAb) disease requires comprehensive knowledge of how this noncommensal organism propagates among at-risk hosts. We molecularly characterized an ongoing surge of CRAb cases among patients in a Midwest US healthcare system, which coincided with sustained reductions in hospital-acquired CRAb infections and falloffs of cases associated with distinctly more resistant antibiotypes. Genome sequencing revealed surge isolates belonged to an emergent Pasteur scheme sequence type 499 and comprised multiple contemporaneous clonal clusters. Detailed query of health records revealed no consistent hospital source but instead identified various outpatient healthcare settings linked to cluster cases. We show that CRAb can rapidly establish a regional presence even without gains in breadth of antibiotic resistance and negligible contribution from sustained intrahospital transmission. As CRAb lineages may sidestep control efforts via outpatient epidemiological niches, our approach can be implemented to investigate outpatient CRAb propagation and inform subsequent local surveillance outside hospital settings.

Antibodies in healthcare personnel following severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) infection.

In a prospective cohort of healthcare personnel (HCP), we measured severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) nucleocapsid IgG antibodies after SARS-CoV-2 infection. Among 79 HCP, 68 (86%) were seropositive 14-28 days after their positive PCR test, and 54 (77%) of 70 were seropositive at the 70-180-day follow-up. Many seropositive HCP (95%) experienced an antibody decline by the second visit.

Assessment of Infectious Diseases Risks From Dental Aerosols in Real-World Settings.

Background: Infectious diseases physicians are leaders in assessing the health risks in a variety of community settings. An understudied area with substantial controversy is the safety of dental aerosols. Previous studies have used in vitro experimental designs and/or indirect measures to evaluate bacteria and viruses from dental surfaces. However, these findings may overestimate the occupational risks of dental aerosols. The purpose of this study was to directly measure dental aerosol composition to assess the health risks for dental healthcare personnel and patients. Methods: We used a variety of aerosol instruments to capture and measure the bacterial, viral, and inorganic composition of aerosols during a variety of common dental procedures and in a variety of dental office layouts. Equipment was placed in close proximity to dentists during each procedure to best approximate the health risk hazards from the perspective of dental healthcare personnel. Devices used to capture aerosols were set at physiologic respiration rates. Oral suction devices were per the discretion of the dentist. Results: We detected very few bacteria and no viruses in dental aerosols-regardless of office layout. The bacteria identified were most consistent with either environmental or oral microbiota, suggesting a low risk of transmission of viable pathogens from patients to dental healthcare personnel. When analyzing restorative procedures involving amalgam removal, we detected inorganic elements consistent with amalgam fillings. Conclusions: Aerosols generating from dental procedures pose a low health risk for bacterial and likely viral pathogens when common aerosol mitigation interventions, such as suction devices, are employed.

Epidemiology of Plasmid Lineages Mediating the Spread of Extended-Spectrum Beta-Lactamases among Clinical Escherichia coli.

The prevalence of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Escherichia coli has been increasing, with this spread driven by ESBL-encoding plasmids. However, the epidemiology of ESBL-disseminating plasmids remains understudied, obscuring the roles of individual plasmid lineages in ESBL spread. To address this, we performed an in-depth genomic investigation of 149 clinical ESBL-like E. coli isolates from a tertiary care hospital. We obtained high-quality assemblies for 446 plasmids, revealing an extensive map of plasmid sharing that crosses time, space, and bacterial sequence type boundaries. Through a sequence-based network, we identified specific plasmid lineages that are responsible for the dissemination of major ESBLs. Notably, we demonstrate that IncF plasmids separate into 2 distinct lineages that are enriched for different ESBLs and occupy distinct host ranges. Our work provides a detailed picture of plasmid-mediated spread of ESBLs, demonstrating the extensive sequence diversity within identified lineages, while highlighting the genetic elements that underlie the persistence of these plasmids within the clinical E. coli population. IMPORTANCE The increasing incidence of nosocomial infections with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a significant threat to public health, given the limited treatment options available for such infections. The rapid ESBL spread is suggested to be driven by localization of the resistance genes on conjugative plasmids. Here, we identify the contributions of different plasmid lineages in the nosocomial spread of ESBLs. We provide further support for plasmid-mediated spread of ESBLs but demonstrate that some ESBL genes rely on dissemination through plasmids more than the others. We identify key plasmid lineages that are enriched in major ESBL genes and highlight the encoded genetic elements that facilitate the transmission and stable maintenance of these plasmid groups within the clinical E. coli population. Overall, our work provides valuable insight into the dissemination of ESBLs through plasmids, furthering our understating of factors underlying the increased prevalence of these genes in nosocomial settings.

Antibiotic-resistant organisms establish reservoirs in new hospital built environments and are related to patient blood infection isolates.

Background: Healthcare-associated infections due to antibiotic-resistant organisms pose an acute and rising threat to critically ill and immunocompromised patients. To evaluate reservoirs of antibiotic-resistant organisms as a source of transmission to patients, we interrogated isolates from environmental surfaces, patient feces, and patient blood infections from an established and a newly built intensive care unit. Methods: We used selective culture to recover 829 antibiotic-resistant organisms from 1594 environmental and 72 patient fecal samples, in addition to 81 isolates from blood cultures. We conducted antibiotic susceptibility testing and short- and long-read whole genome sequencing on recovered isolates. Results: Antibiotic-resistant organism burden is highest in sink drains compared to other surfaces. Pseudomonas aeruginosa is the most frequently cultured organism from surfaces in both intensive care units. From whole genome sequencing, different lineages of P. aeruginosa dominate in each unit; one P. aeruginosa lineage of ST1894 is found in multiple sink drains in the new intensive care unit and 3.7% of blood isolates analyzed, suggesting movement of this clone between the environment and patients. Conclusions: These results highlight antibiotic-resistant organism reservoirs in hospital built environments as an important target for infection prevention in hospitalized patients.

Persisting uropathogenic Escherichia coli lineages show signatures of niche-specific within-host adaptation mediated by mobile genetic elements.

Large-scale genomic studies have identified within-host adaptation as a hallmark of bacterial infections. However, the impact of physiological, metabolic, and immunological differences between distinct niches on the pathoadaptation of opportunistic pathogens remains elusive. Here, we profile the within-host adaptation and evolutionary trajectories of 976 isolates representing 119 lineages of uropathogenic Escherichia coli (UPEC) sampled longitudinally from both the gastrointestinal and urinary tracts of 123 patients with urinary tract infections. We show that lineages persisting in both niches within a patient exhibit increased allelic diversity. Habitat-specific selection results in niche-specific adaptive mutations and genes, putatively mediating fitness in either environment. Within-lineage inter-habitat genomic plasticity mediated by mobile genetic elements (MGEs) provides the opportunistic pathogen with a mechanism to adapt to the physiological conditions of either habitat, and reduced MGE richness is associated with recurrence in gut-adapted UPEC lineages. Collectively, our results establish niche-specific adaptation as a driver of UPEC within-host evolution.

Genomic Surveillance of Clinical Pseudomonas aeruginosa Isolates Reveals an Additive Effect of Carbapenemase Production on Carbapenem Resistance.

Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries is limited. This study establishes the genotypic mechanisms of ß-lactam resistance by whole-genome sequencing (WGS) in 142 P. aeruginosa clinical isolates recovered from three hospitals in Islamabad and Rawalpindi, Pakistan between 2016 and 2017. Isolates were subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion, and their genomes were assembled from Illumina sequencing data. ß-lactam resistance was high, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. Twenty-two percent of isolates were resistant to all ß-lactams tested. WGS revealed that carbapenem resistance was associated with the acquisition of metallo-ß-lactamases (MBLs) or extended-spectrum ß-lactamases (ESBLs) in the blaGES, blaVIM, and blaNDM families, and mutations in the porin gene oprD. These resistance determinants were found in globally distributed lineages, including ST235 and ST664, as well as multiple novel STs which have been described in a separate investigation. Analysis of AST results revealed that acquisition of MBLs/ESBLs on top of porin mutations had an additive effect on imipenem resistance, suggesting that there is a selective benefit for clinical isolates to encode multiple resistance determinants to the same drugs. The strong association of these resistance determinants with phylogenetic background displays the utility of WGS for monitoring carbapenem resistance in P. aeruginosa, while the presence of these determinants throughout the phylogenetic tree shows that knowledge of the local epidemiology is crucial for guiding potential treatment of multidrug-resistant P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is associated with serious infections, and treatment can be challenging. Because of this, carbapenems and ß-lactam/ß-lactamase inhibitor combinations have become critical tools in treating multidrug-resistant (MDR) P. aeruginosa infections, but increasing resistance threatens their efficacy. Here, we used WGS to study the genotypic and phylogenomic patterns of 142 P. aeruginosa isolates from the Potohar region of Pakistan. We sequenced both MDR and antimicrobial susceptible isolates and found that while genotypic and phenotypic patterns of antibiotic resistance correlated with phylogenomic background, populations of MDR P. aeruginosa were found in all major phylogroups. We also found that isolates possessing multiple resistance mechanisms had significantly higher levels of imipenem resistance compared to the isolates with a single resistance mechanism. This study demonstrates the utility of WGS for monitoring patterns of antibiotic resistance in P. aeruginosa and potentially guiding treatment choices based on the local spread of ß-lactamase genes.