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

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

Association Between Clostridium innocuum and Antibiotic-Associated Diarrhea in Adults and Children: A Cross-sectional Study and Comparative Genomics Analysis.

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.

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

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

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

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

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

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

A comparative genomics approach identifies contact-dependent growth inhibition as a virulence determinant.

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.

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

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

Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor.

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.

Aminoglycoside-resistance gene signatures are predictive of aminoglycoside MICs for carbapenem-resistant Klebsiella pneumoniae.

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.

Individual target pharmacokinetic/pharmacodynamic attainment rates among meropenem-treated patients admitted to the ICU with hospital-acquired pneumonia.

OBJECTIVES: Critical illness reduces ß-lactam pharmacokinetic/pharmacodynamic (PK/PD) attainment. We sought to quantify PK/PD attainment in patients with hospital-acquired pneumonia. METHODS: Meropenem plasma PK data (n = 70 patients) were modelled, PK/PD attainment rates were calculated for empirical and definitive targets, and between-patient variability was quantified [as a coefficient of variation (CV%)]. RESULTS: Attainment of 100% T>4×MIC was variable for both empirical (CV% = 92) and directed (CV% = 33%) treatment. CONCLUSIONS: Individualization is required to achieve suggested PK/PD targets in critically ill patients.

Incidence and Clinical Characteristics of Severe Acute Respiratory Syndrome Coronavirus 2 Infection After Vaccination in Children.

The objective of this single-center cohort study was to characterize the frequency, clinical characteristics, and molecular epidemiology of pediatric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection after vaccination. Between May 15, 2021, and January 1, 2022, 171 children experienced SARS-CoV-2 infection postvaccination, 146 (86%) following the Omicron variant predominance. Outcomes were generally mild and comparable before and after Omicron predominance.

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

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

De novo emergence of SARS-CoV-2 spike mutations in immunosuppressed patients.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Generating Genotype-Specific Aminoglycoside Combinations with Ceftazidime/Avibactam for KPC-Producing Klebsiella pneumoniae.

Antibiotic combinations, including ceftazidime/avibactam (CAZ/AVI), are frequently employed to combat KPC-producing Klebsiella pneumoniae (KPC-Kp), though such combinations have not been rationally optimized. Clinical KPC-Kp isolates with common genes encoding aminoglycoside-modifying enzymes (AMEs), aac(6')-Ib' or aac(6')-Ib, were used in static time-kill assays (n = 4 isolates) and the hollow-fiber infection model (HFIM; n = 2 isolates) to evaluate the activity of gentamicin, amikacin, and CAZ/AVI alone and in combinations. A short course, one-time aminoglycoside dose was also evaluated. Gentamicin plus CAZ/AVI was then tested in a mouse pneumonia model. Synergy with CAZ/AVI was more common with amikacin for aac(6')-Ib'-containing KPC-Kp but more common with gentamicin for aac(6')-Ib-containing isolates in time-kill assays. In the HFIM, although the isolates were aminoglycoside-susceptible at baseline, aminoglycoside monotherapies displayed variable initial killing, followed by regrowth and resistance emergence. CAZ/AVI combined with amikacin or gentamicin resulted in undetectable counts 50 h sooner than CAZ/AVI monotherapy against KPC-Kp with aac(6')-Ib'. CAZ/AVI monotherapy failed to eradicate KPC-Kp with aac(6')-Ib and a combination with gentamicin led to undetectable counts 70 h sooner than with amikacin. A one-time aminoglycoside dose with CAZ/AVI provided similar killing to aminoglycosides dosed for 7 days. In the mouse pneumonia model (n = 1 isolate), gentamicin and CAZ/AVI achieved a 6.0-log10 CFU/lung reduction at 24 h, which was significantly greater than either monotherapy (P < 0.005). Aminoglycosides in combination with CAZ/AVI were promising for KPC-Kp infections; this was true even for a one-time aminoglycoside dose. Selecting aminoglycosides based on AME genes or susceptibilities can improve the pharmacodynamic activity of the combination.

Genetic Evaluation of Nosocomial Candida auris Transmission.

Whole-genome sequences of Candida auris isolates from nosocomial and nonnosocomial infections were compared. The average numbers of single nucleotide variations were different between the two groups. The small amount of genetic variability between intra- or interhost isolates suggests recovery of all colonizing or infecting genomes for comparison is required for outbreaks.

Optimizing aminoglycoside selection for KPC-producing Klebsiella pneumoniae with the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib.

OBJECTIVES: KPC-producing Klebsiella pneumoniae (KPC-Kp) isolates commonly co-harbour the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib, which encodes an AME that can confer resistance to some of the commercially available aminoglycosides. We sought to determine the influence of AAC(6')-Ib in KPC-Kp on the pharmacodynamic activity of aminoglycosides. METHODS: Six KPC-Kp clinical isolates, three with and three without aac(6')-Ib, were analysed. Using these isolates, the bacterial killing of amikacin, gentamicin and tobramycin was assessed in static time-kill experiments. The pharmacodynamic activity of the aminoglycosides was then assessed in a dynamic one-compartment infection model over 72 h using simulated human pharmacokinetics of once-daily dosing with amikacin (15 mg/kg), gentamicin (5 mg/kg) and tobramycin (5 mg/kg). RESULTS: At clinically relevant aminoglycoside concentrations in time-kill experiments and the dynamic one-compartment model, gentamicin was more active than amikacin or tobramycin against the isolates harbouring aac(6')-Ib. Amikacin, gentamicin and tobramycin all showed progressively reduced bacterial killing with exposure to repeated doses against most isolates in the dynamic one-compartment model. MIC values were generally not a good predictor of gentamicin pharmacodynamic activity against KPC-Kp, but were more reliable for amikacin and tobramycin. CONCLUSIONS: Gentamicin may be preferred over amikacin or tobramycin for treatment of KPC-Kp infections. However, gentamicin MICs are not a consistent predictor of its pharmacodynamic activity and unexpected treatment failures are possible.

Screening Students and Staff for Asymptomatic Coronavirus Disease 2019 in Chicago Schools.

OBJECTIVES: To assess rates of asymptomatic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) positivity in K-8 schools with risk mitigation procedures in place, and to evaluate SARS-CoV-2 transmission in school and household contacts of these positive individuals. STUDY DESIGN: In this prospective observational study, screening testing for SARS-CoV-2 was performed by oropharyngeal swabbing and polymerase chain reaction (PCR) analysis in students and staff at K-8 private schools in high-risk Chicago ZIP codes. New coronavirus disease 2019 (COVID-19) diagnoses or symptoms among participants, household contacts, and nonparticipants in each school were queried. RESULTS: Among 11 K-8 private schools across 8 Chicago ZIP codes, 468 participants (346 students, 122 staff members) underwent screening testing. At the first school, 17 participants (36%) tested positive, but epidemiologic investigation suggested against in-school transmission. Only 5 participants in the subsequent 10 schools tested positive for an overall 4.7% positivity rate (1.2% excluding school 1). All but 1 positive test among in-person students had high PCR cycle threshold values, suggesting very low SARS-CoV-2 viral loads. In all schools, no additional students, staff, or household contacts reported new diagnoses or symptoms of COVID-19 during the 2 weeks following screening testing. CONCLUSIONS: We identified infrequent asymptomatic COVID-19 in schools in high-risk Chicago communities and did not identify transmission among school staff, students, or their household contacts. These data suggest that COVID-19 mitigation procedures, including masking and physical distancing, are effective in preventing transmission of COVID-19 in schools. These results may inform future strategies for screening testing in K-8 schools.

Natural Clostridioides difficile Toxin Immunization in Colonized Infants.

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.

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

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

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av.