Blue Light Compromises Bacterial ß-Lactamases Activity to Overcome ß-Lactam Resistance.

OBJECTIVE: In this study, we evaluated the effectiveness of antimicrobial blue light (aBL; 410 nm wavelength) against ß-lactamase-carrying bacteria and the effect of aBL on the activity of ß-lactamases. METHODS: Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae strains carrying ß-lactamases as well as a purified ß-lactamase enzymes were studied. ß-lactamase activity was assessed using a chromogenic cephalosporin hydrolysis assay. Additionally, we evaluated the role of porphyrins in the photoreaction, as well as protein degradation by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, we investigated the bactericidal effect of combined aBL-ceftazidime exposure against a metallo-ß-lactamase expressing P. aeruginosa strain. RESULTS: Our study demonstrated that aBL effectively killed ß-lactamase-producing bacteria and reduced ß-lactamase activity. After an aBL exposure of 1.52 J/cm2, a 50% reduction in enzymatic activity was observed in P. aeruginosa. Additionally, we found a 40% decrease in the photoreaction activity of porphyrins following an aBL exposure of 64.8 J/cm2. We also revealed that aBL reduced ß-lactamase activity via protein degradation (after 136.4 J/cm2). Additionally, aBL markedly improved the bactericidal effect of ceftazidime (by >4-log10) in the metallo-ß-lactamase P. aeruginosa strain. CONCLUSION: Our results provide evidence that aBL compromises bacterial ß-lactamase activity, offering a potential approach to overcome ß-lactam resistance in bacteria.

Second Time's the Charm? Assessing the Sensitivity and Yield of Inpatient Diagnostic Algorithms for Pulmonary Tuberculosis in a Low-Prevalence Setting.

Background: For persons with suspected pulmonary tuberculosis, the guidelines of the Centers for Disease Control and Prevention recommend collecting 3 respiratory specimens 8 to 24 hours apart for acid-fast bacilli (AFB) smear and culture, in addition to 1 nucleic acid amplification test (NAAT). However, data supporting this approach are limited. Our objective was to estimate the performance of 1, 2, or 3 AFB smears with or without NAATs to detect pulmonary tuberculosis in a low-prevalence setting. Methods: We conducted a retrospective study of hospitalized persons at 8 Massachusetts acute care facilities who underwent mycobacterial culture on 1 or more respiratory specimens between July 2016 and December 2022. We evaluated percentage positivity and yield on serial AFB smears and NAATs among people with growth of Mycobacterium tuberculosis on mycobacterial cultures. Results: Among 104 participants with culture-confirmed pulmonary tuberculosis, the first AFB smear was positive in 41 cases (39%). A second AFB smear was positive in 11 (22%) of the 49 cases in which it was performed. No third AFB smears were positive following 2 initial negative smears. Of 52 smear-negative cases, 36 had a NAAT performed, leading to 23 additional diagnoses. Overall sensitivity to detect tuberculosis prior to culture positivity was higher in any strategy involving 1 or 2 NAATs (74%-79%), even without AFB smears, as compared with 3 smears alone (60%). Conclusions: Tuberculosis diagnostic testing with 2 AFB smears offered the same yield as 3 AFB smears while potentially reducing laboratory burden and duration of airborne infection isolation. Use of 1 or 2 NAATs increased sensitivity to detect culture-positive pulmonary tuberculosis when added to AFB smear-based diagnostic testing alone.

Multicenter evaluation of the Selux Next-Generation Phenotyping antimicrobial susceptibility testing system.

The Selux Next-Generation Phenotyping (NGP) system (Charlestown, MA) is a new antimicrobial susceptibility testing system that utilizes two sequential assays performed on all wells of doubling dilution series to determine MICs. A multicenter evaluation of the performance of the Selux NGP system compared with reference broth microdilution was conducted following FDA recommendations and using FDA-defined breakpoints. A total of 2,488 clinical and challenge isolates were included; gram-negative isolates were tested against 24 antimicrobials, and gram-positive isolates were tested against 15 antimicrobials. Data is provided for all organism-antimicrobial combinations evaluated, including those that did and did not meet FDA performance requirements. Overall very major error and major error rates were less than 1% (31/3,805 and 107/15,606, respectively), essential agreement and categorical agreement were >95%, reproducibility was ≥95%, and the average time-to-result (from time of assay start to time of MIC result) was 5.65 hours.

Divergent genetic landscapes drive lower levels of AmpC induction and stable de-repression in Serratia marcescens compared to Enterobacter cloacae.

The chromosomally encoded AmpC beta-lactamase is widely distributed throughout the Enterobacterales. When expressed at high levels through transient induction or stable de-repression, resistance to ceftriaxone, a commonly used antibiotic, can develop. Recent clinical guidance suggests, based on limited evidence, that resistance may be less likely to develop in Serratia marcescens compared to the better-studied Enterobacter cloacae and recommends that ceftriaxone may be used if the clinical isolate tests susceptible. We sought to generate additional data relevant to this recommendation. AmpC de-repression occurs predominantly because of mutation in the ampD peptidoglycan amidohydrolase. We find that, in contrast to E. cloacae, where deletion of ampD results in high-level ceftriaxone resistance (with ceftriaxone MIC = 96 µg/mL), in S. marcescens deletion of two amidohydrolases (ampD and amiD2) is necessary for AmpC de-repression, and the resulting ceftriaxone MIC is 1 µg/mL. Two mechanisms for this difference were identified. We find both a higher relative increase in ampC transcript level in E. cloacae ΔampD compared to S. marcescens ΔampDΔamiD2, as well as higher in vivo efficiency of ceftriaxone hydrolysis by the E. cloacae AmpC enzyme compared to the S. marcescens AmpC enzyme. We also observed higher relative levels of transient AmpC induction in E. cloacae vs S. marcescens when exposed to ceftriaxone. In time-kill curves, this difference translates into the survival of E. cloacae but not S. marcescens at clinically relevant ceftriaxone concentrations. In summary, our findings can explain the decreased propensity for on-treatment ceftriaxone resistance development in S. marcescens, thereby supporting recently issued clinical guidance.

Blue Light Potentiates Antibiotics in Bacteria via Parallel Pathways of Hydroxyl Radical Production and Enhanced Antibiotic Uptake.

In the age of antimicrobial resistance, the urgency by which novel therapeutic approaches need to be introduced into the clinical pipeline has reached critical levels. Antimicrobial blue light (aBL), as an alternative approach, has demonstrated promise as a stand-alone therapeutic method, albeit with a limited window of antimicrobial activity. Work by others indicates that treatment with antibiotics increases the production of reactive oxygen species (ROS) which may, in part, contribute to the bactericidal effects of antibiotics. These findings suggest that there may be potential for synergistic interactions with aBL, that similarly generates ROS. Therefore, in this study, the mechanism of aBL is investigated, and the potential for aBL to synergistically promote antibiotic activity is similarly evaluated. Furthermore, the translatability of using aBL and chloramphenicol in combination within a mouse model of Acinetobacter baumanii burn infection is assessed. It is concluded that porphyrins and hydroxyl radicals driven by "free iron" are paramount to the effectiveness of aBL; and aBL is effective at promoting multiple antibiotics in different multidrug-resistant bacteria. Moreover, rROS up-regulation, and promoted antibiotic uptake are observed during aBL+antibiotic exposure. Lastly, aBL combined with chloramphenicol appears to be both effective and safe for the treatment of A. baumannii burn infection. In conclusion, aBL may be a useful adjunct therapy to antibiotics to potentiate their action.

If you don't test, they will not treat: Impact of stopping preoperative screening for asymptomatic bacteriuria.

Objective: Screening for asymptomatic bacteriuria (ASB) is not recommended outside of patients undergoing invasive urological procedures and during pregnancy. Despite national guidelines recommending against screening for ASB, this practice is prevalent. We present outcomes from a quality-improvement intervention targeting patients undergoing cardiac artery bypass grafting surgery (CABG) at Massachusetts General Hospital, a tertiary-care hospital in Boston, Massachusetts, where preoperative testing checklists were modified to remove routine urinalysis and urine culture. This was a before-and-after intervention study. Methods: Prior to the intervention, screening for ASB was included in the preoperative check list for all patients undergoing CABG. We assessed the proportion of patients undergoing screening for ASB in the 6 months prior to and after the intervention. We estimated cost savings from averted laboratory analyses, and we evaluated changes in antibiotic prescriptions. We additionally examined the incidence of postoperative surgical-site infections (SSIs), central-line-associated bloodstream infections (CLABSIs), catheter-associated urinary tract infections (CAUTIs) and Clostridioides difficile infections (CDIs). Results: Comparing the pre- and postintervention periods, urinalyses decreased by 76.5% and urine cultures decreased by 87.0%, with an estimated cost savings of $8,090.38. There were 50% fewer antibiotic prescriptions for bacteriuria after the intervention. Conclusions: Removal of urinalysis and urine culture from preoperative checklists for cardiac surgery led to a statistically significant decrease in testing without an increase in SSIs, CLABSIs, CAUTIs, or CDI. Challenges identified included persistence of checklists in templated order sets in the electronic health record.

Use of expert consensus to develop a shared list of procedures with potential for aerosol generation during the coronavirus disease 2019 (COVID-19) pandemic.

The coronavirus disease 2019 (COVID-19) pandemic highlighted the lack of agreement regarding the definition of aerosol-generating procedures and potential risk to healthcare personnel. We convened a group of Massachusetts healthcare epidemiologists to develop consensus through expert opinion in an area where broader guidance was lacking at the time.

Prospective evaluation of data-driven models to predict daily risk of Clostridioides difficile infection at 2 large academic health centers.

Many data-driven patient risk stratification models have not been evaluated prospectively. We performed and compared the prospective and retrospective evaluations of 2 Clostridioides difficile infection (CDI) risk-prediction models at 2 large academic health centers, and we discuss the models' robustness to data-set shifts.

Distinguishing Severe Acute Respiratory Syndrome Coronavirus 2 Persistence and Reinfection: A Retrospective Cohort Study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection is poorly understood, partly because few studies have systematically applied genomic analysis to distinguish reinfection from persistent RNA detection related to initial infection. We aimed to evaluate the characteristics of SARS-CoV-2 reinfection and persistent RNA detection using independent genomic, clinical, and laboratory assessments. METHODS: All individuals at a large academic medical center who underwent a SARS-CoV-2 nucleic acid amplification test (NAAT) ≥45 days after an initial positive test, with both tests between 14 March and 30 December 2020, were analyzed for potential reinfection. Inclusion criteria required having ≥2 positive NAATs collected ≥45 days apart with a cycle threshold (Ct) value <35 at repeat testing. For each included subject, likelihood of reinfection was assessed by viral genomic analysis of all available specimens with a Ct value <35, structured Ct trajectory criteria, and case-by-case review by infectious diseases physicians. RESULTS: Among 1569 individuals with repeat SARS-CoV-2 testing ≥45 days after an initial positive NAAT, 65 (4%) met cohort inclusion criteria. Viral genomic analysis characterized mutations present and was successful for 14/65 (22%) subjects. Six subjects had genomically supported reinfection, and 8 subjects had genomically supported persistent RNA detection. Compared to viral genomic analysis, clinical and laboratory assessments correctly distinguished reinfection from persistent RNA detection in 12/14 (86%) subjects but missed 2/6 (33%) genomically supported reinfections. CONCLUSIONS: Despite good overall concordance with viral genomic analysis, clinical and Ct value-based assessments failed to identify 33% of genomically supported reinfections. Scaling-up genomic analysis for clinical use would improve detection of SARS-CoV-2 reinfections.

Contact Tracing and Exposure Investigation in Response to the First Case of Monkeypox Virus Infection in the United States During the 2022 Global Monkeypox Outbreak.

BACKGROUND: In May 2022, the first case of monkeypox virus (MPXV) infection in the United States in the current global outbreak was identified. As part of the public health and health care facility response, a contact tracing and exposure investigation was done. OBJECTIVE: To describe the contact tracing, exposure identification, risk stratification, administration of postexposure prophylaxis (PEP), and exposure period monitoring for contacts of the index patient, including evaluation of persons who developed symptoms possibly consistent with MPXV infection. DESIGN: Contact tracing and exposure investigation. SETTING: Multiple health care facilities and community settings in Massachusetts. PARTICIPANTS: Persons identified as contacts of the index patient. INTERVENTION: Contact notification, risk stratification, and symptom monitoring; PEP administration in a subset of contacts. MEASUREMENTS: Epidemiologic and clinical data collected through standard surveillance procedures at each facility and then aggregated and analyzed. RESULTS: There were 37 community and 129 health care contacts identified, with 4 at high risk, 49 at intermediate risk, and 113 at low or uncertain risk. Fifteen health care contacts developed symptoms during the monitoring period. Three met criteria for MPXV testing, with negative results. Two community contacts developed symptoms. Neither met criteria for MPXV testing, and neither showed disease progression consistent with monkeypox. Among 4 persons with high-risk exposures offered PEP, 3 elected to receive PEP. Among 10 HCP with intermediate-risk exposures for which PEP was offered as part of informed clinical decision making, 2 elected to receive PEP. No transmissions were identified at the conclusion of the 21-day monitoring period, despite the delay in recognition of monkeypox in the index patient. LIMITATION: Descriptions of exposures are subject to recall bias, which affects risk stratification. CONCLUSION: In a contact tracing investigation involving 166 community and health care contacts of a patient with monkeypox, no secondary cases were identified. PRIMARY FUNDING SOURCE: None.

Tecovirimat for the Treatment of Human Monkeypox: An Initial Series From Massachusetts, United States.

A large, ongoing multicountry outbreak of human monkeypox has the potential to cause considerable morbidity and mortality. Therapeutics for the treatment of smallpox, a related Orthopoxvirus, may be used and affect the natural history of monkeypox. We present 3 patients from our hospitals treated with tecovirimat, a pan-Orthopoxvirus inhibitor currently available under an expanded access investigational new drug protocol for monkeypox.

Infection Prevention and Control of Severe Acute Respiratory Syndrome Coronavirus 2 in Health Care Settings.

The authors describe infection prevention and control approaches to severe acute respiratory syndrome coronavirus 2 in the health care setting, including a review of the chain of transmission and the hierarchy of controls, which are cornerstones of infection control and prevention. The authors also discuss lessons learned from nosocomial transmission events.

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. METHODS: To overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years. RESULTS: Our CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. CONCLUSIONS: Collectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

A Genome-Scale Antibiotic Screen in Serratia marcescens Identifies YdgH as a Conserved Modifier of Cephalosporin and Detergent Susceptibility.

Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing health care environments and is an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because, in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-MICs of two cephalosporins, cefoxitin, and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single-gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgH in S. marcescens resulted in decreased susceptibility to multiple third-generation cephalosporins and, in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm, and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

Clinical, Laboratory, and Radiologic Characteristics of Patients With Initial False-Negative Severe Acute Respiratory Syndrome Coronavirus 2 Nucleic Acid Amplification Test Results.

BACKGROUND: Concerns about false-negative (FN) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid amplification tests (NAATs) have prompted recommendations for repeat testing if suspicion for coronavirus disease 2019 (COVID-19) infection is moderate to high. However, the frequency of FNs and patient characteristics associated with FNs are poorly understood. METHODS: We retrospectively reviewed test results from 15 011 adults who underwent ≥1 SARS-CoV-2 NAATs; 2699 had an initial negative NAAT and repeat testing. We defined FNs as ≥1 negative NAATs followed by a positive NAAT within 14 days during the same episode of illness. We stratified subjects with FNs by duration of symptoms before the initial FN test (≤5 days versus >5 days) and examined their clinical, radiologic, and laboratory characteristics. RESULTS: Sixty of 2699 subjects (2.2%) had a FN result during the study period. The weekly frequency of FNs among subjects with repeat testing peaked at 4.4%, coinciding with peak NAAT positivity (38%). Most subjects with FNs had symptoms (52 of 60; 87%) and chest radiography (19 of 32; 59%) consistent with COVID-19. Of the FN NAATs, 18 of 60 (30%) were performed early (ie, ≤1 day of symptom onset), and 18 of 60 (30%) were performed late (ie, >7 days after symptom onset) in disease. Among 17 subjects with 2 consecutive FNs on NP NAATs, 9 (53%) provided lower respiratory tract (LRT) specimens for testing, all of which were positive. CONCLUSIONS: Our findings support repeated NAATs among symptomatic patients, particularly during periods of higher COVID-19 incidence. The LRT testing should be prioritized to increase yield among patients with high clinical suspicion for COVID-19.

Yield of Severe Acute Respiratory Syndrome Coronavirus 2 Lower Respiratory Tract Testing After a Negative Nasopharyngeal Test Among Hospitalized Persons Under Investigation for Coronavirus Disease 2019.

Among hospitalized persons under investigation for coronavirus disease 2019 (COVID-19), more repeated severe acute respiratory syndrome coronavirus 2 nucleic acid amplification tests (NAATs) after a negative NAAT were positive from lower than from upper respiratory tract specimens (1.9% vs 1.0%, P = .033). Lower respiratory testing should be prioritized among patients displaying respiratory symptoms with moderate-to-high suspicion for COVID-19 after 1 negative upper respiratory NAAT.

Escherichia coli GyrA Tower Domain Interacts with QnrB1 Loop B and Plays an Important Role in QnrB1 Protection from Quinolone Inhibition.

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo-cross-linking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286 to 298 on the tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286 to 289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.