Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection.

Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.

Impact of FilmArray™ Respiratory Panel testing on the clinical management of pediatric bone marrow transplant patients.

Viral respiratory tract infections cause significant morbidity in bone marrow transplant (BMT) patients. Speed and sensitivity of the FilmArray™ Respiratory Panel (FA-RP) can improve care but may prompt inappropriate testing. Studies describing FA-RP use in pediatric BMT patients are limited; we investigated FA-RP use, results, and clinical management to evaluate clinical significance of testing in pediatric BMT patients. Retrospective analysis of 671 respiratory specimens from 204 unique BMT patients between 01/01/2016 and 01/01/2019 was performed. Age, underlying diagnoses, FA-RP result, reason for FA-RP, and symptoms were abstracted. FA-RP impact on antimicrobial management, scheduled procedures, infection control measures, and hospital admission/discharge were investigated. Impacts of repeat testing were evaluated. Two hundred sixty-nine out of 671 specimens (40%) tested positive; human rhinovirus/enterovirus (hRV/hEV) was the most common (161/269, 60%). The primary reason for FA-RP was URI symptoms (402/671, 60%) with 54% testing positive. One hundred twenty-two out of 671 (18.2%) specimens were from asymptomatic patients; 14 (11.4%) tested positive. FA-RP informed antiviral initiation in 7/19 (36.8%), 7/8 (87.5%), and 5/30 (16.7%) of RSV, influenza, and human parainfluenza cases, respectively. In 11 cases, FA-RP informed azithromycin and ceftriaxone initiation, continuation, or discontinuation. BMT was delayed for three positives (two RSV, one hRV/hEV). In 22 instances, negative FA-RP cleared patients for BMT. In 70% of cases, repeats offered no new clinical information; all negative-to-positive cases had new or worsening respiratory symptoms. FA-RP was ordered on symptomatic and asymptomatic patients, provided rapid diagnosis in > 50% of symptomatic patients, and informed infection control measures for all inpatients and antiviral initiation in > 80% of influenza cases.

Statewide surveillance of carbapenemase-producing carbapenem-resistant Escherichia coli and Klebsiella species in Washington state, October 2012-December 2017.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are common causes of healthcare-associated infections and are often multidrug resistant with limited therapeutic options. Additionally, CRE can spread within and between healthcare facilities, amplifying potential harms. OBJECTIVE: To better understand the burden, risk factors, and source of acquisition of carbapenemase genes in clinical Escherichia coli and Klebsiella spp isolates from patients in Washington to guide prevention efforts. DESIGN: Multicenter prospective surveillance study. METHODS: Escherichia coli and Klebsiella spp isolates meeting the Washington state CRE surveillance case definition were solicited from clinical laboratories and tested at Washington Public Health Laboratories using polymerase chain reaction (PCR) for the 5 most common carbapenemase genes: blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48. Case patients positive by PCR were investigated by the public health department. RESULTS: From October 2012 through December 2017, 363 carbapenem-resistant E. coli and Klebsiella spp isolates were tested. Overall, 45 of 115 carbapenem-resistant K. pneumoniae (39%), 1 of 8 K. oxytoca (12.5%), and 28 of 239 carbapenem-resistant E. coli (11.7%) were carbapenemase positive. Of 74 carbapenemase-positive isolates, blaKPC was most common (47%), followed by blaNDM (30%), blaOXA-48 (22%), and blaIMP (1%). Although all cases had healthcare exposure, blaKPC acquisition was associated with US health care, whereas non-blaKPC acquisition was associated with international health care or travel. CONCLUSIONS: We report that blaKPC, the most prevalent carbapenemase in the United States, accounts for nearly half of carbapenemase cases in Washington state and that most KPC-cases are likely acquired through in-state health care.

Cerebrospinal Fluid Findings Are Poor Predictors of Appropriate FilmArray Meningitis/Encephalitis Panel Utilization in Pediatric Patients.

Molecular testing of cerebrospinal fluid (CSF) using the BioFire FilmArray meningitis/encephalitis (FA-M/E) panel permits rapid, simultaneous pathogen detection. Due to the broad spectrum of targeted organisms, FA-M/E testing may be restricted to patients with abnormal CSF findings. We sought to determine if restriction is appropriate in our previously healthy and/or immunocompromised pediatric patients. FA-M/E was ordered on 1,025 CSF samples from 948 patients; 121 (11.8%) specimens were FA-M/E positive. Of these, 89 (73.6%) were virus positive, and 30 (24.8%) were bacterium positive. The most common targets detected were enterovirus (n = 38), human herpesvirus 6 (HHV-6) (n = 30), and Streptococcus pneumoniae (n = 14). Pleocytosis with white blood cell (WBC) levels of ≥5 cells/mm3 and ≥10 cells/mm3 were found in 33.1% and 24.3% of all specimens, respectively. Using WBC levels of ≥5 cells/mm3, 63.4% (59/93) of positive specimens exhibited pleocytosis, compared to 29.5% (233/789) of negative specimens. Among positive specimens, 54.4% (37/68) of viral and 87% (20/23) of bacterial cases had pleocytosis. The use of a pleocytosis cutoff of ≥10 cells/mm3 would have missed an additional enterovirus, one cytomegalovirus (CMV), and two HHV-6 diagnoses. CSF glucose and protein levels were normal for 83/116 (75.2%) and 51/116 (44%) positive specimens. Abnormal glucose in combination with WBC levels of ≥10 cells/mm3 showed high specificity (94.5%) and was a better predictor of FA-M/E positivity than abnormal protein. Sensitivity and positive predictive values were <90% for all biomarkers. CSF pleocytosis and abnormal glucose/protein were poor predictors of FA-M/E. Restricting FA-M/E orders based on pleocytosis or other abnormal parameters would have resulted in missed diagnostic opportunities, particularly for the detection of viruses in both previously healthy and immunocompromised patients.

Prevalence and clinical associations of Staphylococcus aureus small-colony variant respiratory infection in children with cystic fibrosis (SCVSA): a multicentre, observational study.

BACKGROUND: Staphylococcus aureus is the bacterium cultured most often from respiratory secretions of people with cystic fibrosis. Both meticillin-susceptible S aureus and meticillin-resistant S aureus (MRSA) can adapt to form slow-growing, antibiotic-resistant isolates known as small-colony variants that are not routinely identified by clinical laboratories. We aimed to determine the prevalence and clinical significance of S aureus small-colony variants and their subtypes among children with cystic fibrosis. METHODS: The Small Colony Variant Staphylococcus aureus (SCVSA) study was a 2-year longitudinal study of children aged 6-16 years at five US cystic fibrosis centres, using culture methods sensitive for small-colony variants. Children were eligible if they had a documented diagnosis of cystic fibrosis and a minimum of two cystic fibrosis clinic visits and two respiratory cultures in the previous 12 months at enrolment. Participants attended clinic visits quarterly, at which respiratory tract samples were taken and measures of lung function (percentage of predicted forced expiratory volume in 1 s [FEV1] and frequency of respiratory exacerbations) were recorded. We determined the prevalence of small-colony variants and their subtypes, and assessed their independent associations with lung function and respiratory exacerbations using linear mixed-effects and generalised estimating equation logistic regression models. Analyses included both univariate models (unadjusted) and multivariate models that adjusted for potential confounders, including age, sex, race, baseline microbiology, treatment with CFTR modulator, and CTFR genotype. FINDINGS: Between July 1, 2014, and May 26, 2015, we enrolled 230 children. Participants were followed-up for 2 years, with a mean of 6·4 visits (SD 1·14) per participant (range 2-9 visits) and a mean interval between visits of 3·94 months (SD 1·77). Across the 2-year period, S aureus small-colony variants were detected in 64 (28%) participants. Most (103 [56%] of 185) of the small-colony variants detected in these participants were thymidine dependent. Children with small-colony variants had significantly lower mean percentage of predicted FEV1 at baseline than did children without small-colony variants (85·5 [SD 19] vs 92·4 [SD 18·6]; p=0·0145). Small-colony variants were associated with significantly lower percentage of predicted FEV1 throughout the study in regression models, both in univariate analyses (regression coefficient -7·07, 95% CI -12·20 to -1·95; p=0·0068) and in multivariate analyses adjusting for potential confounders (-5·50, -10·51 to -0·48; p=0·0316). Small colony variants of the thymidine-dependent subtype had the strongest association with lung function in multivariate regression models (regression coefficient -10·49, -17·25 to -3·73; p=0·0024). Compared with children without small-colony variants, those with small-colony variants had significantly increased odds of respiratory exacerbations in univariate analyses (odds ratio 1·73, 95% CI 1·19 to 2·52; p=0·0045). Children with thymidine-dependent small-colony variants had significantly increased odds of respiratory exacerbations (2·81, 1·69-4·67; p=0·0001), even after adjusting for age, sex, race, genotype, CFTR modulator, P aeruginosa culture status, and baseline percentage of predicted FEV1 (2·17, 1·33-3·57; p=0·0021), whereas those with non-thymidine-dependent small-colony variants did not. In multivariate models including small-colony variants and MRSA status, P aeruginosa was not independently associated with lung function (regression coefficient -4·77, 95% CI -10·36 to 0·83; p=0·10) and was associated with reduced odds of exacerbations (0·54, 0·36 to 0·81; p=0·0028). Only the small-colony variant form of MRSA was associated with reduced lung function (-8·44, -16·15 to -0·72; p=0·0318) and increased odds of exacerbations (2·15, 1·24 to 3·71; p=0·0061). INTERPRETATION: Infection with small-colony variants, and particularly thymidine-dependent small-colony variants, was common in a multicentre paediatric population with cystic fibrosis and associated with reduced lung function and increased risk of respiratory exacerbations. The adoption of small-colony variant identification and subtyping methods by clinical laboratories, and the inclusion of small-colony variant prevalence data in cystic fibrosis registries, should be considered for ongoing surveillance and study. FUNDING: The Cystic Fibrosis Foundation and the National Institutes of Health.

Optimized In Vitro Antibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus.

Staphylococcus aureus small-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST of S. aureus SCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86 S. aureus SCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin; mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.

Effect of fullerene intercalation on the conformation and packing of poly-(2-methoxy-5-(3'-7'-dimethyloctyloxy)-1,4-phenylenevinylene).

Photoluminescence (PL) and resonance Raman spectroscopy are used to track changes in the conformations and packing of poly-(2-methoxy-5-(3'-7'-dimethyloctyloxy)-1,4-phenylenevinylene) (MDMO-PPV) chains with the addition of [6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM) molecules. PL lineshapes of MDMO-PPV thin films as a function of annealing time were first measured to determine the spectroscopic signatures of chain conformations and packing in the absence of PCBM. Annealing results in enhanced interchain interactions leading to red-shifts of PL 0-0 transitions by up to ∼300 cm(-1) and apparent increases of the line shape Huang-Rhys factors. Wavelength-dependent PL lifetimes of as-cast and films annealed for short times (∼30 s) are nonexponential with an instrument-limited component of ∼100 ps and a ∼350 ps component. With longer annealing times, decays become single exponential with an average lifetime of ∼1 ns indicating that all excitations efficiently funnel to strongly coupled interchain sites. Addition of PCBM disrupts MDMO-PPV interchain interactions causing PL 0-0 transitions to blue-shift, increases in line width, and decreases in apparent Huang-Rhys factors. Resonance Raman spectra of MDMO-PPV/PCBM thin films with variable PCBM weight fractions (∼50:1 up to 1:8 w/w) were then measured using short (488 nm) and long (568 nm) excitation wavelengths. The out-of-plane vinylene C-H wag mode of MDMO-PPV (∼964 cm(-1)) showed pronounced increases in intensity of up to ∼30% and red-shifts of up to 5 cm(-1) with increasing PCBM content. These changes result from a decrease of planarity between chain segments that suppresses interchain interactions. Furthermore, red-shifts of up to ∼4 cm(-1) were observed for the C═C symmetric stretch of the MDMO-PPV vinylene group (∼1625 cm(-1)) with 488 nm excitation. The sensitivity of the MDMO-PPV vinylene group vibrations with PCBM indicates preferential interactions between these two molecules and is consistent with intercalation of PCBM into the polymer structure. This assignment was confirmed by thermally annealing of MDMO-PPV/PCBM films to remove intercalated PCBM molecules, which partially restores interchain interactions as seen from smaller intensity increases (∼15%) and red-shifts (∼2 cm(-1)) of the ∼964 cm(-1) mode. Overall, the spectroscopic results show that MDMO-PPV chains adopt distorted conformations (i.e., less intrachain order and shorter conjugation lengths) that have important implications for explaining the structural origins for large improvements in charge mobilities in MDMO-PPV/PCBM blends.