Current approaches for computationally analyzing viruses within human microbiomes often rely on databases largely composed of fragmented viral genomes from gastrointestinal samples, limiting identification of viruses exclusively found outside the gastrointestinal tract and analyses requiring high-quality genomes. To address these issues, we created the Unified Human Virome Database (UHVDB), comprising 575,497 high-quality, annotated viral genomes from human gastrointestinal, airway, skin, and urogenital sample metagenomes. We developed an associated toolkit that uses UHVDB to characterize viruses and their potential activity from metagenomes, then applied this toolkit to 1,983 airway sample metagenomes from people with cystic fibrosis. Over half of detected viruses lacked evidence of potential activity and were detected transiently. UHVDB is nearly three times larger than prior viral databases and its ability to identify likely active viruses enables rigorous analysis of viruses from diverse human sample types, expanding the capacity to define virus contributions to health and disease.

ABSTRACT The Burkholderia cepacia complex (BCC) is comprised of 24 species of Gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Sciences Institute (CLSI) to remove disk diffusion (DD) and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild-type phenotype. Here we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 μg/ml for CAZ, 8 μg/ml for LVX, 16 μg/ml for MEM, and 8 μg/ml for MIN. The ECV for TMP-SMX varied depending on the analysis from 2 μg/ml, 8 μg/ml, and 16 μg/ml and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC dataset, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and supported removal of ECVs from the CLSI M100 standards document. IMPORTANCE The Burkholderia cepacia complex is a group of bacterial species that cause difficult-to-treat opportunistic infections. Recently, clinical breakpoints, which are used to determine whether organisms are susceptible to certain antimicrobials, were removed from Clinical and Laboratory Standards Institute (CLSI) standards for these organisms due to problems with antimicrobial susceptibility testing performance. Clinicians are now faced with the challenge of how to treat these complex infections without clinical breakpoints. Here we determine epidemiological cut-off values (ECVs) for relevant antimicrobials for the B. cepacia complex. While we established ECVs for four antimicrobials, we encountered significant challenges in our analyses, including limitations with data for these organisms and high ECVs that are not clinically useful. These challenges limit the practical use of these ECVs in helping guide clinicians on treatment and supported the eventual removal of ECVs from the CLSI M100 standards document.

OBJECTIVES:Stenotrophomonas maltophilia is a multidrug-resistant Gram-negative pathogen causing serious infections in vulnerable populations, including individuals with cystic fibrosis and immunocompromised patients. We evaluated the activity of aztreonam (ATM) and ceftazidime/avibactam (CZA) against S. maltophilia complex (Smc) isolates. METHODS:Interactions between ATM and CZA were evaluated using static concentration time-kill assays across 11 Smc isolates. A mechanism-based pharmacokinetic/pharmacodynamic model was developed to characterize bacterial killing dynamics and guide regimen selection. Selected regimens were then evaluated in the hollow fibre infection model under clinically relevant drug exposures. Emergence of resistance was assessed via population analysis profiles, and scanning electron microscopy visualized antibiotic-induced morphological changes. RESULTS:Clinically relevant ATM + CZA exposures produced 31-74% reduction in area under the log-transformed bacterial count versus time curve across all Smc isolates in static concentration time-kill assays. In the hollow fibre infection model, ATM + CZA achieved ≥3-log10 CFU/mL reduction at 24 hours from an initial ∼7.5-log10 CFU/mL inoculum. This effect was sustained over 168 hours against GG6 strain. Against the non-GG6 strain, both continuous-infusion and standard-dosing regimens showed ∼2-log10 regrowth after 72 hours, but overall suppressed resistant subpopulations more effectively than trimethoprim/sulfamethoxazole. CONCLUSIONS:Smc infections remain challenging because of intrinsic resistance mechanisms. These findings demonstrate the therapeutic potential of ATM + CZA, and support further evaluation using murine pneumonia and bacteraemia models of S. maltophilia.

Change in sputum bacterial density has been used to measure efficacy in clinical trials of novel therapies for cystic fibrosis (CF); however, the baseline degree of day-to-day bacterial density change is not well understood. Recent data show that Pseudomonas density in CF sputum has the potential to vary considerably day-to-day even in the absence of antimicrobial treatment. Here we report daily variability of Burkholderia and Achromobacter density in 288 pairs of sputum samples obtained from eight adults with CF. Compared to similar previous analyses of Pseudomonas density, overall variability of day-to-day change for both Burkholderia and Achromobacter was relatively modest (SD=0.54 and 0.58 log10 16S rRNA gene copies/mL, respectively). Significant inter-person variability was observed among the Burkholderia cohort but not among the Achromobacter cohort (P = 0.001 and P = 0.07, respectively). Density changes for both Burkholderia and Achromobacter were inversely correlated with starting densities. This study provides context for the use of changes in sputum bacterial density as a response biomarker in clinical trials of CF therapeutics.

Ecological guilds are groups of organisms that utilize the same class of resources and occupy similar niches, regardless of their taxonomic identities. Here we propose the Guild Model for Cystic Fibrosis Airway Microbial Ecology, which considers the ecological function and wider role of each microbe in the ecosystem. This model consists of four functional guilds: (i) "Brewers" metabolize host-derived substrates (e.g., mucins) and produce fermentation products; (ii) "Drunkards" exploit the metabolic niche built by Brewers, consuming fermentation products and secreting exopolysaccharides to build biofilms; (iii) "Putrifiers" produce toxic compounds causing inflammation and tissue necrosis; and (iv) "Nihilists" are specialist pathogens characterized by intracellular or lytic life cycles and cytotoxin production. By focusing on microbial function and the broader community context, this model offers a refined framework for interpreting cystic fibrosis airway ecology. Although developed for CF, the Guild Model is adaptable to other diseases influenced by microbial ecology.

The use of bacteriophage (phage) to treat bacterial infection of airways in persons with cystic fibrosis (CF) is gaining interest. However, phenotypic diversification of bacteria during chronic airway infection presents a potential challenge to this therapy. We recovered and subcultured two or three Burkholderia colonies from each of 12 CF sputum samples. All isolates were tested for their susceptibility to a panel of 65 Burkholderia-targeting phages. We observed that 9 (75%) of the 12 colony sets comprised mixtures of isolates that were sensitive or resistant to one or more of the phages tested. The occurrence of mixed populations of phage-sensitive and phageresistant Burkholderia in individuals with CF needs to be considered in the development of phage therapy for this patient population.

BACKGROUND:Chronic lung infections are a hallmark of cystic fibrosis (CF), and the pathogens involved can be transmitted among people with CF (pwCF). The 2013 update of the CF Foundation (CFF) infection prevention and control (IP&C) recommendations emphasized education and monitoring adherence to IP&C practices. Our study aims to assess staff knowledge in outpatient CF clinics, to assess potential barriers to implementation of the 2013 IP&C recommendations, and to identify practice changes resulting from the COVID-19 pandemic. METHODS:In this multicenter study, electronic surveys were developed and sent to 10 pediatric and 10 adult CF centers between May 2022 and November 2022. Local CF center directors distributed the survey to their outpatient clinical care teams, and responses were anonymous. RESULTS:The 174 respondents represented seven different healthcare professions, including respondents from all study sites. Most (72.4%, 126/174) were aware of written CF IP&C policies, but only 50.8% (64/126) knew if the policy specified cleaning roles. Knowledge of the recommended disinfection frequency for specific patient care equipment and high touch surfaces varied strongly by profession (lowest for physicians, highest for certified nursing assistants/medical assistants). Most (77.1%, 215/279) of the responses indicated adherence to recommendations for performing spirometry, with 25.9% (33/127) of respondents reporting no changes in practice during the COVID-19 pandemic. Respondents perceived an increased adherence to use of personal protective equipment and hand hygiene by staff and pwCF since the pandemic. CONCLUSION:While overall awareness of IP&C policies has improved since the 2013 updates, knowledge on specifics should be improved.

Treatment-associated differences in Pseudomonas aeruginosa (Pa) density in sputum have been used as a response biomarker in clinical trials of cystic fibrosis (CF) therapies. Although most studies have included placebo-treated groups as comparators, variability of Pa density in untreated individuals has rarely been reported. We measured day-to-day differences in Pa density in 267 sputum sample pairs collected from 13 adults with CF during days in which no changes in antibiotic therapy occurred. Although the mean sputum Pa density change across all sample pairs was modest (-0.09 log10 16S rRNA gene copies/mL), variability in day-to-day changes were substantial (SD = 1.09) with one-quarter of sample pairs having >1 log10 differences in Pa density; approximately 8 % of pairs had >2 log10 differences in density. Day-to-day variability in Pa density differed substantially between study participants (p = .001). These results will support the design and interpretation of studies using sputum Pa density change as an efficacy biomarker.

Introduction While antibiotics are routinely prescribed for cystic fibrosis (CF) pulmonary exacerbation (PEx) treatment, little evidence exists informing optimal antibiotic selection. This study aimed to determine whether broader-spectrum antibiotics were associated with improved clinical outcomes compared to narrower-spectrum antibiotics for PEx treatment. Methods A secondary analysis of the Standardizing Treatment of Pulmonary Exacerbation-2 (STOP-2) clinical trial was completed. Antibiotic spectrum was defined using the CF antibiotic spectrum index (CF-ASI), a tool that classifies each antibiotic’s expected spectrum of antibacterial activity, and was categorized into quartiles (<14, 14-17, 18-22, and >23). Multivariable generalized linear and inverse probability weighted models were constructed to describe the association between ASI and relevant clinical outcomes, including pre- to post-PEx changes in lung function, weight, and CF symptoms scores. Results A total of 982 people with CF with a mean age of 30.3 years (SD 9.7) were available for analysis. The median CF-ASI score for the entire cohort was 17 (IQR 13-22). The mean ppFEV1 (difference 0.4, 95% CI -1.4, 2.2, p = 0.80) and CRISS (difference 1.3, 95% CI -2.5, 2.5, p = 0.75) changes from baseline did not differ between the lowest and highest CF-ASI quartiles. In the multivariable analysis, a higher CF-ASI quartile score was not statistically significantly associated with improvements in lung function, symptom score or weight compared to a lower CF-ASI quartile score. Adverse events did not differ in frequency between CF-ASI quartiles. Conclusions Opportunities exist to select narrower-spectrum antibiotics for PEx treatment in CF to minimize the risks of antibiotic toxicities.

ABSTRACT The Burkholderia cepacia complex (BCC) is a group of Gram-negative bacteria that cause opportunistic infections, most notably in people with cystic fibrosis (CF), and have been associated with outbreaks caused by contaminated medical products. Antimicrobial susceptibility testing (AST) is often used to guide treatment for BCC infections, perhaps most importantly in people with CF who are being considered for lung transplant. However, recent studies have highlighted problems with AST methods. Here, we address limitations from previous studies to further evaluate BCC AST methods. We assessed the performance of reference broth microdilution (BMD), disk diffusion (DD) using Mueller-Hinton agar (MHA) from three manufacturers, agar dilution (AD), and gradient diffusion (ETEST) for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX) on a set of 205 BCC isolates. The isolate set included 100 isolates from people with CF and 105 isolates from people without CF from a variety of sources, which enabled us to systematically evaluate whether specimen source impacts AST performance. For all BCC isolates, BMD reproducibility was 93%, 98%, 99%, 98%, and 96% for CAZ, LVX, MEM, MIN, and TMP-SMX, respectively. Using BMD as the comparator method, we show that DD, AD, and ETEST perform poorly, with neither MHA manufacturer nor specimen source significantly impacting method performance. Based on our data, we recommend that routine AST should not be performed for BCC isolates. If a provider requests AST, clinical microbiology laboratories should perform Clinical and Laboratory Standards Institute reference methodology for BMD (stored frozen) and report MIC only. IMPORTANCE Antimicrobial susceptibility testing for the Burkholderia cepacia complex (BCC) is often used to determine eligibility for lung transplant in people with cystic fibrosis. However, problems with method performance have been reported. Here, we systematically evaluate the performance of reference broth microdilution, disk diffusion, agar dilution, and gradient diffusion (ETEST) for BCC organisms isolated from people with and without cystic fibrosis. We show that broth microdilution reproducibility is acceptable for levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole, while ceftazidime was just below the acceptability cut-off. Regardless of specimen source, the results from disk diffusion, agar dilution, and ETEST do not correlate with broth microdilution. Based on these findings, we recommend that antimicrobial susceptibility testing should not be routinely performed for BCC, and if requested by the provider, only broth microdilution following Clinical and Laboratory Standards Institute guidelines should be used. Providers should be aware of the significant limitations of antimicrobial susceptibility testing methods for BCC.

Background: Infection control guidelines for cystic fibrosis (CF) stress cleaning of environmental surfaces and patientcare equipment in CF clinics. This multicenter study measured cleanliness of frequently touched surfaces in CF clinics using an ATP bioluminescence assay to assess the effectiveness of cleaning/disinfection and the impact of feedback. Methods: Eight surfaces were tested across 19 clinics (10 pediatric, 9 adult) over 5 rounds of testing. Rounds 1 and 2 served as uncleaned baseline, and Round 3 occurring after routine cleaning. Rounds 4 and 5 were performed after feedback provided to staff and measured after cleaning. Pass rates defined as <250 relative light units were the primary outcome. Results: Of the 750 tests performed, 72% of surfaces passed at baseline, and 79%, 83%, and 85% of surfaces passed in Rounds 3, 4, and 5, respectively. The overall pass-rate was significantly higher in adult compared to pediatric clinics (86% vs 71%; P < 0.001). In pediatric clinics, blood pressure equipment and computer keyboards in the pulmonary function lab consistently passed, but the exam room patient/visitor chairs consistently failed in all rounds. In adult clinics blood pressure equipment, keyboards in exam rooms and exam tables passed in all rounds and no surface consistently failed. Conclusion: We demonstrate the feasibility of an ATP bioluminescence assay to measure cleanliness of patient care equipment and surfaces in CF clinics. Pass rates improved after cleaning and feedback for certain surfaces. We found that surfaces are more challenging to keep clean in clinics taking care of younger patients.

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus, although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices.

In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods. We assessed the groundwater microbiome's taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436-557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety.

A collection of 161 Ralstonia isolates, including 90 isolates from persons with cystic fibrosis, 27 isolates from other human clinical samples, 8 isolates from the hospital environment, 7 isolates from industrial samples, and 19 environmental isolates, was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and yielded confident species level identification scores for only 62 (39%) of the isolates, including four that proved misidentified subsequently. Whole-genome sequence analysis of 32 representative isolates for which no confident MALDI-TOF MS species level identification was obtained revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively, and provided the basis for updating an in-house MALDI-TOF MS database. A reanalysis of all mass spectra with the updated MALDI-TOF MS database increased the percentage of isolates with confident species level identification up to 77%. The antimicrobial susceptibility of 30 isolates mainly representing novel human clinical and environmental Ralstonia species was tested toward 17 antimicrobial agents and demonstrated that the novel Ralstonia species were generally multi-resistant, yet susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline. An analysis of genomic antimicrobial resistance genes in 32 novel and publicly available genome sequences revealed broadly distributed beta-lactam resistance determinants.IMPORTANCEThe present study demonstrated that a commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification database can be tailored to improve the identification of Ralstonia species. It also revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively. An analysis of minimum inhibitory concentration values demonstrated that the novel Ralstonia species were generally multi-resistant but susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline.

Ice machines can harbor water-related organisms, and the use of ice or tap water for clinical care activities has been associated with infections in health care settings. During 2021-2022, a total of 23 cases of infection by Burkholderia multivorans (sequence type ST659) were reported at two southern California hospitals and linked to contaminated ice and water from ice machines. In addition to these 23 cases, this report also includes 23 previously unreported cases of B. multivorans ST659 infections that occurred during 2020-2024: 13 at a northern California hospital, eight at a hospital in Colorado, and two additional cases at one of the southern California hospitals. The same brand of ice machine and brands of filters, descaling, and sanitizing products were used by all four hospitals; B. multivorans was isolated from samples collected from ice machines in two of the hospitals. Whole genome sequencing indicated that all clinical and ice machine isolates were highly genetically similar (0-14 single nucleotide variant differences across 81% of the selected reference genome). Recommendations from public health officials to halt the outbreak included avoiding ice and tap water during clinical care activities. An investigation is ongoing to determine possible sources of ice machine contamination. During outbreaks of water-related organisms in health care facilities, health care personnel should consider avoiding the use of tap water, including ice and water from ice machines, for patient care.

Polymicrobial infection of the airways is a hallmark of obstructive lung diseases such as cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Pulmonary exacerbations (PEx) in these conditions are associated with accelerated lung function decline and higher mortality rates. Understanding PEx ecology is challenged by high inter-patient variability in airway microbial community profiles. We analyze bacterial communities in 880 CF sputum samples collected during an observational prospective cohort study and develop microbiome descriptors to model community reorganization prior to and during 18 PEx. We identify two microbial dysbiosis regimes with opposing ecology and dynamics. Pathogen-governed PEx show hierarchical community reorganization and reduced diversity, whereas anaerobic bloom PEx display stochasticity and increased diversity. A simulation of antimicrobial treatment predicts better efficacy for hierarchically organized communities. This link between PEx, microbiome organization, and treatment success advances the development of personalized clinical management in CF and, potentially, other obstructive lung diseases.

Rationale: Rates of viral respiratory infection (VRI) are similar in people with cystic fibrosis (CF) and the general population; however, the associations between VRI and CF pulmonary exacerbations (PEx) require further elucidation. Objectives: To determine VRI prevalence during CF PEx and evaluate associations between VRI, clinical presentation, and treatment response. Methods: The Standardized Treatment of Pulmonary Exacerbations II (STOP2) study was a multicenter, randomized trial to evaluate different durations of intravenous antibiotic therapy for PEx. In this ancillary study, participant sputum samples from up to three study visits were tested for respiratory viruses using multiplex PCR. Baselines and treatment associated changes in mean lung function (percent predicted forced expiratory volume in one second; ppFEV1), respiratory symptoms (Chronic Respiratory Infection Symptom Score; CRISS), weight, and C-reactive protein (CRP) were compared as a function of virus detection. Odds of PEx retreatment within 30 days and future PEx hazard were modeled by logistic and Cox proportional hazards regression, respectively. Results: A total of 1,254 sputum samples from 621 study participants were analyzed. One or more respiratory viruses were detected in sputum samples from 245 participants (39.5%). Virus-positive participants were more likely to be receiving CF transmembrane conductance regulator (CFTR) modulator therapy (45% vs. 34%) and/or chronic azithromycin therapy (54% vs. 44%), and more likely to have received treatment for nontuberculous mycobacterium infection in the preceding two years (7% vs. 3%). At study visit 1, virus-positive participants were more symptomatic (mean CRISS score 53.8 vs. 51.1), had evidence of greater systemic inflammation (log10CRP concentration: 1.32 log10 mg/L vs. 1.23 log10 mg/L), and had a greater drop in ppFEV1 from the prior 6-month baseline (5.8 vs 3.6). Virus positivity was associated with reduced risk of future PEx (HR: 0.82, 95% CI: 0.69-0.99; p=0.034) and longer median time to next PEx (255 days vs. 172 days, p=0.021) compared to virus-negativity. Conclusions: Over one-third of STOP2 participants treated for a PEx tested positive for a respiratory virus with more symptomatic initial presentation compared to virus-negative participants, but favorable long-term outcomes. More refined phenotyping of PEx, taking VRIs into account, may aid in optimizing personalized management of PEx. Clinical Trial Registration: NCT02781610 Primary Source of Funding: Cystic Fibrosis Foundation

Nontuberculous mycobacteria (NTM) in drinking water are a significant public health concern. However, an incomplete understanding of the factors that influence the occurrence of NTM in drinking water limits our ability to characterize risk and prevent infection. This study sought to evaluate the influence of season and water treatment, distribution, and stagnation on NTM in drinking water. Samples were collected source-to-tap in a full-scale, chloraminated drinking water system approximately monthly from December 2019 to November 2020. NTM were characterized using culture-dependent (plate culture with matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS] isolate analysis) and culture-independent methods (quantitative PCR and genome resolved metagenomics). Sampling locations included source waters, three locations within the treatment plant, and five buildings receiving water from the distribution system. Building plumbing samples consisted of first draw, five-minute flush, and full flush cold-water samples. As the study took place during the COVID-19 pandemic, the influence of reduced water usage in three of the five buildings was also investigated. The highest concentrations of NTM source-to-tap were found in the summer first draw building water samples (107gene copies/L), which also had the lowest chloramine concentrations. Flushing was found to be effective for reducing NTM and restoring disinfectant residuals, though flush times necessary to improve water quality varied by building. Clinically-relevant NTM species, includingMycobacterium avium, were recovered via plate culture, with increased occurrence observed in buildings with higher water age. Four of five NTM metagenome-assembled genomes were identified to the species level and matched identified isolates.

In our curated panel of Burkholderia cepacia complex isolates, Burkholderia multivorans strain AU28442 was unusually highly β-lactam resistant. To explore the molecular mechanisms leading to this phenotype, we performed whole genome sequencing (WGS) and microbiological and biochemical assays. WGS analysis revealed that strain AU28442 produced two β-lactamases, AmpC22 and a novel PenA-like β-lactamase denominated PenA39. Additionally, the strain presented frame-shift mutations in the genes encoding penicillin binding proteins 3 (PBP3) and 4 (PBP4). The antibiotic susceptibilities of the parent AU28442 strain carrying blaPenA39 vs the isogenic E. colistrain producing blaPenA39 were discrepant with ceftazidime MICs of >512 and 1 μg/mL, respectively. Accordingly, PenA39 was found to poorly hydrolyze β-lactams with kcat values of ≤8.8 s-1. An overlay of the crystal structure of PenA39 with PenA1 revealed a shift in the SDN loop in the variant, which may affect the catalytic efficiency of PenA39 toward substrates and inhibitors. Moreover, microscopic examination of AU28442 revealed shortened rod-shaped cells compared to B. multivoransATCC 17616, which carries a full complement of intact PBPs. Further complementation assays confirmed that the loss of PBP3 and PBP4 was the main factor contributing to the high-level β-lactam resistance observed in B. multivoransAU28442. This information allowed us to revert susceptibility by pairing a potent β-lactamase inhibitor with a β-lactam with promiscuous PBP binding. This detailed characterization of B. multivoransprovides an illustration of the myriad ways in which bacteria under antibiotic selection can develop resistance and demonstrates a mechanism to overcome it.

While online monitoring of physicochemical parameters has widely been incorporated into drinking water treatment systems, online microbial monitoring has lagged behind, resulting in the use of surrogate parameters (disinfectant residual, applied dose, concentration × time, CT) to assess disinfection system performance. Online flow cytometry (online FCM) allows for automated quantification of total and intact microbial cells. This study sought to investigate the feasibility of online FCM for full-scale drinking water ozone disinfection system performance monitoring. A water treatment plant with high lime solids turbidity in the ozone contactor influent was selected to evaluate the online FCM in challenging conditions. Total and intact cell counts were monitored for 40 days and compared to surrogate parameters (ozone residual, ozone dose, and CT) and grab sample assay results for cellular adenosine triphosphate (cATP), heterotrophic plate counts (HPC), impedance flow cytometry, and 16S rRNA gene sequencing. Online FCM provided insight into the dynamics of the full-scale ozone system, including offering early warning of increased contactor effluent cell concentrations, which was not observed using surrogate measures. Positive correlations were observed between online FCM intact cell counts and cATP levels (Kendall's tau=0.40), HPC (Kendall's tau=0.20), and impedance flow cytometry results (Kendall's tau=0.30). Though a strong correlation between log intact cell removal and CT was not observed, 16S rRNA gene sequencing results showed that passage through the ozone contactor significantly changed the microbial community (p<0.05). Potential causes of the low overall cell inactivation in the contactor and the significant changes in the microbial community after ozonation include regrowth in the later chambers of the contactor and varied ozone resistance of drinking water microorganisms. This study demonstrates the suitability of direct, online microbial analysis for monitoring full-scale disinfection systems.