Surface area matters: An evaluation of swabs and surface area for environmental surface sampling of healthcare pathogens.

Flocked and foam swabs were used to sample five healthcare pathogens from three sizes of steel and plastic coupons; 26 cm2, 323 cm2, and 645 cm2. As surface area increased, 1-2 log10 decrease in recovered organisms (P < .05) was observed. Sampling 26-cm2 yielded the optimal median percent of pathogens recovered.

An improved workflow for accurate and robust healthcare environmental surveillance using metagenomics.

BACKGROUND: Effective surveillance of microbial communities in the healthcare environment is increasingly important in infection prevention. Metagenomics-based techniques are promising due to their untargeted nature but are currently challenged by several limitations: (1) they are not powerful enough to extract valid signals out of the background noise for low-biomass samples, (2) they do not distinguish between viable and nonviable organisms, and (3) they do not reveal the microbial load quantitatively. An additional practical challenge towards a robust pipeline is the inability to efficiently allocate sequencing resources a priori. Assessment of sequencing depth is generally practiced post hoc, if at all, for most microbiome studies, regardless of the sample type. This practice is inefficient at best, and at worst, poor sequencing depth jeopardizes the interpretation of study results. To address these challenges, we present a workflow for metagenomics-based environmental surveillance that is appropriate for low-biomass samples, distinguishes viability, is quantitative, and estimates sequencing resources. RESULTS: The workflow was developed using a representative microbiome sample, which was created by aggregating 120 surface swabs collected from a medical intensive care unit. Upon evaluating and optimizing techniques as well as developing new modules, we recommend best practices and introduce a well-structured workflow. We recommend adopting liquid-liquid extraction to improve DNA yield and only incorporating whole-cell filtration when the nonbacterial proportion is large. We suggest including propidium monoazide treatment coupled with internal standards and absolute abundance profiling for viability assessment and involving cultivation when demanding comprehensive profiling. We further recommend integrating internal standards for quantification and additionally qPCR when we expect poor taxonomic classification. We also introduce a machine learning-based model to predict required sequencing effort from accessible sample features. The model helps make full use of sequencing resources and achieve desired outcomes. Video Abstract CONCLUSIONS: This workflow will contribute to more accurate and robust environmental surveillance and infection prevention. Lessons gained from this study will also benefit the continuing development of methods in relevant fields.

Epidemiologic Investigation of Two Welder's Anthrax Cases Caused by Bacillus Cereus Group Bacteria: Occupational Link Established by Environmental Detection.

Abstract Bacillus cereus group bacteria containing the anthrax toxin genes can cause fatal anthrax pneumonia in welders. Two welder's anthrax cases identified in 2020 were investigated to determine the source of each patient's exposure. Environmental sampling was performed at locations where each patient had recent exposure to soil and dust. Samples were tested for the anthrax toxin genes by real-time PCR, and culture was performed on positive samples to identify whether any environmental isolates matched the patient's clinical isolate. A total of 185 environmental samples were collected in investigation A for patient A and 108 samples in investigation B for patient B. All samples from investigation B were real-time PCR-negative, but 14 (8%) samples from investigation A were positive, including 10 from patient A's worksite and 4 from his work-related clothing and gear. An isolate genetically matching the one recovered from patient A was successfully cultured from a worksite soil sample. All welder's anthrax cases should be investigated to determine the source of exposure, which may be linked to their worksite. Welding and metalworking employers should consider conducting a workplace hazard assessment and implementing controls to reduce the risk of occupationally associated illnesses including welder's anthrax.

Factors influencing environmental sampling recovery of healthcare pathogens from non-porous surfaces with cellulose sponges.

Results from sampling healthcare surfaces for pathogens are difficult to interpret without understanding the factors that influence pathogen detection. We investigated the recovery of four healthcare-associated pathogens from three common surface materials, and how a body fluid simulant (artificial test soil, ATS), deposition method, and contamination levels influence the percent of organisms recovered (%R). Known quantities of carbapenemase-producing KPC+ Klebsiella pneumoniae (KPC), Acinetobacter baumannii, vancomycin-resistant Enterococcus faecalis, and Clostridioides difficile spores (CD) were suspended in Butterfield's buffer or ATS, deposited on 323cm2 steel, plastic, and laminate surfaces, allowed to dry 1h, then sampled with a cellulose sponge wipe. Bacteria were eluted, cultured, CFU counted and %R determined relative to the inoculum. The %R varied by organism, from <1% (KPC) to almost 60% (CD) and was more dependent upon the organism's characteristics and presence of ATS than on surface type. KPC persistence as determined by culture also declined by >1 log10 within the 60 min drying time. For all organisms, the %R was significantly greater if suspended in ATS than if suspended in Butterfield's buffer (p<0.05), and for most organisms the %R was not significantly different when sampled from any of the three surfaces. Organisms deposited in multiple droplets were recovered at equal or higher %R than if spread evenly on the surface. This work assists in interpreting data collected while investigating a healthcare infection outbreak or while conducting infection intervention studies.

Recovery efficiency of two glove-sampling methods.

Two methods to sample pathogens from gloved hands were compared: direct imprint onto agar and a sponge-wipe method. The sponge method was significantly better at recovering Clostridiodes difficile spores, and no difference was observed between the methods at 101 inoculum for carbapenemase-producing KPC+ Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii.

Sampling efficiency of Candida auris from healthcare surfaces: culture and nonculture detection methods.

Sponges and swabs were evaluated for their ability to recover Candida auris dried 1 hour on steel and plastic surfaces. Culture recovery ranged from <0.1% (sponges) to 8.4% (swabs), and cells detected with an esterase activity assay revealed >50% recovery (swabs), indicating that cells may enter a viable but nonculturable state.

The effect of disinfectants on the microbial community on environmental healthcare surfaces using next generation sequencing.

BACKGROUND: Healthcare-associated infections are a significant economic burden and cause of avoidable morbidity and mortality within healthcare systems. The contribution of environmental contamination to healthcare-associated infection transmission has been recognized, but the mechanisms by which transmission occurs are still being investigated. The objective of this study was to characterize the microbial communities of disinfected, non-critical healthcare surfaces using next generation sequencing technology. METHODS: Composite environmental surface samples were from high-touch surfaces in rooms of patients isolated for infections with multidrug-resistant organisms during their hospitalization. Information on the disinfectant product used and cleaning type (routine or terminal) was collected. 16S rRNA gene amplicon sequencing and analysis were performed. Community analysis was conducted to determine the bacterial composition and compare the detection of target pathogens by culture from 94 Contact Precaution rooms. RESULTS: Overall percent agreement between culture and sequence methods ranged from 52%-88%. A significant difference was observed in bacterial composition between rooms cleaned with bleach and those cleaned with a quaternary ammonium compound for composite 2 (overbed table, intravenous pole, and inner room door handle) (ANOSIM R = 0.66, P = .005) but not composite 1 (bed rails, television remote control unit, call buttons, and telephone). CONCLUSIONS: Surfaces in bleach-cleaned rooms contained a higher proportion of gram-positive microbiota, whereas rooms cleaned with quaternary ammonium compound contained a higher proportion of gram-negative microbiota, suggesting disinfectant products may impact the healthcare environment microbiome.

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

Positive Correlation Between Candida auris Skin-Colonization Burden and Environmental Contamination at a Ventilator-Capable Skilled Nursing Facility in Chicago.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast that contaminates healthcare environments causing healthcare-associated outbreaks. The mechanisms facilitating contamination are not established. METHODS: C. auris was quantified in residents' bilateral axillary/inguinal composite skin swabs and environmental samples during a point-prevalence survey at a ventilator-capable skilled-nursing facility (vSNF A) with documented high colonization prevalence. Environmental samples were collected from all doorknobs, windowsills and handrails of each bed in 12 rooms. C. auris concentrations were measured using culture and C. auris-specific quantitative polymerase chain reaction (qPCR) The relationship between C. auris concentrations in residents' swabs and associated environmental samples were evaluated using Kendall's tau-b (τ b) correlation coefficient. RESULTS: C. auris was detected in 70/100 tested environmental samples and 31/57 tested resident skin swabs. The mean C. auris concentration in skin swabs was 1.22 × 105 cells/mL by culture and 1.08 × 106 cells/mL by qPCR. C. auris was detected on all handrails of beds occupied by colonized residents, as well as 10/24 doorknobs and 9/12 windowsills. A positive correlation was identified between the concentrations of C. auris in skin swabs and associated handrail samples based on culture (τ b = 0.54, P = .0004) and qPCR (τ b = 0.66, P = 3.83e-6). Two uncolonized residents resided in beds contaminated with C. auris. CONCLUSIONS: Colonized residents can have high C. auris burdens on their skin, which was positively related with contamination of their surrounding healthcare environment. These findings underscore the importance of hand hygiene, transmission-based precautions, and particularly environmental disinfection in preventing spread in healthcare facilities.

Environmental Contamination of Contact Precaution and Non-Contact Precaution Patient Rooms in Six Acute Care Facilities.

BACKGROUND: Environmental contamination is an important source of hospital multidrug-resistant organism (MDRO) transmission. Factors such as patient MDRO contact precautions (CP) status, patient proximity to surfaces, and unit type likely influence MDRO contamination and bacterial bioburden levels on patient room surfaces. Identifying factors associated with environmental contamination in patient rooms and on shared unit surfaces could help identify important environmental MDRO transmission routes. METHODS: Surfaces were sampled from MDRO CP and non-CP rooms, nursing stations, and mobile equipment in acute care, intensive care, and transplant units within 6 acute care hospitals using a convenience sampling approach blinded to cleaning events. Precaution rooms had patients with clinical or surveillance tests positive for methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, carbapenem-resistant Enterobacteriaceae or Acinetobacter within the previous 6 months, or Clostridioides difficile toxin within the past 30 days. Rooms not meeting this definition were considered non-CP rooms. Samples were cultured for the above MDROs and total bioburden. RESULTS: Overall, an estimated 13% of rooms were contaminated with at least 1 MDRO. MDROs were detected more frequently in CP rooms (32% of 209 room-sample events) than non-CP rooms (12% of 234 room-sample events). Surface bioburden did not differ significantly between CP and non-CP rooms or MDRO-positive and MDRO-negative rooms. CONCLUSIONS: CP room surfaces are contaminated more frequently than non-CP room surfaces; however, contamination of non-CP room surfaces is not uncommon and may be an important reservoir for ongoing MDRO transmission. MDRO contamination of non-CP rooms may indicate asymptomatic patient MDRO carriage, inadequate terminal cleaning, or cross-contamination of room surfaces via healthcare personnel hands.

Development of a rapid-viability PCR method for detection of Clostridioides difficile spores from environmental samples.

Clostridioides difficile is a common pathogen that is well known to survive for extended periods of time on environmental healthcare surfaces from fecal contamination. During epidemiological investigations of healthcare-associated infections, it is important to be able to detect whether or not there are viable spores of C. difficile on surfaces. Current methods to detect C. difficile can take up to 7 days for culture and in the case of detection by PCR, viability of the spores cannot be ascertained. Prevention of C. difficile infection in healthcare settings includes adequate cleaning and disinfection of environmental surfaces which increases the likelihood of detecting dead organisms from an environmental sample during an investigation. In this study, we were able to adapt a rapid-viability PCR (RV-PCR) method, first developed for detection of viable Bacillus anthracis spores, for the detection of viable C. difficile spores. RV-PCR uses the change in cycle threshold after incubation to confirm the presence of live organisms. Using this modified method we were able to detect viable C. difficile after 22 h of anaerobic incubation in Cycloserine Cefoxitin Fructose Broth (CCFB). This method also used bead beating combined with the Maxwell 16 Casework kit for DNA extraction and purification and a real-time duplex PCR assay for toxin B and cdd3 genes to confirm the identity of the C. difficile spores. Spiked environmental sponge-wipes with and without added organic load were tested to determine the limit of detection (LOD). The LOD from spiked environmental sponge-wipe samples was 104 spores/mL but after incubation initial spore levels of 101 spores/mL were detected. Use of this method would greatly decrease the amount of time required to detect viable C. difficile spores; incubation of samples is only required for germination (22 h or less) instead of colony formation, which can take up to 7 days. In addition, PCR can then quickly confirm or deny the identity of the organism at the same time it would confirm viability. The presence of viable C. difficile spores could be detected at very low levels within 28 h total compared to the 2 to 10-day process that would be needed for culture, identification and toxin detection.

Elution efficiency of healthcare pathogens from environmental sampling tools.

Standardizing healthcare surface sampling requires the evaluation of sampling tools for organism adherence. Here, 7 sampling tools were evaluated to assess their elution efficiencies in the presence of 5 pathogens. Foam sponges (80.6%), microfiber wipes (80.5%), foam swabs (77.9%), and cellulose sponges (66.5%) yielded the highest median elution efficiencies.

Effect of Glove Decontamination on Bacterial Contamination of Healthcare Personnel Hands.

We examined the effect of glove decontamination prior to removal on bacterial contamination of healthcare personnel hands in a laboratory simulation study. Glove decontamination reduced bacterial contamination of hands following removal. However, hand contamination still occurred with all decontamination methods, reinforcing the need for hand hygiene following glove removal.

Survival, Persistence, and Isolation of the Emerging Multidrug-Resistant Pathogenic Yeast Candida auris on a Plastic Health Care Surface.

The emerging multidrug-resistant pathogenic yeast Candida auris represents a serious threat to global health. Unlike most other Candida species, this organism appears to be commonly transmitted within health care facilities and causes health care-associated outbreaks. To better understand the epidemiology of this emerging pathogen, we investigated the ability of C. auris to persist on plastic surfaces common in health care settings compared with that of Candida parapsilosis, a species known to colonize the skin and plastics. Specifically, we compiled comparative and quantitative data essential to understanding the vehicles of spread and the ability of both species to survive and persist on plastic surfaces under controlled conditions (25°C and 57% relative humidity), such as those found in health care settings. When a test suspension of 104 cells was applied and dried on plastic surfaces, C. auris remained viable for at least 14 days and C. parapsilosis for at least 28 days, as measured by CFU. However, survival measured by esterase activity was higher for C. auris than C. parapsilosis throughout the 28-day study. Given the notable length of time Candida species survive and persist outside their host, we developed methods to more effectively culture C. auris from patients and their environment. Using our enrichment protocol, public health laboratories and researchers can now readily isolate C. auris from complex microbial communities (such as patient skin, nasopharynx, and stool) as well as environmental biofilms, in order to better understand and prevent C. auris colonization and transmission.

Assessment of the Overall and Multidrug-Resistant Organism Bioburden on Environmental Surfaces in Healthcare Facilities.

OBJECTIVE To determine the typical microbial bioburden (overall bacterial and multidrug-resistant organisms [MDROs]) on high-touch healthcare environmental surfaces after routine or terminal cleaning. DESIGN Prospective 2.5-year microbiological survey of large surface areas (>1,000 cm2). SETTING MDRO contact-precaution rooms from 9 acute-care hospitals and 2 long-term care facilities in 4 states. PARTICIPANTS Samples from 166 rooms (113 routine cleaned and 53 terminal cleaned rooms). METHODS Using a standard sponge-wipe sampling protocol, 2 composite samples were collected from each room; a third sample was collected from each Clostridium difficile room. Composite 1 included the TV remote, telephone, call button, and bed rails. Composite 2 included the room door handle, IV pole, and overbed table. Composite 3 included toileting surfaces. Total bacteria and MDROs (ie, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci [VRE], Acinetobacter baumannii, Klebsiella pneumoniae, and C. difficile) were quantified, confirmed, and tested for drug resistance. RESULTS The mean microbial bioburden and range from routine cleaned room composites were higher (2,700 colony-forming units [CFU]/100 cm2; ≤1-130,000 CFU/100 cm2) than from terminal cleaned room composites (353 CFU/100 cm2; ≤1-4,300 CFU/100 cm2). MDROs were recovered from 34% of routine cleaned room composites (range ≤1-13,000 CFU/100 cm2) and 17% of terminal cleaned room composites (≤1-524 CFU/100 cm2). MDROs were recovered from 40% of rooms; VRE was the most common (19%). CONCLUSIONS This multicenter bioburden summary provides a first step to determining microbial bioburden on healthcare surfaces, which may help provide a basis for developing standards to evaluate cleaning and disinfection as well as a framework for studies using an evidentiary hierarchy for environmental infection control. Infect Control Hosp Epidemiol 2016;1426-1432.

Evaluation of swabs and transport media for the recovery of Yersinia pestis.

The Government Accountability Office report investigating the surface sampling methods used during the 2001 mail contamination with Bacillus anthracis brought to light certain knowledge gaps that existed regarding environmental sampling with biothreat agents. Should a contamination event occur that involves non-spore forming biological select agents, such as Yersinia pestis, surface sample collection and processing protocols specific for these organisms will be needed. Two Y. pestis strains (virulent and avirulent), four swab types (polyester, macrofoam, rayon, and cotton), two pre-moistening solutions, six transport media, three temperatures, two levels of organic load, and four processing methods (vortexing, sonicating, combined sonicating and vortexing, no agitation) were evaluated to determine the conditions that would yield the highest percent of cultivable Y. pestis cells after storage. The optimum pre-moistening agent/transport media combination varied with the Y. pestis strain and swab type. Directly inoculated macrofoam swabs released the highest percent of cells into solution (93.9% recovered by culture) and rayon swabs were considered the second best swab option (77.0% recovered by culture). Storage at 4°C was found to be optimum for all storage times and transport media. In a worst case scenario, where the Y. pestis strain is not known and sample processing and analyses could not occur until 72h after sampling, macrofoam swabs pre-moistened with PBS supplemented with 0.05% Triton X-100 (PBSTX), stored at 4°C in neutralizing buffer (NB) as a transport medium (PBSTX/NB) or pre-moistened with NB and stored in PBSTX as a transport medium (NB/PBSTX), then vortexed 3min in the transport medium, performed significantly better than all other conditions for macrofoam swabs, regardless of strain tested (mean 12 - 72h recovery of 85.9-105.1%, p<0.001). In the same scenario, two combinations of pre-moistening medium/transport medium were found to be optimal for rayon swabs stored at 4°C (p<0.001), then sonicated 3min in the transport medium; PBSTX/PBSTX and NB/PBSTX (mean 12-72h recovery of 83.7-110.1%).

Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters.

The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p≤0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p>0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident.

Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores.

In this study, four commonly-used sampling devices (vacuum socks, 37 mm 0.8 µm mixed cellulose ester (MCE) filter cassettes, 37 mm 0.3 µm polytetrafluoroethylene (PTFE) filter cassettes, and 3M™ forensic filters) were comparatively evaluated for their ability to recover surface-associated spores. Aerosolized spores (~10(5)CFUcm(-2)) of a Bacillus anthracis surrogate were allowed to settle onto three material types (concrete, carpet, and upholstery). Ten replicate samples were collected using each vacuum method, from each material type. Stainless steel surfaces, inoculated simultaneously with test materials, were sampled with pre-moistened wipes. Wipe recoveries were utilized to normalize vacuum-based recoveries across trials. Recovery (CFUcm(-2)) and relative recovery (vacuum recovery/wipe recovery) were determined for each method and material type. Recoveries and relative recoveries ranged from 3.8 × 10(3) to 7.4 × 10(4)CFUcm(-2) and 0.035 to 1.242, respectively. ANOVA results indicated that the 37 mm MCE method exhibited higher relative recoveries than the other methods when used for sampling concrete or upholstery. While the vacuum sock resulted in the highest relative recoveries on carpet, no statistically significant difference was detected. The results of this study may be used to guide selection of sampling approaches following biological contamination incidents.

Storage Effects on Sample Integrity of Environmental Surface Sampling Specimens with Bacillus anthracis Spores.

The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis. Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T0) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 102, p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.