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Environmental monitoring of antimicrobial resistant bacteria in North Carolina water and wastewater using the WHO Tricycle protocol in combination with membrane filtration and compartment bag test met [1]
['K. Clark Appling', 'Gillings School Of Global Public Health', 'Department Of Environmental Sciences', 'Engineering', 'University Of North Carolina At Chapel Hill', 'Chapel Hill', 'Nc', 'United States Of America', 'Mark D. Sobsey', 'Lisa M. Durso']
Date: 2023-09
All farms and wastewater treatment facilities participated voluntarily and gave their informed consent for inclusion before samples were collected. Written permission was obtained to collect samples from the North Carolina State University Lake Wheeler Road Field Laboratory, 3720 Lake Wheeler Rd, Raleigh, NC) (supplementary info), and verbal permission was obtained by the lead author from the farm manager of the privately owned farm, and from the appropriate administrators or staff persons for the utilities visited (Orange Water and Sewer Authority, 400 Jones Ferry Rd, Carrboro, NC; Neuse River Resource Recovery Facility, 8545, 8500 Battle Bridge Rd, Raleigh, NC). Apart from this permission, provided directly by each facility owner or representative, no formal permits were required or obtained for the collection of anonymous environmental samples. Likewise, approval and permits were not required or obtained for sampling publicly accessible surface water in public parks and public lands, such as water samples obtained at the Morgan Creek trailhead. Animal waste samples were collected without any animal contact, from ground-deposited material. No human or animal subjects were included in this work and no data were collected from any human or animal subjects, only data on environmental microbes; as a result, IRB approval was not applicable or sought.
Sampling was conducted in the NC Piedmont region, in and around a large city (Raleigh; estimated population of 480,000) and a smaller “sentinel” city (Chapel Hill; estimated population ~63,000), as specified in the WHO TP. The study areas include sources of chicken agriculture in addition to urban wastewaters and surface waters.
Using sterile polypropylene wide-mouth bottles (Thermo-Fisher Scientific, Waltham, MA), 0.5-liter grab samples were collected from Raleigh’s Neuse River Resource Recovery (wastewater) Facility and 24-hour composite samples were taken from the autosampler at the Orange Water and Sewer Authority’s Mason Farm Road Wastewater Treatment Plant in Chapel Hill and transferred to sterile 0.5-liter bottles. Surface water grab samples were collected from the Neuse River in Raleigh and from Morgan Creek in Chapel Hill, both up- and downstream of the respective wastewater treatment plants (WWTPs) in each location using sterile 0.5-liter sample bottles. These surface waters serve as receiving waters for effluent from their respective WWTPs. Chicken waste samples were collected in sterile 300-mL Whirl-Pak® (Whirl-Pak, Madison, WI) bags from farms in Raleigh and Chapel Hill by inverting the sterile bags with gloved hands and collecting samples by directly grabbing with the inverted bag (then un-inverting and sealing) or by scooping with a sterile scoop and depositing in the bag. Approximately 5 chicken droppings per visit were collected at the Chapel Hill site and approximately one cup of fecally soiled chicken litter was collected per visit at the Raleigh site. Samples were stored and transported on ice at 4°C and analyzed within 24–48 hours of collection. Not every sampling location was visited on each sampling trip due to challenges with regular access to some sites, specifically wastewater and poultry agriculture sites.
Samples were collected between July 2021 and March 2022 from surface water, wastewater, and chicken agriculture sites in Raleigh and Chapel Hill, NC on sampling visits conducted approximately every two weeks (Tables 1 & 2 , Table A in S1 Text ; note that not all sites could be sampled on each visit due to logistical constraints).
Sample processing and analysis
For chicken waste samples collected at the Chapel Hill site, 1 g of chicken feces was suspended in 10 mL of sterile distilled (DI) water and vortex mixed until homogenous. For chicken feces-contaminated litter samples (collected at Raleigh site), 5 grams of each sample were added to 50 mL of sterile DI water, vortex mixed, and the supernatant decanted for analysis. Chicken litter samples contained both bedding and feces, and therefore required a larger mass than chicken feces samples to ensure uniformity of mixed samples.
Municipal wastewater and chicken waste suspension/supernatant samples were diluted with sterile DI water to bring concentrations to quantifiable levels of 10–200 CFU/100mL or MPN/100 mL. Suitable dilution factors were estimated based on previous results or else based on an initial dilution test (preparing and culturing serial dilutions of sample to determine the most suitable dilution factor) performed on the day of sample collection, prior to full sample analysis the following day. Surface water samples were typically analyzed undiluted or diluted 1:10 (10−1 dilution), while wastewater and chicken waste samples were typically diluted to between 10−3 and 10−5. Samples were mixed and diluted immediately before analysis, and dilution factors were recorded. Results were then normalized to initial undiluted sample volume or mass prior to data analysis.
Replicate 100-mL aliquots of processed samples were analyzed by membrane filtration (MF) followed by incubation on TBX agar and using the compartment bag test (CBT), both with and without added cefotaxime (4 mg/L). MF samples (100-mL) were filtered through 0.45-μm cellulose nitrate filters followed by incubation (at 44°C for 24 hours) on 47-mm diameter TBX agar plates with or without 4 mg/L added Cefotaxime (CTX) according to the Tricycle protocol. Blue-green colonies were counted as presumptively positive ESBL-Ec. CBT samples (100-mL) were analyzed according to the manufacturer’s instructions. For CBT assays with added cefotaxime, 4 mg/L cefotaxime was added to the proprietary media by spiking each sample with 1 mL/100 mL of 100x stock solution. Apart from this modification, CBT assays were performed as described in the manufacturer’s instructions (Aquagenx LLC n.d.). A new CBT2 (containing the same proprietary growth medium as the CBT but pre-formulated to include 4 mg/L cefotaxime) was also used; apart from the differerence in media formulation, the method was performed identically to the standard CBT. All 100-mL CBT samples were incubated at 35°C for 24 hours. If a CBT compartment exhibited a blue-green color after incubation, that compartment was counted as positive [13]. Results were reported as presumptive E. coli and ESBL-Ec for assays without/with cefotaxime, respectively. Results from membrane filtration (MF) and agar medium plating assays for colonies on TBX were reported in units of CFU/100 mL, and in units of most probable number (MPN/100 mL) concentrations for CBT assays. For MF and agar medium plating assays, all colonies meeting standard assay criteria for E. coli colony appearance were counted as presumptively positive [7].
A subset of presumptive ESBL organisms were isolated from MF+TBX agar medium plates for further characterization. Specifically, 1 to 5 presumptive ESBL-Ec colonies (identified as described above) were selected from positive TBX/cefotaxime plates using a sterile inoculating loop, and streaked to isolation on TBX media with 4 mg/L cefotaxime (isolation plates) before incubation (as described above). Following incubation, a single presumptive positive colony was picked with a sterile loop from each isolation plate, cultured overnight in Tryptic Soy Broth (TSB), diluted 1:1 with sterile glycerol, and stored at -20°C or -80°C in sterile 2mL cryovials [7]. Presumptive ESBL-Ec organisms were isolated from CBTs by a similar method. The exteriors of 1–5 positive compartments of each CBT were swabbed with 70% ethanol, the compartments were then pierced using a sterile syringe and needle and a drop (approximately 20–50 μL) of medium was withdrawn, then spotted onto a TBX plate with cefotaxime, streaked to isolation, and then incubated at 44°C for 24 hours. Following incubation, a single presumptive target colony was picked from each such isolation plate with a sterile loop, incubated overnight in TSB at 35°C, diluted with glycerol, and stored as described above.
Stored isolates were thawed and further characterized by Enteropluri® (Liofilchem, Roseto degli Abruzzi (TE), Italy) biochemical testing according to the manufacturer’s instructions [14] and by Kirby-Bauer antibiotic sensitivity testing (for cefotaxime [CTX], Imipenem [IMP], ampicillin [AMP], Ceftazidime [CAZ], and vancomycin [VAN]) using Oxoid™ antimicrobial susceptibility testing disks (Thermo Scientific™, Waltham, MA) according to standard methods
https://sciwheel.com/work/citation?ids=12673339,11872598&pre=&pre=&suf=&suf=&sa=0,0 [7, 15]. Isolates were confirmed as ESBL by Kirby Bauer testing using the criteria defined in the Tricycle Protocol using CTX, CAZ, CTX + clavulanic acid (CLA), and CAZ + CLA paper discs. Where synergy between CLA and beta lactams was observed (Eqs 1–2, Fig 1), strains were scored as ESBL. When the CTX zone of inhibition minus the CTX + CLA zone of inhibition was > = 5 mm or the CAZ zone of inhibition minus the CAZ + CLA zone of inhibition was > = 5 mm, the isolate was scored as a positive ESBL result [7].
Eq 1
Eq 2
Where (CTX + CLA) = the zone of inhibition (in mm) for the area equidistant from the CTX and CLA discs as shown in Fig 1, (CAZ + CLA) = the zone of inhibition (in mm) for the area equidistant from the CAZ and CLA discs as shown in Fig 1, (CTX) = the zone of inhibition (in mm) for the area surrounding the CTX paper disc but furthest from the CLA paper disc, and (CAZ) = the zone of inhibition (in mm) for the area surrounding the CAZ paper disc and furthest from the CLA paper disc.
The distributions of presumptive and confirmed E. coli and ESBL-Ec CFU and MPN concentrations were characterized separately for each environmental sample type (surface water, wastewater, chicken waste), each assay format (MF, CBT), and antibiotic absence or presence (without CTX, on media amended with CTX, or on proprietary media formulated with CTX). E. coli concentrations were subjected to Shapiro–Wilk normality tests and the geometric mean, arithmetic standard deviation, and range were calculated. As with all culture-based methods, the minimum limit of detection (MLOD) of all methods used corresponded to the detection of one colony forming unit or MPN in the sample volume analyzed (100 mL). Because non-detects may represent any concentration between this MLOD and 0, it is useful to introduce a continuity correction to minimize bias in calculations and enable the inclusion of non-detects in calculations relying on log-transformed values. Non-detects were therefore assigned a nominal value of one half the minimum limit of detection (MLOD/2) for all calculations. Thus, a non-detect for a 100 mL undiluted sample would be scored as 0.5/100 mL. Presumptive resistance proportion was calculated as the ratio of ESBL-Ec/total Ec quantified in each sample. Confirmed ESBL-Ec proportion was calculated as the ratio of confirmed ESBL-Ec isolates to total isolates tested, adjusted for the numbers of isolates collected from each sample type (wastewater/ surface water/chicken waste) and location. Both presumptive and confirmed results were included in analyses and reported. Presumptive ESBL-Ec results are considered less robust indicators of ESBL status than confirmed ESBL phenotype, but have the advantage of quantifying all culturable organisms in each sample aliquot capable of growing on the differential and selective culture media and additives used. By contrast, confirmed ESBL phenotype is a more robust indicator of resistance, but was only able to be determined at the isolate level for the smaller subset of isolates subjected to further characterization. Difference in median log-transformed, continuity-corrected values between sample type and assay type (Table 3) was assessed by non-parametric Wilcoxon signed ranked-paired tests. All analyses were conducted in GraphPad Prism version 9.5 (Graphpad Software, Boston, MA).
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[1] Url:
https://journals.plos.org/water/article?id=10.1371/journal.pwat.0000117
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