Development of Wastewater Pooled Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from Congregate Living Settings.

Wastewater-based monitoring for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the individual building level could be an efficient, passive means of early detection of new cases in congregate living settings, but this approach has not been validated. Preliminary samples were collected from a hospital and a local municipal wastewater treatment plant. Molecular diagnostic methods were compared side by side to assess feasibility, performance, and sensitivity. Refined sample collection and processing protocols were then used to monitor two occupied dormitory complexes (n = 105 and 66) over 8 weeks. Wastewater results were validated using known case counts from external clinical testing of building occupants. Results confirm that ultracentrifugation from a 24-h composite collection had a sensitivity of 96.2% and a specificity of 100%. However, the method could not distinguish new infectious cases from persistent convalescent shedding of SARS-CoV-2 RNA. If the detection of convalescent shedding is considered a false positive, then the sensitivity is 100% and specificity drops to 45%. It was determined that the proposed approach constitutes a highly sensitive wastewater surveillance method for detecting SARS-CoV-2, but it could not distinguish new infectious cases from persistent convalescent shedding. Future work must focus on approaches to distinguish new infections from convalescent shedding to fully realize the potential of building wastewater as a surveillance tool for congregate living. IMPORTANCE Some of the most severe outbreaks of COVID-19 have taken place in places where persons live together, such as nursing homes. Wastewater testing from individual buildings could be used for frequent pooled surveillance of virus from all occupants, including those who are contagious, with or without symptoms. This work provides a sensitive practical method for detecting infected individuals, as validated in two building complexes housing occupants who underwent frequent clinical testing performed by external entities. Although this sensitive method could be deployed now for pooled surveillance as an early warning system to limit outbreaks, the study shows that the approach will require further refinement to differentiate contagious, newly infected individuals from persons who have persistent viral fragments shedding in their stool outside the contagious period.

Risk Factors Associated with Carbapenemase-Producing Enterobacterales (CPE) Positivity in the Hospital Wastewater Environment.

Hospital wastewater is an increasingly recognized reservoir for resistant Gram-negative organisms. Factors involved in establishment and persistence of Klebsiella pneumoniae carbapenemase-producing organisms (KPCOs) in hospital wastewater plumbing are unclear. This study was conducted at a hospital with endemic KPCOs linked to wastewater reservoirs and robust patient perirectal screening for silent KPCO carriage. Over 5 months, both rooms occupied and rooms not occupied by KPCO-positive patients were sampled at three wastewater sites within each room (sink drain, sink P-trap, and toilet or hopper). Risk factors for KPCO positivity were assessed using logistic regression. Whole-genome sequencing (WGS) identified environmental seeding by KPCO-positive patients. A total of 219/475 (46%) room sampling events were KPCO positive in at least one wastewater site. KPCO-positive patient exposure was associated with increased risk of environmental positivity for the room and toilet/hopper. Previous positivity and intensive care unit room type were consistently associated with increased risk. Tube feeds were associated with increased risk for the drain, while exposure to patients with Clostridioides difficile was associated with decreased risk. Urinary catheter exposure was associated with increased risk of P-trap positivity. P-trap heaters reduced risk of P-trap and sink drain positivity. WGS identified genomically linked environmental seeding in 6 of 99 room occupations by 40 KPCO-positive patients. In conclusion, KPCO-positive patients seed the environment in at least 6% of opportunities; once positive for KPCOs, wastewater sites are at greater risk of being positive subsequently. Increased nutrient exposure, e.g., due to tube food disposal down sinks, may increase risk; frequent flushing may be protective.IMPORTANCEKlebsiella pneumoniae carbapenemase-producing organisms (KPCOs) are bacteria that are resistant to most antibiotics and thus are challenging to treat when they cause infections in patients. These organisms can be acquired by patients who are hospitalized for other reasons, complicating their hospital stay and even leading to death. Hospital wastewater sites, such as sink drains and toilets, have played a role in many reported outbreaks over the past decade. The significance of our research is in identifying risk factors for environmental positivity for KPCOs, which will facilitate further work to prevent transmission of these organisms to patients from the hospital environment.

Klebsiella quasipneumoniae Provides a Window into Carbapenemase Gene Transfer, Plasmid Rearrangements, and Patient Interactions with the Hospital Environment.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae (n = 23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

Managing All the Genotypic Knowledge: Approach to a Septic Patient Colonized by Different Enterobacteriales with Unique Carbapenemases.

The recent development of new antimicrobials active against carbapenemase-producing Enterobacteriales (CPE) has brought new hope for the treatment of infections due to these organisms. However, the evolving epidemiology of bacteria with carbapenemases may complicate management, as providers are faced with treating patients colonized by bacteria producing multiple carbapenemases. Here, we present the clinical course and treatment of Raoultella planticola bacteremia in a cirrhotic patient known to be colonized with both blaKPC- and blaOXA-48-carrying organisms.

The Role of fosA in Challenges with Fosfomycin Susceptibility Testing of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Clinical Isolates.

With multidrug-resistant (MDR) Enterobacterales on the rise, a nontoxic antimicrobial agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-Escherichia coli isolates in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with 96 KPC-producing clinical isolates of multiple strains and species collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole-genome sequencing (WGS) data using NCBI's AMRFinder and custom HMM search. Susceptibility testing was performed using a glucose-6-phosphate-supplemented fosfomycin Etest and Kirby-Bauer disk diffusion (DD) assays, and the results were compared to those obtained by agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by Etest, 70% (67/96) by DD, and 88% (84/96) by agar dilution. fosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA positive compared to those that were fosA negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.

Don't overlook the little guy: An evaluation of the frequency of small plasmids co-conjugating with larger carbapenemase gene containing plasmids.

As the spread of antimicrobial resistance (AMR) genes becomes an increasing global threat, improved understanding of mobile genetic elements which contribute to the spread of antimicrobial resistance genes, becomes more critical. We created transconjugants from the mating of three chromosomally isogenic Klebsiella pneumoniae carbapenemase (blaKPC) positive Citrobacter freundii isolates with a laboratory strain of Escherichia coli and evaluated the movement of small cryptic plasmids (SCPs), p3223 and p1916, when larger blaKPC-plasmids were transferred. In all of the 143 transconjugants, multiple plasmids, both large and small, transferred with each mating. When two blaKPC-plasmids were present in the host, frequently (87%; 98/113) both would be transferred during mating. p3223 is found in a wide range of bacterial hosts that harbor AMR genes; p1916 has been identified in only a limited number of publicly available sequences to date. From our evaluation, there is still much to learn about SCPs, and the high rate of co-transfer of multiple plasmids from real-world carbapenemase-producing Enterobacteriales.

Intensive Care Unit Wastewater Interventions to Prevent Transmission of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Organisms.

Background: The increasing prevalence of nosocomial carbapenemase-producing Enterobacteriaceae is a concern. However, the role of the environment in multispecies outbreaks remains poorly understood. There is increasing recognition that hospital wastewater plumbing may play a role. Methods: Covers were installed on all hoppers (a "toilet-like" waste disposal system) in adult intensive care units (ICUs) of a university hospital; additionally in the surgical ICU, sink trap heating and vibration devices were also installed. Patient acquisitions of Klebsiella pneumoniae carbapenemase-producing organisms (KPCOs) for patients who were admitted to an intervention unit were compared for 18-month preintervention and intervention periods. Results: Sixty hopper covers and 23 sink trap devices were installed. Fifty-six new multispecies KPCO acquisitions occurred preintervention compared to 30 during the intervention. Decreases for all KPCO acquisitions (odds ratio [OR], 0.51; 95% confidence interval [CI], 0.31-0.81; P = .003) and KPCO-positive clinical cultures (OR, 0.29; 95% CI, 0.17-0.48; P < .001) per admission in patients exposed to an intervention unit were observed. The incidence rate ratio was 0.51-fold (95% CI, 0.43-0.61) lower for all KPCO acquisitions during the intervention. The effect of the sink trap devices alone could not be determined, although the proportion of sink drain cultures positive for KPCO decreased (12/15 [80%] sites sampled preintervention vs 40/840 [5%] sampled during the intervention; P = .001). Conclusions: An intervention targeting wastewater plumbing fixtures, by installation of hopper covers, demonstrated a decrease in patient KPCO acquisitions. Considering wastewater reservoirs in nosocomial transmission of multispecies carbapenemase-producing Enterobacteriaceae may be critical.

Tracking Klebsiella pneumoniae carbapenemase gene as an indicator of antimicrobial resistance dissemination from a hospital to surface water via a municipal wastewater treatment plant.

Antibiotic-resistant bacteria originating from hospitals are ultimately discharged to municipal wastewater treatment plants (WWTP), which may serve as important reservoirs for the spread of antibiotic resistant genes. This study traced and quantified the presence of a rare but clinically relevant antimicrobial resistance gene; Klebsiella pneumoniae carbapenamase (KPC)-and the viable organisms (KPCO) which carried this gene in hospital, non-hospital wastewater discharges, various compartments within a municipal WWTP, receiving water and sediment samples. High concentration of the gene, blaKPC harbored in viable and multispecies KPCO was detected in the hospital wastewater and in the forepart stages of the WWTP, but was not detected in the final effluent following UV disinfection. KPCO were not detected in multiple non-hospital sources of wastewater discharges tested. The treatment train used in the sampled WWTP was found to help remove and reduce KPCO load. Using whole-genome sequencing, a KPC-producing Klebsiella oxytoca strain identical to strains seen in the patients and hospital environment was isolated from the downstream receiving water on one sampling event. KPCO were also found to persist in the biosolids throughout the WWTP, but were not detected in the processed compost-products made from WWTP-biosolids. This study systematically demonstrates dissemination of KPCO from hospital point source to environment via municipal WWTP. Understanding hospitals as the origin and source of spread of some of the most clinically urgent antimicrobial-resistant organisms may help direct interventions that target rate at which antibiotic resistant bacteria evolve and spread via enhancement of wastewater treatment and mitigation of dissemination at source.