Environmental surveillance for COVID-19 using SARS-CoV-2 RNA concentration in wastewater - a study in District East, Karachi, Pakistan.

Background: Wastewater-based surveillance is used to track the temporal patterns of the SARS-CoV-2 virus in communities. Viral RNA particle detection in wastewater samples can indicate an outbreak within a catchment area. We describe the feasibility of using a sewage network to monitor SARS-CoV-2 trend and use of genomic sequencing to describe the viral variant abundance in an urban district in Karachi, Pakistan. This was among the first studies from Pakistan to demonstrate the surveillance for SARS-CoV-2 from a semi-formal sewage system. Methods: Four sites draining into the Lyari River in District East, Karachi, were identified and included in the current study. Raw sewage samples were collected early morning twice weekly from each site between June 10, 2021 and January 17, 2022, using Bag Mediated Filtration System (BMFS). Secondary concentration of filtered samples was achieved by ultracentrifugation and skim milk flocculation. SARS-CoV-2 RNA concentrations in the samples were estimated using PCR (Qiagen ProMega kits for N1 & N2 genes). A distributed-lag negative binomial regression model within a hierarchical Bayesian framework was used to describe the relationship between wastewater RNA concentration and COVID-19 cases from the catchment area. Genomic sequencing was performed using Illumina iSeq100. Findings: Among the 151 raw sewage samples included in the study, 123 samples (81.5%) tested positive for N1 or N2 genes. The average SARS-CoV-2 RNA concentrations in the sewage samples at each lag (1-14 days prior) were associated with the cases reported for the respective days, with a peak association observed on lag day 10 (RR: 1.15; 95% Credible Interval: 1.10-1.21). Genomic sequencing showed that the delta variant dominated till September 2022, while the omicron variant was identified in November 2022. Interpretation: Wastewater-based surveillance, together with genomic sequencing provides valuable information for monitoring the community temporal trend of SARS-CoV-2. Funding: PATH, Bill & Melinda Gates Foundation, and Global Innovation Fund.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) RNA and viable virus contamination of hospital emergency department surfaces and association with patient coronavirus disease 2019 (COVID-19) status and aerosol-generating procedures.

Emergency departments are high-risk settings for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) surface contamination. Environmental surface samples were obtained in rooms with patients suspected of having COVID-19 who did or did not undergo aerosol-generating procedures (AGPs). SARS-CoV-2 RNA surface contamination was most frequent in rooms occupied by coronavirus disease 2019 (COVID-19) patients who received no AGPs.

Carbapenem-resistant Enterobacteriaceae in sink drains of 40 healthcare facilities in Sindh, Pakistan: A cross-sectional study.

In Pakistan, antimicrobial resistance (AMR) is expected to greatly increase the already high mortality and morbidity rates attributed to infections, making AMR surveillance and prevention a priority in the country. The aims of the project were to characterize the prevalence of carbapenem-resistant Enterobacteriaceae (CRE) in healthcare facility sink drains in Pakistan and to characterize how physical characteristics of sinks and healthcare facility rooms were associated with CRE in those sinks. The study took place in 40 healthcare facilities in Jamshoro Pakistan. Swabs were collected from sink drains in each facility that had a sink, and structured observations of sinks and facilities were performed at each facility. Swabs were plated on CHROMagar KPC to screen for carbapenem-resistant Enterobacteriaceae, which were then isolated on Mueller-Hinton agar plates. Antibiotic susceptibility was determined using the disk diffusion method to assess resistance to carbapenems, cephalosporins, and fluoroquinolones. Thirty-seven of the healthcare facilities had at least one sink, and thirty-nine total sinks were present and sampled from those healthcare facilities. Sinks in these facilities varied in quality; at the time of sampling 68% had water available, 51% had soap/alcohol cleanser at the sink, 28% appeared clean, and 64% drained completely. Twenty-five (64%) of the sink samples grew Enterobacteriaceae on CHROMagar KPC, sixteen (41%) of which were clinically non-susceptible to ertapenem. Seven of the 39 sampled sinks (18%) produced Enterobacteriaceae that were resistant to all three antibiotic classes tested. Several facilities and sink characteristics were associated with CRE. Sinks and drains can serve as undetected reservoirs for carbapenem-resistant Enterobacteriaceae. Control and remediation of such environments will require both systemic strategies and physical improvements to clinical environments.

Differences in chlorine and peracetic acid disinfection kinetics of Enterococcus faecalis and Escherichia fergusonii and their susceptible strains based on gene expressions and genomics.

This study was conducted to investigate mechanisms of cross-resistance to chlorine and peracetic acid (PAA) disinfectants by antibiotic-resistant bacteria. Our study evaluated chlorine and PAA based disinfection kinetics of erythromycin-resistant Enterococcus faecalis, meropenem-resistant Escherichia fergusonii, and susceptible strains of these species. Using the integrated second-order disinfectant decay model and first-order Chick-Watson's Law, it was found that the meropenem-resistant Escherichia fergusonii strain showed significantly less log inactivation compared to the susceptible E. fergusonii strain in response to both chlorine and PAA disinfection (p-value = 0.059, 3.5 × 10-6). On the other hand, the susceptible Enterococcus faecalis strain showed similar log inactivation compared to the erythromycin-resistant strain in response to either treatment (p-value = 0.075, 0.28). Meropenem-resistant E. fergusonii showed an increase in gene expression of New Delhi metallo-ß-lactamase (blaNDM-1) gene to chlorine, but there was no increase in expression to PAA. Whole genome sequencing (WGS) was then conducted to elucidate the differences in genes among both resistant and susceptible table E. fergusonii strains. The average nucleotide identity (ANI) analysis of the draft genomes (>97% similarity) suggests that meropenem-resistant E. fergusonii (S1) and meropenem-susceptible E. fergusonii (S2) are the same species but different strains. Both strains have the same genes for oxidative stress pathways, oxidative scavenger genes, and nearly 40 different antibiotic efflux pump genes. The chromosomal and plasmid draft genomes of meropenem-resistant and susceptible E. fergusonii strains each have 65 and 52 antibiotic resistance genes, respectively. Of these, the resistant E. fergusonii strain harbored the extended-spectrum beta-lactamases blaCTX-M-15 and blaTEM-1 genes located on the chromosome, and a blaTEM-1 gene on the plasmid. The overall findings of this study are significant, as they reveal that antibiotic-resistant and susceptible strains of E. fergusonii exhibit different responses towards chlorine and PAA disinfection.

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.

Localized effect of treated wastewater effluent on the resistome of an urban watershed.

BACKGROUND: Wastewater treatment is an essential tool for maintaining water quality in urban environments. While the treatment of wastewater can remove most bacterial cells, some will inevitably survive treatment to be released into natural environments. Previous studies have investigated antibiotic resistance within wastewater treatment plants, but few studies have explored how a river's complete set of antibiotic resistance genes (the "resistome") is affected by the release of treated effluent into surface waters. RESULTS: Here we used high-throughput, deep metagenomic sequencing to investigate the effect of treated wastewater effluent on the resistome of an urban river and the downstream distribution of effluent-associated antibiotic resistance genes and mobile genetic elements. Treated effluent release was found to be associated with increased abundance and diversity of antibiotic resistance genes and mobile genetic elements. The impact of wastewater discharge on the river's resistome diminished with increasing distance from effluent discharge points. The resistome at river locations that were not immediately downstream from any wastewater discharge points was dominated by a single integron carrying genes associated with resistance to sulfonamides and quaternary ammonium compounds. CONCLUSIONS: Our study documents variations in the resistome of an urban watershed from headwaters to a major confluence in an urban center. Greater abundances and diversity of antibiotic resistance genes are associated with human fecal contamination in river surface water, but the fecal contamination effect seems to be localized, with little measurable effect in downstream waters. The diverse composition of antibiotic resistance genes throughout the watershed suggests the influence of multiple environmental and biological factors.

A microbiological survey of handwashing sinks in the hospital built environment reveals differences in patient room and healthcare personnel sinks.

Handwashing sinks and their associated premise plumbing are an ideal environment for pathogen-harboring biofilms to grow and spread throughout facilities due to the connected system of wastewater plumbing. This study was designed to understand the distribution of pathogens and antibiotic resistant organisms (ARO) within and among handwashing sinks in healthcare settings, using culture-dependent methods to quantify Pseudomonas aeruginosa, opportunistic pathogens capable of growth on a cefotaxime-containing medium (OPP-C), and carbapenem-resistant Enterobacteriaceae (CRE). Isolates from each medium identified as P. aeruginosa or Enterobacteriaceae were tested for susceptibility to aztreonam, ceftazidime, and meropenem; Enterobacteriaceae were also tested against ertapenem and cefotaxime. Isolates exhibiting resistance or intermediate resistance were designated ARO. Pathogens were quantified at different locations within handwashing sinks and compared in quantity and distribution between healthcare personnel (HCP) and patient room (PR) sinks. ARO were compared between samples within a sink (biofilm vs planktonic samples) and between sink types (HCP vs. PR). The drain cover was identified as a reservoir within multiple sinks that was often colonized by pathogens despite daily sink cleaning. P. aeruginosa and OPP-C mean log10 CFU/cm2 counts were higher in p-trap and tail pipe biofilm samples from HCP compared to PR sinks (2.77 ± 2.39 vs. 1.23 ± 1.62 and 5.27 ± 1.10 vs. 4.74 ± 1.06) for P. aeruginosa and OPP-C, respectively. P. aeruginosa and OPP-C mean log10 CFU/ml counts were also higher (p < 0.05) in HCP compared to PR sinks p-trap water (2.21 ± 1.52 vs. 0.89 ± 1.44 and 3.87 ± 0.78 vs. 3.21 ± 1.11) for P. aeruginosa and OPP-C, respectively. However, a greater percentage of ARO were recovered from PR sinks compared to HCP sinks (p < 0.05) for Enterobacteriaceae (76.4 vs. 32.9%) and P. aeruginosa (25.6 vs. 0.3%). This study supports previous work citing that handwashing sinks are reservoirs for pathogens and ARO and identifies differences in pathogen and ARO quantities between HCP and PR sinks, despite the interconnected premise plumbing.

Environmental Contamination with Candida Species in Multiple Hospitals Including a Tertiary Care Hospital with a Candida auris Outbreak.

BACKGROUND: Environmental sources have been implicated as a potential source for exogenous acquisition of Candida species, particularly the emerging multidrug-resistant Candida auris. However, limited information is available on environmental reservoirs of Candida species in healthcare facilities. METHODS: During a 6-month period, cultures for Candida species were collected from high-touch surfaces in patient rooms and from portable equipment in 6 US acute care hospitals in 4 states. Additional cultures were collected from sink drains and floors in one of the hospitals and from high-touch surfaces, portable equipment, and sink drains in a hospital experiencing an outbreak due to C. auris. Candida species were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectometry. RESULTS: Candida species were recovered from patient rooms in 4 of the 6 hospitals. Seven of 147 patient room cultures (4.8%) and 1 of 57 (1.8%) portable equipment cultures were positive, with the most common species being C. parapsilosis. For the hospital where additional sites were sampled, Candida species were recovered from 8 of 22 (36.4%) hospital room floors and 4 of 17 (23.5%) sink drains. In the facility with a C. auris outbreak, Candida species were frequently recovered from sink drains (20.7%) and high-touch surfaces (15.4%), but recovery of C. auris was uncommon (3.8% of high-touch surfaces, 3.4% of sink drains, and 0% of portable equipment) and only present in rooms that currently or recently housed a patient with C. auris. CONCLUSION: Candida species often contaminate surfaces in hospitals and may be particularly common on floors and in sink drains. However, C. auris contamination was uncommon in a facility experiencing an outbreak, suggesting that current cleaning and disinfection practices can be effective in minimizing environmental contamination.

Microbiome and Antimicrobial Resistance Gene Dynamics in International Travelers.

We used metagenomic next-generation sequencing to longitudinally assess the gut microbiota and antimicrobial resistomes of international travelers to clarify global exchange of resistant organisms. Travel resulted in an increase in antimicrobial resistance genes and a greater proportion of Escherichia species within gut microbial communities without impacting diversity.

Multi-state study of Enterobacteriaceae harboring extended-spectrum beta-lactamase and carbapenemase genes in U.S. drinking water.

Community-associated acquisition of extended-spectrum beta-lactamase- (ESBL) and carbapenemase-producing Enterobacteriaceae has significantly increased in recent years, necessitating greater inquiry into potential exposure routes, including food and water sources. In high-income countries, drinking water is often neglected as a possible source of community exposure to antibiotic-resistant organisms. We screened coliform-positive tap water samples (n = 483) from public and private water systems in six states of the United States for blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, and blaOXA-48-type genes by multiplex PCR. Positive samples were subcultured to isolate organisms harboring ESBL or carbapenemase genes. Thirty-one samples (6.4%) were positive for blaCTX-M, ESBL-type blaSHV or blaTEM, or blaOXA-48-type carbapenemase genes, including at least one positive sample from each state. ESBL and blaOXA-48-type Enterobacteriaceae isolates included E. coli, Kluyvera, Providencia, Klebsiella, and Citrobacter species. The blaOXA-48-type genes were also found in non-fermenting Gram-negative species, including Shewanella, Pseudomonas and Acinetobacter. Multiple isolates were phenotypically non-susceptible to third-generation cephalosporin or carbapenem antibiotics. These findings suggest that tap water in high income countries could serve as an important source of community exposure to ESBL and carbapenemase genes, and that these genes may be disseminated by non-Enterobacteriaceae that are not detected as part of standard microbiological water quality testing.

Horizontal transfer of the blaNDM-1 gene to Pseudomonas aeruginosa and Acinetobacter baumannii in biofilms.

Horizontal gene transfer has contributed to the global spread of the blaNDM-1 gene. Multiple studies have demonstrated plasmid transfer of blaNDM-1 between Gram-negative bacteria, primarily Enterobacteriaceae species, but conjugational transfer of natural blaNDM-1 plasmids from Enterobacteriaceae into Pseudomonas aeruginosa and Acinetobacter baumannii has not previously been shown. As P. aeruginosa and A. baumannii are both typically strong biofilm formers, transfer of natural blaNDM-1 plasmids could potentially occur more readily in this environment. To determine whether natural blaNDM-1 plasmids could transfer to P. aeruginosa or A. baumannii in biofilms, three clinical and environmental Enterobacteriaceae strains carrying NDM-1-encoding plasmids of different incompatibility types were mated with E. coli J53, producing E. coli J53- blaNDM-1 transconjugants. Subsequently, dual-species biofilms were created using the E. coli J53 transconjugants as plasmid donors and either P. aeruginosa or A. baumannii as recipients. Biofilm transfer of NDM-encoding plasmids to P. aeruginosa and A. baumannii was successful from one and two E. coli J53- blaNDM-1 transconjugants, respectively. This demonstrates the potential for the spread of blaNDM-1, genes to P. aeruginosa and A. baumannii in clinical and environmental settings.

Estimates of the risk of large or long-lasting outbreaks of Middle East respiratory syndrome after importations outside the Arabian Peninsula.

We quantify outbreak risk after importations of Middle East respiratory syndrome outside the Arabian Peninsula. Data from 31 importation events show strong statistical support for lower transmissibility after early transmission generations. Our model projects the risk of ≥10, 100, and 500 transmissions as 11%, 2%, and 0.02%, and ≥1, 2, 3, and 4 generations as 23%, 14%, 0.9%, and 0.05%, respectively. Our results suggest tempered risk of large, long-lasting outbreaks with appropriate control measures.

Vital Signs: Estimated Effects of a Coordinated Approach for Action to Reduce Antibiotic-Resistant Infections in Health Care Facilities - United States.

BACKGROUND: Treatments for health care-associated infections (HAIs) caused by antibiotic-resistant bacteria and Clostridium difficile are limited, and some patients have developed untreatable infections. Evidence-supported interventions are available, but coordinated approaches to interrupt the spread of HAIs could have a greater impact on reversing the increasing incidence of these infections than independent facility-based program efforts. METHODS: Data from CDC's National Healthcare Safety Network and Emerging Infections Program were analyzed to project the number of health care-associated infections from antibiotic-resistant bacteria or C. difficile both with and without a large scale national intervention that would include interrupting transmission and improved antibiotic stewardship. As an example, the impact of reducing transmission of one antibiotic-resistant infection (carbapenem-resistant Enterobacteriaceae [CRE]) on cumulative prevalence and number of HAI transmission events within interconnected groups of health care facilities was modeled using two distinct approaches, a large scale and a smaller scale health care network. RESULTS: Immediate nationwide infection control and antibiotic stewardship interventions, over 5 years, could avert an estimated 619,000 HAIs resulting from CRE, multidrug-resistant Pseudomonas aeruginosa, invasive methicillin-resistant Staphylococcus aureus (MRSA), or C. difficile. Compared with independent efforts, a coordinated response to prevent CRE spread across a group of inter-connected health care facilities resulted in a cumulative 74% reduction in acquisitions over 5 years in a 10-facility network model, and 55% reduction over 15 years in a 102-facility network model. CONCLUSIONS: With effective action now, more than half a million antibiotic-resistant health care-associated infections could be prevented over 5 years. Models representing both large and small groups of interconnected health care facilities illustrate that a coordinated approach to interrupting transmission is more effective than historical independent facilitybased efforts. IMPLICATIONS FOR PUBLIC HEALTH: Public health-led coordinated prevention approaches have the potential to more completely address the emergence and dissemination of these antibiotic-resistant organisms and C. difficile than independent facility-based efforts.

Development and field evaluation of a method for detecting carbapenem-resistant bacteria in drinking water.

In this study, a fluorogenic heterotrophic plate count test for drinking water was modified in order to detect the presence of carbapenem-resistant bacteria. Antimicrobial agents and concentrations were selected based on recoveries of known carbapenem-resistant and carbapenem-susceptible strains inoculated into simulated samples. The modified method was field-tested on 19 drinking water samples from the New Delhi, India distribution system. Samples exhibiting fluorescence indicated bacterial growth in the presence of the supplemented antimicrobial agents, and organisms from these samples were cultured. Twenty-one Gram-negative isolates were identified from nine of the 19 samples and the meropenem minimum inhibitory concentrations were determined. Ultimately, eight carbapenem-resistant organisms were isolated from five sampling sites within the New Delhi water distribution system.

Does universal active MRSA surveillance influence anti-MRSA antibiotic use? A retrospective analysis of the treatment of patients admitted with suspicion of infection at Veterans Affairs Medical Centers between 2005 and 2010.

OBJECTIVES: After the implementation of an active surveillance programme for MRSA in US Veterans Affairs (VA) Medical Centers, there was an increase in vancomycin use. We investigated whether positive MRSA admission surveillance tests were associated with MRSA-positive clinical admission cultures and whether the availability of surveillance tests influenced prescribers' ability to match initial anti-MRSA antibiotic use with anticipated MRSA results from clinical admission cultures. METHODS: Analyses were based on barcode medication administration data, microbiology data and laboratory data from 129 hospitals between January 2005 and September 2010. Hospitalized patient admissions were included if clinical cultures were obtained and antibiotics started within 2 days of admission. Mixed-effects logistic regression was used to examine associations between positive MRSA admission cultures and (i) admission MRSA surveillance test results and (ii) initial anti-MRSA therapy. RESULTS: Among 569,815 included admissions, positive MRSA surveillance tests were strong predictors of MRSA-positive admission cultures (OR 8.5; 95% CI 8.2-8.8). The negative predictive value of MRSA surveillance tests was 97.6% (95% CI 97.5%-97.6%). The diagnostic OR between initial anti-MRSA antibiotics and MRSA-positive admission cultures was 3.2 (95% CI 3.1-3.4) for patients without surveillance tests and was not significantly different for admissions with surveillance tests. CONCLUSIONS: The availability of nasal MRSA surveillance tests in VA hospitals did not seem to improve the ability of prescribers to predict the necessity of initial anti-MRSA treatment despite the high negative predictive value of MRSA surveillance tests. Prospective trials are needed to establish the safety and effectiveness of using MRSA surveillance tests to guide antibiotic therapy.