Assessing cross-laboratory performance for quantifying coliphage using EPA Method 1642.

AIMS: Widespread adoption of the new U.S. Environmental Protection Agency (USEPA) Method 1642 for enumeration of coliphage in recreational water requires demonstration that laboratories consistently meet internal method performance goals and yield results that are consistent across laboratories. METHODS AND RESULTS: Here we assess the performance of six laboratories processing a series of blind wastewater- and coliphage-spiked samples along with laboratory blanks. All laboratories met the method-defined recovery requirements when performance was averaged across samples, with the few failures on individual samples mostly occurring for less-experienced laboratories on the initial samples processed. Failures that occurred on later samples were generally attributed to easily correctable activities. Failure rates were higher for somatic vs. F+ coliphage, attributable to the more stringent performance criteria associated with somatic coliphage. There was no difference in failure rate between samples prepared in a marine water matrix compared to that in phosphate-buffered saline. CONCLUSIONS: Variation among laboratories was similar to that previously reported for enterococci, the current bacterial indicator used for evaluating beach water quality for public health protection. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that laboratory performance is not an inhibitor to the adoption of coliphage as a new indicator for assessing recreational health risk.

A method to quantify viable carbapenem resistant gram-negative bacteria in treated and untreated wastewater.

Carbapenem resistance, particularly in Enterobacteriaceae, is an urgent threat to public health worldwide. Wastewater treatment plants are a critical control point for the spread of antimicrobial resistance into the environment yet, due in part to the lack of appropriate methods, the occurrence, identification and removal of carbapenem resistant bacteria has not been well characterized in wastewater matrices. This project was designed to provide a method for quantification of viable carbapenem resistant (CR) gram-negative bacteria (GNB) in raw sewage and treated wastewater effluents. A two-step procedure using membrane filtration and selective media supplemented with each of four carbapenems (doripenem, meropenem, imipenem, and ertapenem) was established for the quantification of CR GNB in wastewater matrices. Carbapenemase production was also assessed on individual bacterial colonies using two separate methods. Vitek®2 antimicrobial susceptibility test and disk diffusion assays were used to verify results from the supplemented media test and provide taxonomic identification. Treated and untreated wastewater samples from secondary and tertiary-stage wastewater treatment plants were analyzed for CR bacteria using the supplemented media procedure. Over 98% of all isolates selected from the carbapenem-supplemented media were verified as CR GNB. Carbapenemase production was observed in 80% of these isolates and 88% were multidrug resistant. All Enterobacteriaceae isolates from the supplemented media were verified as CR and 97% tested positive for carbapenemase production. The highest concentrations of CR GNB in wastewater were observed using the ertapenem-supplemented media. Doripenem-supplemented media showed the greatest specificity and selectivity for carbapenemase-producing CRE. Overall, the cumulative CR GNB in wastewater were reduced by approximately three- and five-log10 by the secondary and tertiary-stage WWTPs, respectively. This study establishes a method for characterization of viable CR GNB in wastewater matrices and demonstrates that current wastewater treatment technologies effectively reduce CR bacteria, including CRE, in sewage.