Wastewater-based monitoring reveals geospatial-temporal trends for antibiotic-resistant pathogens in a large urban community.

Antimicrobial resistance (AMR) is one of the top ten global health threats, and current surveillance programs rarely monitor it outside healthcare settings. This limits our ability to understand and manage the spread of AMR. Wastewater testing has the potential to simply, reliably and continuously survey trends in AMR outside the healthcare settings, as it captures biological material from the entire community. To establish and evaluate such a surveillance, we monitored wastewater for four clinically significant pathogens across the urban area of Greater Sydney, Australia. Untreated wastewater from 25 wastewater treatment plants (WWTPs) covering distinct catchment regions of 5.2 million residents was sampled between 2017 and 2019. Isolates for extended-spectrum ß-lactamases-producing Enterobacteriaceae (ESBL-E) were consistently detected, suggesting its endemicity in the community. Isolates for carbapenem-resistant Enterobacteriaceae (CRE), vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA) were only occasionally detected. The flow normalized relative (FNR) ESBL-E load was positively correlated with the proportion of the population between 19 and 50 years of age, completion of vocational education and the average length of hospital stay. Collectively, these variables explained only a third of the variance of the FNR ESBL-E load, indicating further, yet-unidentified factors as a contributor to the distribution. About half of the variation in the FNR CRE load was explained by the average length of hospital stay, showing healthcare-related drivers. Interestingly, variation in the FNR VRE load was not correlated to healthcare-related parameters but to the number of schools per 10,000 population. Our study provides insight into how routine wastewater surveillance can be used to understand the factors driving the distribution of AMR in an urban community. Such information can help to manage and mitigate the emergence and spread of AMR in important human pathogens.

Utility of matrix-assisted laser desorption ionization-time of flight mass spectrometry following introduction for routine laboratory bacterial identification.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated prospectively in a diagnostic laboratory. Nine hundred twenty-seven organisms were tested in triplicate; 2,351/2,781 (85%) species and 2,681/2,781 (96%) genus identifications were correct. Known issues such as the misidentification of alpha-hemolytic streptococci as Streptococcus pneumoniae were easily corrected. Identifications cost AUD$0.45 per isolate and were available in minutes. MALDI-TOF MS is rapid, accurate, and inexpensive.

Whole genome sequence analysis of the first Australian OXA-48-producing outbreak-associated Klebsiella pneumoniae isolates: the resistome and in vivo evolution.

Whole genome sequencing was used to characterize the resistome of intensive care unit (ICU) outbreak-associated carbapenem-resistant K. pneumoniae isolates. Importantly, and of particular concern, the carbapenem-hydrolyzing ß-lactamase gene bla(OXA-48) and the extended-spectrum ß-lactamase gene bla(CTX-M-14), were identified on a single broad host-range conjugative plasmid. This represents the first report of bla(OXA-48) in Australia and highlights the importance of resistance gene surveillance, as such plasmids can silently spread amongst enterobacterial populations and have the potential to drastically limit treatment options. Furthermore, the in vivo evolution of these isolates was also examined after 18 months of intra-abdominal carriage in a patient that transited through the ICU during the outbreak period. Reflecting the clonality of K. pneumoniae, only 11 single nucleotide polymorphisms (SNPs) were accumulated during this time-period and many of these were associated with genes involved in tolerance/resistance to antibiotics, metals or organic solvents, and transcriptional regulation. Collectively, these SNPs are likely to be associated with changes in virulence (at least to some extent) that have refined the in vivo colonization capacity of the original outbreak isolate.