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This paper aims to examine the multiresidue enantiomeric profiling of (fluoro)quinolones and their metabolites in solid and liquid environmental matrices using chiral HPLC-MS/MS method and a CHIRALCEL OZ-RH column. Simultaneous chiral separation was obtained for chiral ofloxacin and its main metabolites ofloxacin-N-oxide and desmethyl-ofloxacin; moxifloxacin; the prodrug prulifloxacin and its active compound ulifloxacin; flumequine; nadifloxacin and R-(+)-besifloxacin. Achiral antibiotics (ciprofloxacin, norfloxacin and nalidixic acid) were also included in the method to enable the analysis of all targeted quinolones within one analytical run. Satisfactory enantiomeric resolution (Rs ≥ 1) was obtained for five out of eight chiral drugs enabling quantitative analysis. The overall performance of the method was satisfactory with a method precision <20%, relative recoveries >70% for most of the analytes and method detection limits (MDL) at low ng L levels (0.1 < MDL (ng L)< 6.4, 0.1 < MDL (ng L)< 6.6 and 0.1 < MDL (ng L)< 7.0 in influent, effluent and river waters for 83% compounds, 0.01 < MDL (ng g)< 4.9 in solids for 91% compounds). Enantiomeric profiling from a week-long monitoring campaign in the UK showed that (±)-ofloxacin was found to be racemic in upstream waters but it was enriched with S-(-)-enantiomer in wastewater and in receiving waters. This could be due to the fact that ofloxacin can be used both as a racemate and as a S-(-)-enantiomer. Its consumption was further confirmed by the chiral signature of the investigated ofloxacin metabolites. As a result, alterations in the enantiomeric composition of antibiotics could influence not only their activity and toxicity in the environment, but also could induce changes in the microbial communities constantly exposed to them.

This study provides an insight into the prevalence of (fluoro)quinolones (FQs) and their specific quinolone qnrS resistance gene in the aquatic environment from the Avon river catchment area receiving treated wastewater from 5 wastewater treatment plants (WWTPs), serving 1.5 million people and accounting for 75% of inhabitants living in the catchment area in the South West of England. FQs were analysed by stereoselective chiral chromatography and tandem mass spectrometry and their specific qnrS resistance gene was measured with digital PCR, which allowed for spatiotemporal evaluation of the prevalence of FQs and qnrS across the catchment. Ofloxacin, ciprofloxacin, nalidixic acid and norfloxacin were found to be ubiquitous with daily loads reaching a few hundred g/day in wastewater influent and tens of g/day in receiving waters. This was in contrast to other FQs analysed: flumequine, nadifloxacin, lomefloxacin, ulifloxacin, prulifloxacin, besifloxacin and moxifloxacin, which were hardly quantified. Enantiomeric profiling revealed that ofloxacin was enriched with the S-(-)-enantiomer, likely deriving from its prescription as the more potent enantiomerically pure levofloxacin, alongside racemic ofloxacin. While ofloxacin's AS might be facilitating antimicrobial resistance (AMR) prevalence to higher extent than TF.Wastewater-based epidemiology (WBE) was also applied to monitor any potential misuse (e.g. direct disposal) of FQs in the catchment. In most cases higher use of antibiotics with respect to official statistics (i.e. ciprofloxacin, ofloxacin) was observed, which suggests that FQs management practice require further attention.

Klebsiella species occupy a wide range of environmental and animal niches, and occasionally cause opportunistic infections that are resistant to multiple antibiotics. In particular, Klebsiella pneumoniae (Kpne) has gained notoriety as a major nosocomial pathogen, due principally to the rise in non-susceptibility to carbapenems and other beta-lactam antibiotics. Whilst it has been proposed that the urban water cycle facilitates transmission of pathogens between clinical settings and the environment, the level of risk posed by resistant Klebsiella strains in hospital wastewater remains unclear. We used whole genome sequencing (WGS) to compare Klebsiella species in contemporaneous samples of wastewater from an English hospital and influent to the associated wastewater treatment plant (WWTP). As we aimed to characterize representative samples of Klebsiella communities, we did not actively select for antibiotic resistance (other than for ampicillin), nor for specific Klebsiella species. Two species, Kpne and K. ( Raoultella ) ornithinolytica (Korn), were of equal dominance in the hospital wastewater, and four other Klebsiella species were present in low abundance in this sample. In contrast, despite being the species most closely associated with healthcare settings, Kpne was the dominant species within the WWTP influent. In total, 29 % of all isolates harboured the bla OXA-48 gene on a pOXA-48-like plasmid, and these isolates were almost exclusively recovered from the hospital wastewater. This gene was far more common in Korn (68 % of isolates) than in Kpne (3.4 % of isolates). In general plasmid-borne, but not chromosomal, resistance genes were significantly enriched in the hospital wastewater sample. These data implicate hospital wastewater as an important reservoir for antibiotic-resistant Klebsiella , and point to an unsuspected role of species within the Raoultella group in the maintenance and dissemination of plasmid-borne bla OXA-48. This article contains data hosted by Microreact.

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Increasing understanding and awareness of antimicrobial resistance (AMR) is critical in tackling this growing global crisis. Wastewater-based epidemiology (WBE) is a promising approach to monitoring a range of AMR targets in communities through analysis of wastewater. This longitudinal study provides insight into antimicrobial (AA) usage within two communities in the South West of the UK, one city (Bath) and one town (Keynsham). AAs, including metabolites, from a range of different classes were quantified over the study period. Average loads of AAs were higher in Bath than for Keynsham (difference on average was 88 ± 6%) which reflected the larger population. Several AAs experienced seasonal fluctuations, such as the macrolides erythromycin and clarithromycin that were found in higher loads in the winter, whilst other AA levels, including sulfamethoxazole and sulfapyridine, stayed consistent over the study period. Several antimicrobial resistant genes (ARGs) were also studied within the city area, in order to determine how closely the abundance of these genes correlates with the levels of relevant AAs. Several genes including ermB, sul1 and intI1 were not found at statistically significant different loads in winter 2018/19 when compared to summer 2019. Due to relatively stable AA and ARG levels across 13 months monitoring time, no clear correlation was observed between absolute loads of ARGs and total loads of associated AAs by class. Hospital effluent within the city catchment was also investigated for AAs and ARGs. Several AAs were more common in hospital wastewater than in community wastewater, including sulfamethoxazole and trimethoprim. This work can help establish baselines for AA usage in communities, providing community-wide surveillance and evidence for informing public health interventions.

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