Epidemiological Description and Detection of Antimicrobial Resistance in Various Aquatic Sites in Marseille, France.

Antibiotic resistance is a worldwide public health concern and has been associated with reports of elevated mortality. According to the One Health concept, antibiotic resistance genes are transferrable to organisms, and organisms are shared among humans, animals, and the environment. Consequently, aquatic environments are a possible reservoir of bacteria harboring antibiotic resistance genes. In our study, we screened water and wastewater samples for antibiotic resistance genes by culturing samples on different types of agar media. Then, we performed real-time PCR to detect the presence of genes conferring resistance to beta lactams and colistin, followed by standard PCR and gene sequencing for verification. We mainly isolated Enterobacteriaceae from all samples. In water samples, 36 Gram-negative bacterial strains were isolated and identified. We found three extended-spectrum ß-lactamase (ESBL)-producing bacteria-Escherichia coli and Enterobacter cloacae strains-harboring the CTX-M and TEM groups. In wastewater samples, we isolated 114 Gram-negative bacterial strains, mainly E. coli, Klebsiella pneumoniae, Citrobacter freundii and Proteus mirabilis strains. Forty-two bacterial strains were ESBL-producing bacteria, and they harbored at least one gene belonging to the CTX-M, SHV, and TEM groups. We also detected carbapenem-resistant genes, including NDM, KPC, and OXA-48, in four isolates of E. coli. This short epidemiological study allowed us to identify new antibiotic resistance genes present in bacterial strains isolated from water in Marseille. This type of surveillance shows the importance of tracking bacterial resistance in aquatic environments. IMPORTANCE Antibiotic-resistant bacteria are involved in serious infections in humans. The dissemination of these bacteria in water, which is in close contact with human activities, is a serious problem, especially under the concept of One Health. This study was done to survey and localize the circulation of bacterial strains, along with their antibiotic resistance genes, in the aquatic environment in Marseille, France. The importance of this study is to monitor the frequency of these circulating bacteria by creating and surveying water treatments.

SARS-CoV-2 Testing of Aircraft Wastewater Shows That Mandatory Tests and Vaccination Pass before Boarding Did Not Prevent Massive Importation of Omicron Variant into Europe.

BACKGROUND: Most new SARS-CoV-2 epidemics in France occurred following the importation from abroad of emerging viral variants. Currently, the risk of new variants being imported is controlled based on a negative screening test (PCR or antigenic) and proof of up-to-date vaccine status, such as the International Air Transport Association travel pass. METHODS: The wastewater from two planes arriving in Marseille (France) from Addis Ababa (Ethiopia) in December 2021 was tested by RT-PCR to detect SARS-CoV2 and screen for variants. These tests were carried out between landing and customs clearance and were then sequenced by MiSeq Illumina. Antigenic tests and sequencing by NovaSeq were carried out on respiratory samples collected from the 56 passengers on the second flight. RESULTS: SARS-CoV-2 RNA suspected of being from the Omicron BA.1 variant was detected in the aircraft's wastewater. SARS-CoV2 RNA was detected in 11 [20%) passengers and the Omicron BA.1 variant was identified. CONCLUSION: Our work shows the efficiency of aircraft wastewater testing to detect SARS-CoV-2 cases among travellers and to identify the viral genotype. It also highlights the low efficacy of the current control strategy for flights entering France from outside Europe, which combines a requirement to produce a vaccine pass and proof of a negative test before boarding.

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France.

The monitoring of SARS-CoV-2 RNA in sewage has been proposed as a simple and unbiased means of assessing epidemic evolution and the efficiency of the COVID-19 control measures. The past year has been marked by the emergence of variants that have led to a succession of epidemic waves. It thus appears that monitoring the presence of SARS-CoV-2 in wastewater alone is insufficient, and it may be important in the future to also monitor the evolution of these variants. We used a real-time RT-PCR screening test for variants in the wastewater of our city to assess the effectiveness of direct SARS-CoV-2 sequencing from the same wastewater. We compared the genome sequencing results obtained over the large RS network and the smaller B7 network with the different distributions of the variants observed by RT-PCR screening. The prevalence of the "UK variant" in the RS and B7 networks was estimated to be 70% and 8% using RT-PCR screening compared to 95% and 64% using genome sequencing, respectively. The latter values were close to the epidemiology observed in patients of the corresponding area, which were 91% and 58%, respectively. Genome sequencing in sewage identified SARS-CoV-2 of lineage B.1.525 in B7 at 27% (37% in patients), whereas it was completely missed by RT-PCR. We thus determined that direct sequencing makes it possible to observe, in wastewater, a distribution of the variants comparable to that revealed by genomic monitoring in patients and that this method is more accurate than RT-PCR. It also shows that, rather than a single large sample, it would be preferable to analyse several targeted samples if we want to more appropriately assess the geographical distribution of the different variants. In conclusion, this work supports the wider surveillance of SARS-CoV-2 variants in wastewater by genome sequencing and targeting small areas on the condition of having a sequencing capacity and, when this is not the case, to developing more precise screening tests based on the multiplexed detection of the mutations of interest.

Viral RNA in City Wastewater as a Key Indicator of COVID-19 Recrudescence and Containment Measures Effectiveness.

In recent years, and more specifically at the beginning of the COVID-19 crisis, wastewater surveillance has been proposed as a tool to monitor the epidemiology of human viral infections. In the present work, from July to December 2020, the number of copies of SARS-CoV-2 RNA in Marseille's wastewater was correlated with the number of new positive cases diagnosed in our Institute of Infectious Disease, which tested about 20% of the city's population. Number of positive cases and number of copies of SARS-CoV-2 RNA in wastewater were significantly correlated (p = 0.013). During the great epidemic peak, from October to December 2020, the curves of virus in the sewers and the curves of positive diagnoses were perfectly superposed. During the summer period, the superposition of curves was less evident as subject to many confounding factors that were discussed. We also tried to correlate the effect of viral circulation in wastewater with containment measures, probably the most unbiased correlation on their potential inflection effect of epidemic curves. Not only is this correlation not obvious, but it also clearly appears that the drop in cases as well as the drop in the viral load in the sewers occur before the containment measures. In fact, this suggests that there are factors that initiate the end of the epidemic peak independently of the containment measure. These factors will therefore need to be explored more deeply in the future.