Viral variant-resolved wastewater surveillance of SARS-CoV-2 at national scale.

SARS-CoV-2 surveillance by wastewater-based epidemiology is poised to provide a complementary approach to sequencing individual cases. However, robust quantification of variants and de novo detection of emerging variants remains challenging for existing strategies. We deep sequenced 3,413 wastewater samples representing 94 municipal catchments, covering >59% of the population of Austria, from December 2020 to February 2022. Our system of variant quantification in sewage pipeline designed for robustness (termed VaQuERo) enabled us to deduce the spatiotemporal abundance of predefined variants from complex wastewater samples. These results were validated against epidemiological records of >311,000 individual cases. Furthermore, we describe elevated viral genetic diversity during the Delta variant period, provide a framework to predict emerging variants and measure the reproductive advantage of variants of concern by calculating variant-specific reproduction numbers from wastewater. Together, this study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without extensive individual monitoring.

Emergence of SARS-CoV-2 Alpha lineage and its correlation with quantitative wastewater-based epidemiology data.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gave rise to an international public health emergency in 3 months after its emergence in Wuhan, China. Typically for an RNA virus, random mutations occur constantly leading to new lineages, incidental with a higher transmissibility. The highly infective alpha lineage, firstly discovered in the UK, led to elevated mortality and morbidity rates as a consequence of Covid-19, worldwide. Wastewater surveillance proved to be a powerful tool for early detection and subsequent monitoring of the dynamics of SARS-CoV-2 and its variants in a defined catchment. Using a combination of sequencing and RT-qPCR approaches, we investigated the total SARS-CoV-2 concentration and the emergence of the alpha lineage in wastewater samples in Vienna, Austria linking it to clinical data. Based on a non-linear regression model and occurrence of signature mutations, we conclude that the alpha variant was present in Vienna sewage samples already in December 2020, even one month before the first clinical case was officially confirmed and reported by the health authorities. This provides evidence that a well-designed wastewater monitoring approach can provide a fast snapshot and may detect the circulating lineages in wastewater weeks before they are detectable in the clinical samples. Furthermore, declining 14 days prevalence data with simultaneously increasing SARS-CoV-2 total concentration in wastewater indicate a different shedding behavior for the alpha variant. Overall, our results support wastewater surveillance to be a suitable approach to spot early circulating SARS-CoV-2 lineages based on whole genome sequencing and signature mutations analysis.

KPC-50 Confers Resistance to Ceftazidime-Avibactam Associated with Reduced Carbapenemase Activity.

KPC-50 is a KPC-3 variant identified from a Klebsiella pneumoniae clinical isolate recovered in Switzerland in 2019. Compared to KPC-3, KPC-50 shows (i) a three-amino-acid insertion (Glu-Ala-Val) between amino acids 276 and 277, (ii) an increased affinity to ceftazidime, (iii) a decreased sensitivity to avibactam, explaining the ceftazidime-avibactam resistance, and (iv) an association with a sharp reduction of its carbapenemase activity.

Ongoing dissemination of OXA-244 carbapenemase-producing Escherichia coli in Switzerland and their detection.

OXA-244 is a derivative of OXA-48 showing weaker carbapenemase activity, compromising the detection of corresponding producers in clinical laboratories. Since 2017, the Swiss National Reference Center for Emerging Antibiotic Resistance noticed an increased identification of OXA-244-producing Escherichia coli (n=15) within the country. Different methods (biochemical and immunoassay tests, screening culture media) were tested for the detection of OXA-244 producers. Whole genome sequencing was used to investigate the genetic relatedness between the isolates and the genetic structures at the origin of the acquisition of the blaOXA-244 gene. The mSuperCARBA® medium and the NG-Test CARBA5 assay were found to be suitable tools for detecting all OXA-244-producing isolates. Other selective media did not perform optimally. Among the fifteen strains, five sequence types were identified, with ST38 being predominant. The blaOXA-244 gene was located on the chromosome for all isolates. Overall, detection of OXA-244 producers is challenging and specific guidelines must be followed.

PFM-Like Enzymes Are a Novel Family of Subclass B2 Metallo-ß-Lactamases from Pseudomonas synxantha Belonging to the Pseudomonas fluorescens Complex.

A carbapenem-resistant Pseudomonas synxantha isolate recovered from chicken meat produced the novel carbapenemase PFM-1. That subclass B2 metallo-ß-lactamase shared 71% amino acid identity with ß-lactamase Sfh-1 from Serratia fonticola The blaPFM-1 gene was chromosomally located and likely acquired. Variants of PFM-1 sharing 90% to 92% amino acid identity were identified in bacterial species belonging to the Pseudomonas fluorescens complex, including Pseudomonas libanensis (PFM-2) and Pseudomonas fluorescens (PFM-3), highlighting that these species constitute reservoirs of PFM-like encoding genes.

Wide spread of carbapenemase-producing bacterial isolates in a Nigerian environment.

OBJECTIVES: The presence of carbapenemase-producing bacterial isolates is found not only in hospital and community settings but also in the environment. Carbapenemase production may be related to acquired, usually plasmid-borne, ß-lactamase genes or to chromosomal genes intrinsic to various species. The aim of this study was to evaluate the occurrence of such carbapenemase-producing bacterial isolates among environmental samples from Nigeria. METHODS: A total of 122 environmental samples were plated on carbapenem-containing media. A total of 259 isolates were recovered, among which 124 were carbapenemase-producers according to the results of the Rapidec® Carba NP test. RESULTS: The majority of isolates (n=112) recovered corresponded to natural producers of carbapenemases, i.e. Stenotrophomonas maltophilia (n=108), Burkholderia cepacia (n=1), Shewanella sp. (n=1), Sphingobacterium sp. (n=1) and Chryseobacterium gleum (n=1). Ten isolates (mainly Enterobacteriaceae and Acinetobacter baumannii) produced an acquired carbapenemase, most commonly of the NDM type. In addition, two Pseudomonas otitidis isolates were identified as producing the Ambler class B carbapenemase POM-1, further confirming that this carbapenemase is naturally produced in this environmental species. Finally, several isolates co-producing 16S rRNA methylases (ArmA, RmtC) and/or extended-spectrum ß-lactamases (CTX-M-9, CTX-M-15) were also identified. CONCLUSION: This study revealed the presence and diversity of clinically-relevant antimicrobial-resistant bacteria in the environment in Nigeria.

Phenotypic, biochemical and genetic analysis of KPC-41, a KPC-3 variant conferring resistance to ceftazidime-avibactam and exhibiting reduced carbapenemase activity.

A novel KPC variant, KPC-41, was identified in a Klebsiella pneumoniae clinical isolate from Switzerland. This ß-lactamase possessed a three amino-acid insertion (Pro-Asn-Lys) located between amino acids 269 and 270 compared to the KPC-3 amino acid sequence. Cloning and expression of the bla KPC-41 gene in Escherichia coli, followed by determination of MIC values and kinetic parameters, showed that KPC-41, compared to KPC-3, has an increased affinity to ceftazidime and a decreased sensitivity to avibactam, leading to resistance to ceftazidime-avibactam once produced in K. pneumoniae Furthermore, KPC-41 exhibited a drastic decrease of its carbapenemase activity. This report highlights that a diversity of KPC variants conferring resistance to ceftazidime-avibactam already circulate in Europe.