Carbapenem-resistant Escherichia coli in Black-headed gulls, the Danube, and human clinical samples: A One Health comparison of contemporary isolates.

OBJECTIVES: Our aim was to characterize and compare contemporary carbapenem-resistant Enterobacterales (CRE) isolates from gulls, the River Danube, and humans in Hungary, Budapest. METHODS: Multiresistant Enterobacterales were sought for in 227 gull faecal and 24 Danube water samples from 2019 to 2020. Eosin-methylene blue agar containing 2 mg/L cefotaxime and Colilert-test containing 10 mg/L cefotaxime were used for gull and water samples, respectively. Isolates were characterized by polymerase chain reactions (PCRs); acquired carbapenemase producers were further analysed by whole-genome sequencing, together with 21 Hungarian human CR Escherichia coli (CREc) isolates. RESULTS: Gull and water samples exhibited a CRE prevalence of 7.4% (9/122) and 6.7% (7/105), none and 5/12 water samples yielded CRE from 2019 and 2020, respectively; CRE were found only in samples taken downstream of Budapest. The dominant species was Escherichia coli and the most prevalent carbapenemase was blaNDM-1. High-risk CREc clones were found both in gulls (ST224, ST372, ST744) and the Danube (ST10, ST354, ST410); the closest associations were between ST410 from humans and the Danube, among ST1437 among gulls, and between ST1437 in gulls and the Danube (46, 0, and 22-24 allelic distances, respectively). Direct links between human and gull isolates were not demonstrated. CONCLUSION: The study demonstrates potential epidemiological links among humans, a river crossing a city, and urbanised birds, suggesting a local transmission network. Water bodies receiving influent wastewater, together with animals using such habitats, may serve as a local reservoir system for CRE, highlighting the importance of One Health in CRE transmission, even in a country with a low CRE prevalence in humans.

SARS-CoV-2 variant detection from wastewater: rapid spread of B.1.1.7 lineage in Hungary.

Wastewater-based epidemiology (WBE) is a recognised tool for tracking community transmission of COVID-19. From the second half of 2020, the emergence of new, highly infective, more pathogenic or vaccine-escape SARS-CoV-2 variants is the major public health concern. Variant analysis in sewage might assist the early detection of new mutations. Weekly raw sewage samples from 22 wastewater treatment plants (WWTPs) in Hungary (representing 40% of the population) were analysed between December 2020 and March 2021 for signature mutations N501Y and del H69/V70 of B.1.1.7 lineage by melting point genotyping and RT-digital droplet PCR (RT-ddPCR). The latter method proved to be more efficient in parallel detection of different variants and also provides quantitative information. Wastewater surveillance indicated that the B.1.1.7 variant first emerged in Budapest in early January 2021 and rapidly became dominant in the entire country. Results are in close agreement with the available clinical data (Pearson's correlation coefficient, R = 0.9153). RT-ddPCR was confirmed to be a reliable tool for tracking emerging variant ratios in wastewaters. It is a rapid and cost-effective method compared to whole-genome sequencing, but only applicable for the detection of known mutations. Efficient variant surveillance might require the combination of multiple methods.

Ahead of the second wave: Early warning for COVID-19 by wastewater surveillance in Hungary.

Wastewater based epidemiology is a potential early warning tool for the detection of COVID-19 outbreak. Sewage surveillance for SARS-CoV-2 RNA was introduced in Hungary after the successful containment of the first wave of the pandemic to forecast the resurge of infections. Three wastewater treatment plants servicing the entire population (1.8 million) of the capital, Budapest were sampled weekly. 24 h composite (n = 44) and grab samples (n = 21) were concentrated by an in-house flat sheet membrane ultrafiltration method. The efficiency and reproducibility of the method was comparable to those previously published. SARS-CoV-2 RNA was quantified using RT-qPCR of the N gene. The first positive signal in sewage was detected 2 weeks before the rise in case numbers. Viral concentration and volume-adjusted viral load correlated to the weekly new cases from the same week and the rolling 7-day average of active cases in the subsequent week. The correlation was more pronounced in the ascending phase of the outbreak, data was divergent once case numbers plateaued. Wastewater surveillance was found to be effective in predicting the second wave of the outbreak in Hungary. Data indicated that even relatively low frequency (weekly) sampling is useful and at the same time, cost effective tool in outbreak detection.

Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain.

Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A-D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR-RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica.

Flagellin typing of Bordetella bronchiseptica strains originating from different host species.

Bordetella bronchiseptica is a widespread Gram-negative pathogen occurring in different mammal species. It is known to play a role in the aetiology of infectious atrophic rhinitis of swine, canine kennel cough, respiratory syndromes of cats, rabbits and guinea pigs, and sporadic human cases have also been reported. In this study, 93 B. bronchiseptica strains were examined from a broad range of host species and different geographical regions using restriction fragment length polymorphism analysis of polymerase chain reaction products of flaA to reveal the possible host-specificity of the flagellin. Eight types (A-H) of flaA were identified, including five newly described ones (D-H). All but one of the 22 B. bronchiseptica strains from swine showed type B fragment pattern. The eighteen Hungarian isolates of canine origin were uniform (type A) while in other countries type B and D were also present in dogs. The sequence and phylogenetic analysis of 36 representative strains of flaA types revealed four clusters. These clusters correlated with flaA PCR-RFLP types and host species, especially in pigs and dogs. The revealed diversity of the strains isolated from human cases indicated possible zoonotic transmissions from various animal sources.

Detection of urease-negative Bordetella bronchiseptica from the field.

Four urease-negative Bordetella bronchiseptica isolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs™ assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific and ureC PCR, and all strains showed uniform bands in PCR-RFLP studies of flaA genes. The reason for the lack of urease activity, a characteristic considered species specific for B. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification of B. bronchiseptica seems questionable.