Prevalence, risk factors, and characterisation of extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) in horses entering an equine hospital and description of longitudinal excretion.

BACKGROUND: Extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) are important zoonotic pathogens that can cause serious clinical infections, also in horses. Preventing the spread of ESBL-E, especially in the equine hospital environment, is key to reducing the number of difficult-to-treat infections. Estimating the local prevalence of ESBL-E in horses is crucial to establish targeted infection control programs at equine hospitals. We conducted a prevalence and risk factor study in equine patients on admission to an equine teaching hospital in Finland through a rectal ESBL-E screening specimen of the horse and a questionnaire. RESULTS: The prevalence of ESBL-E in admitted horses was 3% (5/161, 95% CI 1-7%); none of the tested factors remained statistically significant in multivariate analysis, although antimicrobial treatment within three months was borderline significant (p = 0.052). Extended-spectrum ß-lactamase -producing Klebsiella pneumoniae ST6179:CTX-M-15 was detected in three horses using whole-genome sequencing, which in combination with patient records suggested nosocomial transmission. Escherichia coli isolates were ST1250:CTX-M-1 (n = 1), ST1079:CTX-M-1 (n = 1), and ST1245:CTX-M-14 (n = 1). Multiple virulence genes were detected in the ESBL-E isolates. In the ESBL-E positive horses enrolled in a one-year follow-up study, ESBL-E were unlikely to be isolated in rectal screening specimens after the initial positive specimen. CONCLUSIONS: The prevalence of ESBL-E in horses visiting a veterinary teaching hospital in Finland is low, indicating an overall low prevalence estimate in the country's equine population. No statistically significant risk factors were identified, likely due to the low number of cases. The duration of ESBL-E carriage is likely to be very short in horses.

Bacterial quality and safety of raw beef: A comparison between Finland and Nigeria.

Beef can easily be contaminated with bacteria during the meat production chain. In this work, we studied the contamination levels of mesophilic aerobic bacteria (MAB) and thermotolerant coliform bacteria (TCB) on raw beef surfaces from small shops in Helsinki, Finland and meat markets in Benin City, Nigeria. We also investigated the prevalence of Salmonella, Campylobacter, Yersinia, Shiga toxin-producing Escherichia coli (STEC), Listeria, and cephalosporin-resistant E. coli (CREC). In total, one hundred unpacked raw beef samples from Finland and Nigeria were collected in 2019. The median MAB and TCB counts were significantly (P < 0.001) higher on beef from Nigeria than from Finland. The median MAB and TCB counts in Nigeria were 7.5 and 4.0 log10 cfu/cm2, respectively, and 6.5 and 2.8 log10 cfu/cm2 in Finland, respectively. Most (94%) Nigerian samples were unacceptable according to limits set by the EU. Beef samples from meat markets in Benin City were significantly (P < 0.05) more frequently contaminated with Salmonella, STEC, and CREC than beef samples from small shops in Helsinki. Salmonella, STEC, and CREC were isolated from 30, 36, and 96% of Nigerian samples, respectively, and from <2, 12, and 2% of Finnish samples, respectively. Our study demonstrates a significant difference between the bacterial contaminations of raw beef in Nigeria and Finland, along with a possible misuse of cephalosporins in animal husbandry in Nigeria.

Foodborne pathogenic bacteria in wild European hedgehogs (Erinaceus europaeus).

BACKGROUND: European hedgehogs (Erinaceus europaeus) are widely distributed across Europe. They may play an important role by spreading zoonotic bacteria in the environment and to humans and animals. The aim of our work was to study the prevalence and characteristics of the most important foodborne bacterial pathogens in wild hedgehogs. RESULTS: Faecal samples from 148 hospitalised wild hedgehogs originating from the Helsinki region in southern Finland were studied. Foodborne pathogens were detected in 60% of the hedgehogs by PCR. Listeria (26%) and STEC (26%) were the most common foodborne pathogens. Salmonella, Yersinia, and Campylobacter were detected in 18%, 16%, and 7% of hedgehogs, respectively. Salmonella and Yersinia were highly susceptible to the tested antimicrobials. Salmonella Enteritidis and Listeria monocytogenes 2a were the most common types found in hedgehogs. All S. Enteritidis belonged to one sequence type (ST11), forming four clusters of closely related isolates. L. monocytogenes was genetically more diverse than Salmonella, belonging to 11 STs. C. jejuni ST45 and ST677, Y. pseudotuberculosis O:1 of ST9 and ST42, and Y. enterocolitica O:9 of ST139 were also found. CONCLUSIONS: Our study shows that wild European hedgehogs should be considered an important source of foodborne pathogens, and appropriate hygiene measures after any contact with hedgehogs and strict biosecurity around farms are therefore important.

Enteropathogenic Yersinia with Public Health Relevance Found in Dogs and Cats in Finland.

Yersiniosis is a common zoonotic enteric disease among humans, which has been linked to pigs and contaminated food, especially pork. The epidemiology of yersiniosis is still obscure, and studies on yersiniosis in pets are very scarce. In this study, we performed pheno- and genotypic characterisation of 50 Yersinia strains isolated from pets in Finland between 2012 and 2023. Y. enterocolitica 4/O:3/ST135, the most common type in human yersiniosis, was also the most common type (68%) found in clinical faecal samples in our study. Also, human pathogenic Y. enterocolitica 2/O:9/ST139 and Y. pseudotuberculosis O:1/ST9 and O:1/ST42 strains carrying all essential pathogenic genes were identified. Three Y. enterocolitica 4/O:3/ST9 strains were multi-drug-resistant and two of them were highly related, showing one allelic difference (AD) with core genome multi-locus sequence typing. Non-pathogenic, genotypically highly diverse Y. enterocolitica 1A strains, showing more than 1000 ADs and missing the essential virulence genes, were also recognised in dogs and cats. Our study demonstrates that pets can excrete human pathogenic Yersinia in their faeces and may serve as an infection source for human yersiniosis, especially in families with small children in close contact with their pets.

Wastewater and environmental sampling holds potential for antimicrobial resistance surveillance in food-producing animals - a pilot study in South African abattoirs.

Antimicrobial resistance (AMR) poses a significant global One Health challenge that causes increased mortality and a high financial burden. Animal production contributes to AMR, as more than half of antimicrobials are used in food-producing animals globally. There is a growing body of literature on AMR in food-producing animals in African countries, but the surveillance practices across countries vary considerably. This pilot study aims to explore the potential of wastewater and environmental surveillance (WES) of AMR and its extension to the veterinary field. Floor drainage swab (n = 18, 3/abattoir) and wastewater (n = 16, 2-3/abattoir) samples were collected from six South African abattoirs that handle various animal species, including cattle, sheep, pig, and poultry. The samples were tested for Extended-Spectrum Beta-Lactamase (ESBL) and Carbapenemase-producing Enterobacterales, Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), and Candida auris by using selective culturing and MALDI-TOF MS identification. The phenotype of all presumptive ESBL-producing Escherichia coli (n = 60) and Klebsiella pneumoniae (n = 24) isolates was confirmed with a disk diffusion test, and a subset (15 and 6 isolates, respectively), were further characterized by whole-genome sequencing. In total, 314 isolates (0-12 isolates/sample) withstood MALDI-TOF MS, from which 37 species were identified, E. coli and K. pneumoniae among the most abundant. Most E. coli (n = 48/60; 80%) and all K. pneumoniae isolates were recovered from the floor drainage samples, while 21 presumptive carbapenem-resistant Acinetobacter spp. isolates were isolated equally from floor drainage and wastewater samples. MRSA, VRE, or C. auris were not found. All characterized E. coli and K. pneumoniae isolates represented ESBL-phenotype. Genomic analyses revealed multiple sequence types (ST) of E. coli (n = 10) and K. pneumoniae (n = 5), including STs associated with food-producing animals globally, such as E. coli ST48 and ST10 and K. pneumoniae ST101. Common beta-lactamases linked to food-producing animals, such as bla CTX-M-55 and bla CTX-M-15, were detected. The presence of food-production-animal-associated ESBL-gene-carrying E. coli and K. pneumoniae in an abattoir environment and wastewater indicates the potential of WES in the surveillance of AMR in food-producing animals. Furthermore, the results of this pilot study encourage studying the topic further with refined methodologies.

Simultaneous biomass concentration and subsequent quantitation of multiple infectious disease agents and antimicrobial resistance genes from community wastewater.

Wastewater-based surveillance (WBS) of infectious disease agents is increasingly seen as a reliable source of population health data. To date, wastewater-based surveillance efforts have largely focused on individual pathogens. However, given that wastewater contains a broad range of pathogens circulating in the population, a more comprehensive approach could enhance its usability. We focused on the simultaneous detection of SARS-CoV-2, sapovirus, Campylobacter jejuni, Campylobacter coli, Salmonella spp., pathogenic Escherichia coli, Cryptosporidium spp., Giardia spp. and antimicrobial resistance genes (ARGs) of clinical relevance. To achieve this goal, biomass concentration and nucleic acid extraction methods were optimized, and samples were analyzed by using a set of (RT)-qPCR and (HT)-qPCR methods. We determined the prevalence and the spatial and temporal trends of the targeted pathogens and collected novel information on ARGs in Finnish wastewater. In addition, the use of different wastewater concentrates, namely the ultrafiltered concentrate of the supernatant and the centrifuged pellet, and the effect of freezing and thawing wastewater prior to sample processing were investigated with the indicator microbe crAssphage. Freeze-thawing of wastewater decreased the gene copy count of crAssphage in comparison to analyzing fresh samples (p < 0.001). Campylobacters were most abundant in two of the four studied summer months (30 % detection rate) and in wastewaters from regions with intensive animal farming. Salmonella, however, was detected in 40 % of the samples without any clear seasonal trends, and the highest gene copy numbers were recorded from the largest wastewater treatment plants. Beta-lactamase resistance genes that have commonly been detected in bacteria isolated from humans in Finland, namely blaCTX-M, blaOXA48, blaNDM, and blaKPC, were also frequently detected in wastewaters (100, 98, 98, and 70 % detection rates, respectively). These results confirm the reliability of using wastewater in public health surveillance and demonstrate the possibility to simultaneously perform WBS of multiple pathogens.

Comparison of Different Reverse Transcriptase-Polymerase Chain Reaction-Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study.

BACKGROUND: Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required. OBJECTIVE: This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings. METHODS: We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively. RESULTS: Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater. CONCLUSIONS: As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.

Characterization of methicillin resistant Staphylococcus Aureus in municipal wastewater in Finland.

Wastewater-based surveillance (WBS) of multidrug-resistant bacteria could complement clinical data, serving as a population-level early warning tool. This study evaluated WBS as a pandemic preparedness tool, by selectively isolating and culturing methicillin-resistant Staphylococcus aureus (MRSA) with CHROMagar MRSA. Some 24-h composite wastewater samples (n = 80) were collected from ten treatment plants across Finland between February 2021 and January 2022. MRSA prevalence in wastewater samples was 27.5% (n = 22/80), showing seasonal and temporal variations. Phenotypic antimicrobial susceptibility testing (AST) with microdilution showed that over 80% of isolates were drug-resistant to clindamycin, sulfamethoxazole/trimethoprim, tetracycline, fusidic acid, and erythromycin. Four isolates (18.2%) were vancomycin-resistant. WGS revealed that 31.8% (n = 7) of the isolates belonged to the ST8-t008 and ST6-t304 spa types, respectively. In addition, two spa types (t011 and t034) belong to the CC398 complex. The mecA gene was found in all isolates (n = 22) and three tetracycline resistance determinants (tet38, tetK, and tetM) were detected with tet38 being the most abundant (81.8%, n = 18/22). Three isolates harboured the plasmid-mediated sat4 gene that confers resistance to Streptothricin. In addition, resistance determinants to macrolide antibiotics (mph (C)/msr (A) and fosfomycin (fosB) were detected in the seven isolates that belonged to spa type t008. All isolates except one harboured the SCCmec_type_IVa(2B). Six ST8 isolates harboured the LukS/F-PV genes encoding the Panton-Valentine leukocidin (PVL) and were also positive for the Arginine Catabolic Mobile Element (ACME), suggesting they belong to the USA300 clone. The Inc18 plasmid was the most abundant as it was detected in 72.7% (n = 16/22) of the isolates. Other plasmid replicons detected were the rep_trans and repA_N which were detected in 45.4% (n = 10/22) and 40.9% (n = 9/22) of the isolates respectively. Ten isolates harboured at least three plasmid replicons and no plasmid replicons were detected in four isolates (ST6/t304). The cgMLST revealed that some isolates aggregated into two genomically indistinguishable clusters: ST6/t304 belonging to cluster type CT12405 (≤20 allelic differences) and ST8/t008 belonging to cluster type CT1925 (<8 allelic differences). Overall, we found a high genotypic concordance with the national clinical bacterial resistance data. Our study demonstrates the sensitivity of culture-based wastewater surveillance for MRSA using clinical media following pre-enrichment, reliably predicting pathogen occurrence at the population level.

Comparison of IncK-blaCMY-2 Plasmids in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Isolated from Poultry and Humans in Denmark, Finland, and Germany.

Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0-60 and 13-90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

Wastewater-based surveillance is an efficient monitoring tool for tracking influenza A in the community.

Around the world, influenza A virus has caused severe pandemics, and the risk of future pandemics remains high. Currently, influenza A virus surveillance is based on the clinical diagnosis and reporting of disease cases. In this study, we apply wastewater-based surveillance to monitor the amount of the influenza A virus RNA at the population level. We report the influenza A virus RNA levels in 10 wastewater treatment plant catchment areas covering 40 % of the Finnish population. Altogether, 251 monthly composite influent wastewater samples (collected between February 2021 and February 2023) were analysed from supernatant fraction using influenza A virus specific RT-qPCR method. During the study period, an influenza A virus epidemic occurred in three waves in Finland. This study shows that the influenza A virus RNA can be detected from the supernatant fraction of 24 h composite influent wastewater samples. The influenza A virus RNA gene copy number in wastewater correlated with the number of confirmed disease cases in the Finnish National Infectious Diseases Register. The median Kendall's τ correlation strength was 0.636 (min= 0.486 and max=0.804) and it was statistically significant in all 10 WTTPs. Wastewater-based surveillance of the influenza A virus RNA is an independent from individual testing method and cost-efficiently reflects the circulation of the virus in the entire population. Thus, wastewater monitoring complements the available, but often too sparse, information from individual testing and improves health care and public health preparedness for influenza A virus pandemics.

Clinically relevant sequence types of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae detected in Finnish wastewater in 2021-2022.

BACKGROUND: Antimicrobial resistance (AMR) is a critical threat to human health. Escherichia coli and Klebsiella pneumoniae are clinically the most important species associated with AMR and are the most common carbapenemase-producing (CP) Enterobacterales detected in human specimens in Finland. Wastewater surveillance has emerged as a potential approach for population-level surveillance of AMR, as wastewater could offer a reflection from a larger population with one sample and minimal recognized ethical issues. In this study, we investigated the potential of wastewater surveillance to detect CP E. coli and K. pneumoniae strains similar to those detected in human specimens. METHODS: Altogether, 89 composite samples of untreated community wastewater were collected from 10 wastewater treatment plants across Finland in 2021-2022. CP E. coli and K. pneumoniae were isolated using selective culture media and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed using disk diffusion test and broth microdilution method, and a subset of isolates was characterized using whole-genome sequencing. RESULTS: CP E. coli was detected in 26 (29.2%) and K. pneumoniae in 25 (28.1%) samples. Among E. coli, the most common sequence type (ST) was ST410 (n = 7/26, 26.9%), while ST359 (n = 4/25, 16.0%) predominated among K. pneumoniae. Globally successful STs were detected in both E. coli (ST410, ST1284, ST167, and ST405) and K. pneumoniae (ST512, ST101, and ST307). K. pneumoniae carbapenemases (KPC) were the most common carbapenemases in both E. coli (n = 11/26, 42.3%) and K. pneumoniae (n = 13/25, 52.0%), yet also other carbapenemases, such as blaNDM-5, blaOXA-48, and blaOXA-181, were detected. We detected isolates harboring similar ST and enzyme type combinations previously linked to clusters in Finland, such as E. coli ST410 with blaKPC-2 and K. pneumoniae ST512 with blaKPC-3. CONCLUSIONS: Our study highlights the presence of clinically relevant strains of CP E. coli and K. pneumoniae in community wastewater. The results indicate that wastewater surveillance could serve as a monitoring tool for CP Enterobacterales. However, the specificity and sensitivity of the methods should be improved, and technologies, like advanced sequencing methods, should be utilized to distinguish data with public health relevance, harness the full potential of wastewater surveillance, and implement the data in public health surveillance.

Detection of SARS-COV-2 variants and their proportions in wastewater samples using next-generation sequencing in Finland.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants may have different characteristics, e.g., in transmission, mortality, and the effectiveness of vaccines, indicating the importance of variant detection at the population level. Wastewater-based surveillance of SARS-CoV-2 RNA fragments has been shown to be an effective way to monitor the COVID-19 pandemic at the population level. Wastewater is a complex sample matrix affected by environmental factors and PCR inhibitors, causing insufficient coverage in sequencing, for example. Subsequently, results where part of the genome does not have sufficient coverage are not uncommon. To identify variants and their proportions in wastewater over time, we utilized next-generation sequencing with the ARTIC Network's primer set and bioinformatics pipeline to evaluate the presence of variants in partial genome data. Based on the wastewater data from November 2021 to February 2022, the Delta variant was dominant until mid-December in Helsinki, Finland's capital, and thereafter in late December 2022 Omicron became the most common variant. At the same time, the Omicron variant of SARS-CoV-2 outcompeted the previous Delta variant in Finland in new COVID-19 cases. The SARS-CoV-2 variant findings from wastewater are in agreement with the variant information obtained from the patient samples when visually comparing trends in the sewerage network area. This indicates that the sequencing of wastewater is an effective way to monitor temporal and spatial trends of SARS-CoV-2 variants at the population level.

Developing wastewater-based surveillance schemes for multiple pathogens: The WastPan project in Finland.

Wastewater comprises multiple pathogens and offers a potential for wastewater-based surveillance (WBS) to track the prevalence of communicable diseases. The Finnish WastPan project aimed to establish wastewater-based pandemic preparedness for multiple pathogens (viruses, bacteria, parasites, fungi), including antimicrobial resistance (AMR). This article outlines WastPan's experiences in this project, including the criteria for target selection, sampling locations, frequency, analysis methods, and results communication. Target selection relied on epidemiological and microbiological evidence and practical feasibility. Within the WastPan framework, wastewater samples were collected between 2021 and 2023 from 10 wastewater treatment plants (WWTPs) covering 40 % of Finland's population. WWTP selection was validated for reported cases of Extended Spectrum Beta-lactamase-producing bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) from the National Infectious Disease Register. The workflow included 24-h composite influent samples, with one fraction for culture-based analysis (bacteria and fungi) and the rest of the sample was reserved for molecular analysis (viruses, bacteria, antibiotic resistance genes, and parasites). The reproducibility of the monitoring workflow was assessed for SARS-CoV-2 through inter-laboratory comparisons using the N2 and N1 assays. Identical protocols were applied to same-day samples, yielding similar positivity trends in the two laboratories, but the N2 assay achieved a significantly higher detection rate (Laboratory 1: 91.5 %; Laboratory 2: 87.4 %) than the N1 assay (76.6 %) monitored only in Laboratory 2 (McNemar, p < 0.001 Lab 1, = 0.006 Lab 2). This result indicates that the selection of monitoring primers and assays may impact monitoring sensitivity in WBS. Overall, the current study recommends that the selection of sampling frequencies and population coverage of the monitoring should be based on pathogen-specific epidemiological characteristics. For example, pathogens that are stable over time may need less frequent annual sampling, while those that are occurring across regions may require reduced sample coverage. Here, WastPan successfully piloted WBS for monitoring multiple pathogens, highlighting the significance of one-litre community composite wastewater samples for assessing community health. The infrastructure established for COVID-19 WBS is valuable for monitoring various pathogens. The prioritization of the monitoring targets optimizes resource utilization. In the future legislative support in target selection, coverage determination, and sustained funding for WBS is recomended.

Prevalence of AmpC, ESBL, and colistin resistance genes in Enterobacterales isolated from ready-to-eat food in Algeria.

Antimicrobial resistance among bacteria present in ready-to-eat foods is an emerging concern. Hence, this study investigated the presence of extended-spectrum and AmpC ß-lactamases (ESBL/AmpC)-producing Enterobacterales (ESBL-E) and the dissemination of mcr-1 in ESBL-E from ready-to-eat food samples (RTE) in Algeria. RTE food samples (n = 204) were aseptically collected and selectively cultured using MacConkey agar. The isolates were screened for ESBL production using the DDST test, confirmed ESBL-E isolates were identified using different conventional methods and MALDI-TOF MS, antibiotic susceptibility was determined using the disc diffusion and broth microdilution assay, ESBL-E isolates were analyzed for colistin and ESBL/AmpC encoding genes by PCR, and food samples were analyzed by univariate and multiple logistic regression. Overall, 48 (17.4%) of the 276 Enterobacterales were confirmed as ESBL producers, with a high prevalence in soups (40%), salads (25%), and cream-filled pastries (23.8%). Antibiotic susceptibility testing revealed that all the ESBL-E isolates were found multi-drug resistant. PCR revealed that blaTEM, blaCTX-M, blaCMY-2, blaOXA-1, and blaSHV were the most frequently detected. blaCTX-M-9 and blaCTX-M-1 were the predominant CTX-M types. Furthermore, four isolates were positive for mcr-1; three of them harbored the colistin resistance gene and ESBL/AmpC genes (2 E. cloacae and 1 S. enterica). To the best of our knowledge, this is the first report that detects the presence of the mcr-1 gene in ESBL-E strains isolated from RTE foods in Algeria. These findings suggest an urgent need for strict policies that prevent the spread and transmission of ESBL-E in food.

Phenotypic and genotypic characteristics of beta-lactamase dominant with CARBA, AmpC, and ESBL-producing bacteria in municipal wastewater influent in Helsinki, Finland.

OBJECTIVES: Analysing samples of municipal wastewater influent (before treatment) can help to map the status of antibiotic-resistant bacteria (ARB) at the population level in sewershed communities and may also help in predicting the public health risks of ARB in surface water because of the outfall of wastewater. In this study, we investigated the bacterial isolates carrying beta-lactamase genes in wastewater and compared their genotypic and phenotypic characteristics. METHODS: A total of 399 bacterial isolates grown on CHROMagarESBL (n = 207) and CHROMagarKPC (n = 192) from composite wastewater influent samples (n = 7) from the Viikinmäki wastewater treatment plant (Helsinki) were subcultured, nucleic acid was extracted, and the prevalence of different beta-lactamase genes was screened with multiplex polymerase chain reaction (PCR). All PCR-positive isolates were identified with MALDI-TOF. RESULTS: A total of 32.6% of isolates (130 of 399) were PCR positive for at least one resistance gene, and 13% of these positive isolates out of 130 had at least three resistance genes. Among the 22 detected genes, blaGES group was the most prevalent, at 25.8% (n = 198; many isolates carried multiple genes), followed by blaMOX (13.1%) and blaTEM (10.1%) as most frequently detected. Furthermore, out of 18 different bacterial species/genera detected as carrying beta-lactamase genes, A. hydrophila/caviae (28.5%), Enterobacter spp. (16.9%), and E. coli (14.6%) were the most prevalent. Enterobacter spp., Aeromonas spp., and K. cryocescens potentially carried AmpC genes, and E. coli carried ESBL genes. CONCLUSION: We recorded a huge variety of beta-lactamases (blaAmpC, blaESBL, and blaCARBA) genes in many potential pathogens that probably originated from both enteric and environmental sources.

Antibiotic resistance genes, mobile elements, virulence genes, and phages in cultivated ESBL-producing Escherichia coli of poultry origin in Kwara State, North Central Nigeria.

The paucity of information on the genomic diversity of drug-resistant bacteria in most food-producing animals, including poultry in Nigeria, has led to poor hazard characterization and the lack of critical control points to safeguard public health. Hence, this study used whole genome sequencing (WGS) to assess the presence and the diversity of antibiotic resistance genes, mobile genetic elements, virulence genes, and phages in Extended Spectrum Beta Lactamase producing Escherichia coli (ESBL - E. coli) isolates obtained from poultry via the EURL guideline of 2017 in Ilorin, Nigeria. The prevalence of ESBL - E. coli in poultry was 10.5 % (n = 37/354). The phenotypic antibiotic susceptibility testing showed that all the ESBL- E. coli isolates were multi-drug resistant (MDR). The in-silico analysis of the WGS raw-read data from 11 purposively selected isolates showed that the isolates had a wide array of ARGs that conferred resistance to beta-lactam antibiotics, and 8 other classes of antibiotics (fluoroquinolones, foliate pathway antagonists, aminoglycoside, phenicol, tetracycline, epoxide, macrolides, and rifamycin). All the ARGs were in the bacterial chromosome except in two isolates where plasmid-mediated quinolone resistance (PMQR) was detected. Two isolates carried the gyrAp.S83L mutation which confers resistance to certain fluoroquinolones. The mobilome consisted of several Col-plasmids and the predominant IncF plasmids belonged to the IncF64:A-:B27 sequence type. The virulome consisted of genes that function as adhesins, iron acquisition genes, toxins, and protectins. Intact phages were found in 8 of the 11 isolates and the phageome consisted of representatives of four families of viruses: Myoviridae (62.5 %, n = 5/8), Siphoviridae (37.5 %, n = 3/8), Inoviridae (12.5 %, n = 1), and Podoviridae (12.5 %, n = 1/8). ESBL - E. coli isolates harboured 1-5 intact phages and no ARGs were identified on any of the phages. Although five of the isolates belonged to phylogroup A, the isolates were diverse as they belonged to different serotype and sequence types. Our findings demonstrate the high genomic diversity of ESBL - E. coli of poultry origin in Ilorin, Nigeria. These diverse isolates harbor clinically relevant ARGs, mobile elements, virulence genes, and phages that may have detrimental zoonotic potentials on human health.

Genomic epidemiology of nosocomial carbapenemase-producing Citrobacter freundii in sewerage systems in the Helsinki metropolitan area, Finland.

Multi-drug resistance is emerging in Citrobacter freundii, which is the third most common carbapenemase-producing (CP) Enterobacteriaceae in humans in Finland due to recent outbreaks. The objective of this study was to determine if wastewater surveillance (WWS) could detect CP C. freundii strains causing infections in humans. Selective culturing was used to isolate CP C. freundii from the hospital environment, hospital wastewater, and untreated municipal wastewater in Helsinki, Finland, between 2019 and 2022. Species were identified using MALDI-TOF, and presumptive CP C. freundii isolates were subjected to antimicrobial susceptibility testing and further characterized by whole genome sequencing. A genomic comparison was conducted to compare isolates collected from the hospital environment, untreated municipal wastewater, and a selection of isolates from human specimens from two hospitals in the same city. We also examined the persistence of CP C. freundii in the hospital environment and the impact of our attempts to eradicate it. Overall, 27 blaKPC - 2-carrying C. freundii were detected in the hospital environment (ST18; n = 23 and ST8; n = 4), while 13 blaKPC - 2-carrying C. freundii (ST8) and five blaVIM - 1-carrying (ST421) C. freundii were identified in untreated municipal wastewater. CP C. freundii was not identified in hospital wastewater. We found three clusters (cluster distance threshold ≤ 10 allelic difference) after comparing the recovered isolates and a selection of isolates from human specimens. The first cluster consisted of ST18 isolates from the hospital environment (n = 23) and human specimens (n = 4), the second consisted of ST8 isolates from the hospital environment (n = 4), untreated municipal wastewater (n = 6), and human specimens (n = 2), and the third consisted of ST421 isolates from the untreated municipal wastewater (n = 5). Our results support previous studies suggesting that the hospital environment could act as a source of transmission of CP C. freundii in clinical settings. Furthermore, the eradication of CP Enterobacteriaceae from the hospital environment is challenging. Our findings also showed that CP C. freundii is persistent throughout the sewerage system and demonstrate the potential of WWS for detecting CP C. freundii.

Monkeypox outbreak: Wastewater and environmental surveillance perspective.

Monkeypox disease (MPXD), a viral disease caused by the monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022; 92 % (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS) has been extensively utilized to monitor communicable diseases, particularly during the ongoing COVID-19 pandemic. It helped in monitoring infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in lesion materials (e.g. skin, vesicle fluid, crusts), skin rashes, and various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of the MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.

Towards monitoring of antimicrobial resistance in the environment: For what reasons, how to implement it, and what are the data needs?

Antimicrobial resistance (AMR) is a global threat to human and animal health and well-being. To understand AMR dynamics, it is important to monitor resistant bacteria and resistance genes in all relevant settings. However, while monitoring of AMR has been implemented in clinical and veterinary settings, comprehensive monitoring of AMR in the environment is almost completely lacking. Yet, the environmental dimension of AMR is critical for understanding the dissemination routes and selection of resistant microorganisms, as well as the human health risks related to environmental AMR. Here, we outline important knowledge gaps that impede implementation of environmental AMR monitoring. These include lack of knowledge of the 'normal' background levels of environmental AMR, definition of high-risk environments for transmission, and a poor understanding of the concentrations of antibiotics and other chemical agents that promote resistance selection. Furthermore, there is a lack of methods to detect resistance genes that are not already circulating among pathogens. We conclude that these knowledge gaps need to be addressed before routine monitoring for AMR in the environment can be implemented on a large scale. Yet, AMR monitoring data bridging different sectors is needed in order to fill these knowledge gaps, which means that some level of national, regional and global AMR surveillance in the environment must happen even without all scientific questions answered. With the possibilities opened up by rapidly advancing technologies, it is time to fill these knowledge gaps. Doing so will allow for specific actions against environmental AMR development and spread to pathogens and thereby safeguard the health and wellbeing of humans and animals.

Genomic characterization of ESBL/AmpC-producing and high-risk clonal lineages of Escherichia coli and Klebsiella pneumoniae in imported dogs with shelter and stray background.

OBJECTIVES: Extended spectrum ß-lactamase (ESBL)- and ampicillinase C (AmpC)-carrying Enterobacteriaceae have been widely reported among companion animals. According to previous studies, dogs with a shelter or stray background might be at risk of carrying such bacteria. The aim of this study was to explore, with whole-genome sequencing (WGS), the genomic characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from imported dogs with a stray or shelter background. METHODS: E. coli (n = 58) and K. pneumoniae (n = 2) isolates from imported dogs originating from seven countries were included. Phenotypic resistance was investigated by selective isolation and antibiotic susceptibility testing. Whole-genome sequencing was used to study the genomic characteristics and the presence of antimicrobial resistance genes (ARGs) and virulence determinants of the ESBL/AmpC-producing E. coli and K. pneumoniae isolates. RESULTS: A high diversity of different ARGs (n = 56) and sequence types (STs) (n = 32), including high-risk clonal lineages ST410 (n = 3) and ST307 (n = 1), was identified in E. coli and K. pneumoniae isolates, respectively. Genes encoding resistance to ß-lactams accounted for the majority, with the most frequent being blaCTX-M-15. Moreover, 17 (29%) E. coli isolates qualified as presumptive extraintestinal pathogenic and/or uropathogenic E. coli. CONCLUSIONS: Our results highlight the multiplicity of genetic backgrounds disseminating ESBL/AmpC-genes in the studied dogs, calling for further investigation of possible drivers responsible for the dissemination of ARGs in animal shelters and amongst stray dogs. From a public health perspective, enhanced genomic surveillance of ESBL/AmpC-producing Enterobacteriaceae in dogs is needed in Finland.