Friederike Hilbert

Background/Objectives: Microbiomes surrounding mining sites have been found to harbor both antibiotic resistance genes and metal resistance genes. Within the “One Health” framework, which spans human, veterinary and environmental health, it is crucial to determine whether bacterial metal resistance (MR) genes can independently trigger antimicrobial resistance (AMR) or if they are linked to AMR genes and co-transferred horizontally. Methods and Results: Bacteria were isolated from an active and an inactive mining site in the alpine region of Austria. Most of the isolated bacteria harbored antimicrobial and metal resistance genes (88%). MALDI-TOF and whole genome sequencing (WGS) revealed that species from the Pseudomonadaceae family were the most identified, accounting for 32.5%. All Pseudomonas spp. carried AMR genes from the mex family, which encode multidrug efflux pumps. β-lactamase production encoded by bla genes were detected as the second most common (26%). The same AMR genes have often been detected within a particular bacterial genus. No tetracycline resistance gene has been identified. Among metal resistance genes, rufB (tellurium resistance) was the most prevalent (33%), followed by recGM (selenium resistance, 30%), copA (copper resistance, 26%), and mgtA (magnesium and cobalt resistance, 26%). Notably, the mer gene family (mercury resistance) was found exclusively in isolates from the inactive mining site (n = 6). In addition, genes associated with both antimicrobial and metal resistance, including arsBM, acrD, and the mer operon, were identified in 19 out of the 43 isolates. Conclusions: Bacteria isolated from mine water harbored both MR and AMR genes. Given the exceptional diversity of bacterial species in these settings, 16S rRNA gene sequence analysis is the recommended method for accurate species identification. Moreover, the presence of multi-drug transporters and transferable resistance genes against critically important antimicrobials such as fluoroquinolones and colistin identified in these environmental bacteria emphasizes the importance of retrieving environmental data within the “One Health” framework.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre‐evaluation of the safety of microorganisms intended for use in the food or feed chains. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by ‘qualifications’ which should be assessed at strain and/or product level by EFSA's Scientific Panels. The generic qualification ‘the strains should not harbour any acquired antimicrobial resistance (AMR) genes to clinically relevant antimicrobials’ applies to all QPS bacterial TUs. The different EFSA risk assessment areas use the same approach to assess the qualification related to AMR genes. In this statement, the terms ‘intrinsic’ and ‘acquired’ AMR genes were defined for the purpose of EFSA's risk assessments, and they apply to bacteria used in the food and feed chains. A bioinformatic approach is proposed for demonstrating the ‘intrinsic’/’acquired’ nature of an AMR gene. All AMR genes that confer resistance towards ‘critically important’, ‘highly important’ and ‘important’ antimicrobials, as defined by the World Health Organisation (WHO), found as hits, need to be considered as hazards (for humans, animals and environment) and need further assessment. Genes identified as responsible for ‘intrinsic’ resistance could be considered as being of no concern in the frame of the EFSA risk assessment. ‘Acquired’ AMR genes resulting in a resistant phenotype should be considered as a concern. If the presence of the ‘acquired’ AMR gene is not leading to phenotypic resistance, further case‐by‐case assessment is necessary.

Salmonella enterica subsp. enterica is a zoonotic bacterial pathogen that causes foodborne outbreaks in humans. Lytic bacteriophages to control Salmonella in food production are already being used in scientific studies and some are commercially available. However, phage application is still controversial. In addition to virulent phages, which are used in phage therapy and lyse the bacterial host, lysogenic phages coexist in the environment and can reside as prophages in the bacterial host. Therefore, information about Salmonella prophages is essential to understand successful phage therapy. In 100 Salmonella food isolates of the serovars Enteritidis and Typhimurium, we propagated prophages by oxidative stress. In isolates of the serovars Typhimurium and Enteritidis, 80% and 8% prophages could be activated, respectively. In the phage lysates from the serovar Typhimurium, the following antibiotic resistance genes or gene fragments were detected by PCR: sul1, sul2, bla_TEM, strA and cmlA; however, no tetA,B,C, bla_OXA, bla_CMY, aadA1, dfr1,2 or cat were detected. In contrast, no resistance genes were amplified in the phage lysates of the serovar Enteritidis. None of the phage lysates was able to transduce phenotypic resistance to WT 14028s. Most of the prophage lysates isolated were able to infect the various Salmonella serovars tested. The high abundance of prophages in the genome of the serovar Typhimurium may counteract phage therapy through phage resistance and the development of hybrid phages.

American foulbrood is caused by the spore-forming Paenibacillus larvae. Although the disease effects honey bee larvae, it threatens the entire colony. Clinical signs of the disease are seen at a very late stage of the disease and bee colonies are often beyond saving. Therefore, through active monitoring based on screening, an infection can be detected early and bee colonies can be protected with hygiene measures. As a result, the pressure to spread in an area remains low. The cultural and molecular biological detection of P. larvae is usually preceded by spore germination before detection. In this study, we compared the results of two methods, the culture detection and RT-PCR detection of DNA directly isolated from spores. Samples of honey and cells with honey surrounding the brood were used in a five-year voluntary monitoring program in a western part of Lower Austria. DNA-extraction from spores to speed up detection involved one chemical and two enzymatic steps before mechanical bashing-beat separation and additional lysis. The results are comparable to culture-based methods, but with a large time advantage. Within the voluntary monitoring program, the proportion of bee colonies without the detection of P. larvae was high (2018: 91.9%, 2019: 72.09%, 2020: 74.6%, 2021: 81.35%, 2022: 84.5%), and in most P. larvae-positive bee colonies, only a very low spore content was detected. Nevertheless, two bee colonies in one apiary with clinical signs of disease had to be eradicated.

Campylobacter (C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the “One Health” approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a β-lactamase resistance gene bla_OXA-184, a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments.

Escherichia coli isolated from meat of different animal species may harbour antimicrobial resistance genes and may thus be a threat to human health. The objectives of this study were to define antimicrobial resistance genes in E. coli isolates from pork, beef, chicken- and turkey meat and analyse whether their resistance genotypes associated with phylogenetic groups or meat species. A total number of 313 E. coli samples were isolated using standard cultural techniques. In 98% of resistant isolates, a dedicated resistance gene could be identified by PCR. Resistance genes detected were tet(A) and tet(B) for tetracycline resistance, strA and aadA1 for streptomycin resistance, sulI and sulII for resistance against sulphonamides, dfr and aphA for kanamycin resistance and bla_TEM for ampicillin resistance. One stx1 harbouring E. coli isolated from pork harboured the tet(A) gene and belonged to phylogenetic group B2, whilst another stx1 positive isolate from beef was multi-resistant and tested positive for bla_TEM,aphA, strA–B, sulII, and tet(A) and belonged to phylogenetic group A. In conclusion, the distribution of resistance elements was almost identical and statistically indifferent in isolates of different meat species. Phylogenetic groups did not associate with the distribution of resistance genes and a rather low number of diverse resistance genes were detected. Most E. coli populations with different resistance genes against one drug often revealed statistically significant different MIC values.

Antimicrobial resistance is on the rise in certain pathogens that infect pets and their owners. This has raised concerns about the use of antibiotics and the transfer of resistance elements in small animal clinics. We sampled a surgery unit, diagnostic rooms after disinfection, and a dog with chronic urinary tract infection (UTI), in a small animal clinic in Austria, and isolated/characterized phages and Escherichia (E.) coli for antimicrobial resistance, resistance genes and transduction ability. Neither the coliphages nor E. coli were isolated in the 20 samples of the surgery units and diagnostic rooms. From the urinary tract of the dog, we recovered 57 E. coli isolates and 60 coliphages. All of the E. coli isolates were determined as resistant against nalidixic acid, 47 against ampicillin, 34 against sulfonamides, and 33 against streptomycin. No isolate held resistance against tetracycline, trimethoprim, kanamycin, or chloramphenicol. Among the 60 phages, 29 tested positive for one or more resistance gene(s) by PCR, but none was able to transduce it to a laboratory strain or to an E. coli isolated from samples. Nevertheless, six phages out of 60 were able to transduce ampicillin resistance (bla gene) after being grown on a puc19 harboring E. coli strain.

The qualified presumption of safety (QPS) was developed to provide a safety pre‐assessment within EFSA for microorganisms. Strains belonging to QPS taxonomic units (TUs) still require an assessment b...

Qualified presumption of safety (QPS) was developed to provide a generic safety evaluation for biological agents to support EFSA's Scientific Panels. The taxonomic identity, body of knowledge, safety...