Abstract
Despite advances in dairy husbandry, mastitis-associated Escherichia (E.) coli (MAEC) remains a primary causative agent of bovine mastitis, contributing significantly to economic losses in the cattle industry. This study characterized 50 E. coli isolates from severe, moderate, mild, and subclinical mastitis by antimicrobial susceptibility testing and genotypic analysis, including PCR, DNA microarray, E. coli phylotyping and whole-genome sequencing of selected strains. Antimicrobial resistance rates were highest for ampicillin (30%), tetracycline (20%), amoxicillin-clavulanate (20%), and trimethoprim-sulfamethoxazole (14%); The majority of isolates (66%) was susceptible to antibiotics. One isolate displayed an ESBL phenotype, carrying blaCTX-M-3 and blaTEM-1 genes. Phylogenetic analysis indicated that members of phylogroups B1 (50%) and A (16%) are predominant among the isolates, followed by group C (12%), with the remaining isolates belonging to groups D, E, and F, and two unassigned. The most prevalent virulence-associated genes detected were fimH (100%), and genes of the ferric dicitrate uptake locus (fecIRABCDE). Overall, MAEC isolates exhibited diverse and heterogeneous genetic profiles. The proliferation of ESBL-producing E. coli poses a threat within a One Health framework, given the risk of multidrug-resistant strains at the human-animal-environment interface, compromising critical antimicrobials in both veterinary and human medicine.
Keywords: bovine mastitis, E. coli, MAEC, microarray-based diagnostics, resistance genes, virulence genes, whole genome sequencing (WGS)
1. Introduction
Mastitis is a leading cause of economic loss and health problems in dairy cattle and is a major reason for early culling (1). Antimicrobial treatment is often required to maintain udder health, animal welfare, and economic stability. However, the use of antimicrobials in food-producing livestock is a critically discussed issue due to the increasing development of antimicrobial resistance (2, 3).
Escherichia (E.) coli is an important and prevalent cause of environmental mastitis (4), which can cause intramammary infections ranging from severe but temporary inflammation to subclinical or mild clinical infections that are more chronic (1). E. coli strains are categorized as intestinal pathogenic (InPEC) or extraintestinal pathogenic (ExPEC), depending on their ability to colonize different anatomic sites (5, 6). E. coli isolates from bovine mastitis belong to the ExPEC group, which are part of the intestinal microflora where they colonize asymptomatically the gut. However, if they reach niches outside, they can act as pathogens. Some ExPEC are considered to share characteristic virulence factors and are therefore grouped into pathotypes (7, 8). In recent studies, molecular characterization of bovine mastitis-associated E. coli strains did not identify any specific virulence factors clearly associated with bovine mastitis (6, 8). The main virulence factors that have been identified so far in mastitis-associated E. coli strains include genes encoding adhesins/fimbriae (fimH, fimA, sfaDE, papC, afaBcIII, iha), invasins (ibe), toxins/hemolysins (hlyA, vat, cnf, cdt), and mechanisms for serum resistance (kpsM), which participates in the biosynthesis of group 2 and group 3 capsules, and siderophores (iroN, iucD, iutA) (9, 10).
The common feature of MAEC is rather an enrichment in fitness capabilities for survival and rapid adaptation in the mammary gland, which are referred to as intramammary ecotypes and is considered as mastitis-associated E. coli (6). The E. coli strains have a great intraspecific diversity and are classified into eight phylogenetic groups (A, B1, B2, C, D, E, F, and clade) (11, 12). The majority of mastitis-associated E. coli strains are clustered in group A and B1 (6). Regarding antimicrobial resistance, prevalence in mastitis strains is strongly influenced by strain origin and therefore varies considerably (6). Antimicrobial resistance is a significant threat to global food security, human health, and the future of livestock production. Overall, evidence indicates that antimicrobial resistance in mastitis-associated E. coli is a reactive process in response to antibiotic use (13). Thus, monitoring the resistance situation and the use of antibiotic agents in the dairy industry are essential. Changes in resistance patterns can occur unpredictably and pose a challenge for the entire ecosystem (14). Although previous studies have identified virulence-associated genes in MAEC, there is a notable research gap concerning these genes in Austria. The present study aims to characterize a collection of MAEC isolates from various locations within Austria. This study presents the first characterization of Austrian MAEC isolates and employs a One Health approach to address the interconnected risks these pathogens pose to animal welfare, economic stability, and public health.
2. Materials and methods
2.1. E. coli isolates
Fifty E. coli isolates were recovered from subclinical and clinical mastitis cases in dairy cows, using aseptically collected quarter milk samples. A mastitis case was defined as subclinical if milk was macroscopically normal, accompanied by an elevated somatic cell count. Clinical mastitis cases were defined as mild (Severity Score 1), if only milk secretions were abnormal. Cases with alterations of the milk and local inflammatory reactions (e.g., swelling of the affected quarter) defined a moderate (Score 2); anamnestic reports of systemic inflammatory reactions (e.g., fever) were defined as severe (Score 3) clinical mastitis, and subclinical mastitis (Score 4) (15). Isolates were identified as presumptive E. coli based on colony characteristics, and species identification was performed with MALDI-TOF mass spectrometry (Bruker Daltonik, Heidelberg, Germany). Isolates were stored at −80 °C for further analyses.
2.2. Antimicrobial susceptibility testing
Antimicrobial susceptibility was determined by the agar disk diffusion method (16), using the following agents: ampicillin (10 μg), amoxicillin-clavulanate (20/10 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefoxitin (30 μg), meropenem (10 μg), gentamicin (10 μg), tobramycin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), tetracycline (30 μg), chloramphenicol (30 μg), fosfomycin (200 μg), and nitrofurantoin (300 μg) (all from Becton Dickinson, Heidelberg, Germany). Escherichia coli ATCC® 25922™ was used as a quality control strain. Extended-spectrum beta-lactamase (ESBL) production was assessed using combination disk tests with cefotaxime and ceftazidime (with or without clavulanic acid; Becton, Dickinson, Heidelberg, Germany). AmpC beta-lactamase production was screened using cefoxitin (30 μg).
2.3. Molecular characterization
All isolates underwent characterized as follows. E. coli phylogroups were determined as previously described (11, 12). A set of virulence-associated genes (Table 1) was determined using a custom-made DNA microarray-based technology from INTER-ARRAY (INTER-ARRAY by fzmb GmbH, Bad Langensalza, Germany) (17, 18). In addition, the following genes were screened via PCRs: sfaDE, ihA, and afaBC III (19, 20). The genes fecI and fecE of fec locus (fecIRABCDE), usually present in MAEC, were screened using primers designed during the present study (fecI primer: fecI-fwd: 5´-ATCACGGCTGGTTGAAAAG-3′ and fecI-rev: 5´-CAATCTCGCTGTATGTCAG-3′ (381-bp amplicon), and fecE primer: fecE-fwd: 5´-TGCCTCAGCACCATTTAAC-3′ and fecE-rev: 5´-CATTACCACCAGTTGATCG-3′ (392-bp amplicon)). Twenty E. coli isolates were selected for whole-genome sequencing according to clinical presentation (mostly severe clinical mastitis with fever, sometimes accompanied by gastrointestinal disorders) and resistance profile. Genomic DNA isolation, whole-genome sequencing, and subsequent genome assembly was conducted as described previously (21). Strain typing and serogenotype prediction were done in SeqSphere+ (Ridom, Münster, Germany), while phylogroups were assigned with the Clermont Typing tool (http://clermontyping.iame-research.center/, accessed on 2025 November 30) (22). Sequence types (STs) were identified in EnteroBase (23). SeqSphere + software (Ridom, Münster, Germany) was used for core-genome multilocus sequence typing (cgMLST) analysis. Resistance/virulence genes were identified using ABRicate (24) with ResFinder (25), Comprehensive Antibiotic Resistance Database CARD (26), and Virulence Factor Database (27). Additional gene prediction and annotation were performed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (28).
Table 1.
A set of virulence-associated genes tested using DNA-microarray.
| Virulence factor | Gene | Function |
|---|---|---|
| paa | acfC | Porcine attaching-effacing associated protein Paa/adherence factor adfo |
| aidA | aidA | Responsible for bacterial autoaggregation |
| bfpB | bfpB | Bundle forming pilus B |
| escV | escV | Type III secrection inner membrane protein |
| ent | espL | Ent protein |
| fimH | fimH | Typ I fimbrial protein |
| F5 | fimK99/fanC | Fimbrial protein |
| exhA | hlyA-var1 | Haemolysin |
| hlyA | hlyA-var2 | Haemolysin |
| iucD | iucD | Aerobactin biosynthesis protein |
| papC | papC | Outer membrane usher P fimbriae |
| invE | virB | Enteroinvasive protein |
| aggR | virF-aggR | Regulator of enteroaggregative E. Coli |
| astA | astA | Heat-resistant agglutinin, EAST |
| bfpA | bfpA | Bundle forming pilus A |
| cnf1 | cnf | Cytotoxic necrotizing factor |
| eaeA | eae | Intimin for attaching and effacing |
| F17 | f17aG | Fimbrial protein for adhesion |
| F6 | fasA | Fimbrial protein for adhesion |
| F18 | fedA | Fimbrial protein for adhesion |
| F41 | fim-41a | Fimbrial protein for adhesion |
| F4 | fim-K88ab/faeG | Fimbrial protein for adhesion |
| gad | gad | Transcriptional regulator, important in acid environment |
| elt, LT (eltA) | ltcA-var1 | Heat-labile enterotoxin A subunit |
| pic | pic | Serine protease of enteroaggregative E. Coli |
| estIa | sta1 | Heat-stabile enterotoxin ST-Ia |
| estIb | sta2 | Heat-stabile enterotoxin II |
| stx1 | stx1 | Shigatoxin |
| stx2 | stx2 | Shigatoxin |
| Stx2e | stx2e | Shigatoxin |
3. Results
3.1. Mastitis score
All 50 isolates were confirmed as E. coli by MALDI TOF MS. The majority of isolates (n = 39) originated from severe mastitis cases (Score 3), while the remaining eleven originated from subclinical (n = 3), mild (Score 1, n = 3), or moderate (Score 2, n = 5).
3.2. Antimicrobial susceptibility testing
All 50 E. coli isolates were susceptible to carbapenems, fosfomycin, and amikacin. Seventeen (34%) displayed resistance to at least one antimicrobial, and seven (14%) were classified as multidrug-resistant (29). No isolate showed AmpC beta-lactamase production, and one isolate (P22) exhibited an ESBL phenotype. Overall, 33 of 50 isolates (66%) were fully susceptible to all tested antimicrobials. Antimicrobial resistance rates were highest for ampicillin (30%), tetracycline (20%), amoxicillin-clavulanate (20%), and trimethoprim-sulfamethoxazole (14%). Tables 2 and 3 summarize these results.
Table 2.
Phenotypic and genotypic characterization of E. coli isolates from bovine mastitis using whole-genome sequencing.
| Sample number | Mastitis score | Phylogroup | Sequence type | Serotype | ResistancePhenotyp1 | Resistance genotype | Virulence genes | Mutations QRDR2 GyrA | Mutations QRDR | Mutations QRDR | Biocide resistance genes |
|---|---|---|---|---|---|---|---|---|---|---|---|
| ParC | ParE | ||||||||||
| P1 | 4 | B1 | 2,598 | O175: H16 | fim Operon, | ||||||
| fecIRABCDE | |||||||||||
| P3 | 3 | B1 | 1,125 | ONT: H19 | NR | fim Operon astA, hlyA-var2, fecIRABCDE | |||||
| P5 | 3 | C | 410 | ONT: H9 | AMP, AMC, FQR, TET, CHL, SXT | blaOXA-1, aadA, aadA5, tet(A), floR, sul1, sul2, dfrA17, dfrA36 | fim Operon, astA,. papC1, papC2, incD1, iucD2, fecIRABCDE, sitABCD | gyrA D87N, gyrA S83L | parCS80I | parE | qacE |
| S458A | |||||||||||
| P7 | 3 | B1 | 18,185 | ONT: H21 | NR | fim Operon, cnf1, f17, papC1, papC2, hlyA-var2, incD1, iucD2, iha, cdt, iutA, fecIRABCDE | |||||
| P10 | 3 | D | 69 | O15: H18 | AMP, TET | blaTEM-1, | fim Operon, fecIRABCDE sitABCD | ||||
| tet(A) | |||||||||||
| P12 | 4 | B1 | 1,396 | ONT: H7 | NR | fim Operon, fecI, fecIRAB | |||||
| P14 | 3 | F | 117 | ONT: H4 | AMP, AMC, TET | blaTEM-1, aph(3″)-Ib, aph(6)-Id, tet(A), sul2 |
fim Operon, astA, pic, papC1, papC2, iucD1, iucD2, vat, iroN, fecIRABCDE, sitABCD |
gyrA S83L | D | ||
| P17 | 3 | A | 744 | O101: H9 | AMP, AMC, FQR, TET, CHL, SXT | blaOXA-1, aph(3′)-Ia, |
fim Operon, papC1, papC2, iucD2, iha, fecIRABCDE, sitABCD |
gyrA D87N, gyrA S83L | parCA56T, parCS80I | D, qacE | |
| aadA, aph(3″)-Ib, aph(6)-Id, tet(B), catA1, floR, sul1, | |||||||||||
| dfrA36 | |||||||||||
| P20 | 1 | E | 1,508 | ONT: H45 | NR | fim Operon, fecIRABCDE | |||||
| P22 | 3 | C | 88 | O8: H25 | ESBL, GEN, TET, SXT | blaCTX-M-3, blaTEM-1, aac(3)-IId, aph(3′)-Ia, aadA1, aadA5, tet(B), | fim Operon, astA, papC1, papC2 afaA-VIII/afaA-VII, afaD-VIII, afaE-VIII, fecIRABCDE, sitABCD | , qacE | |||
| sul1, dfrA17 | |||||||||||
| P27 | 3 | B2 | 547 | ONT: H5 | NR | fim Operon, vat, iroN, ibe, kpsM, kpsFEDUCS, fecI, fecE, | |||||
| P28 | 3 | A | 10 | ONT: H16 | NR | fim Operon, astA, cnf1, papC1, papC2, hlyA-var2, iucD1, iucD2, iha, cdt, afaA-VIII/afaA-VII, afaD-VIII, afaE-VIII, iutA, fecIRABCDE | |||||
| P31 | 3 | B1 | 58 | ONT: H25 | AMP, AMC, GEN, TOB, TET, CHL, SXT | blaTEM-1, ant(2″)-Ia |
fim Operon, cnf1, papC1, papC2, hlyA-var2, iucD1, iucD2, iroN, fecIRABCDE, sitABCD |
, qacE | |||
| ant(2″)-Ia, aadA1, aph(3″)-Ib, aph(6)-Id | |||||||||||
| ant(2″)-Ia, tet(A), | |||||||||||
| floR, sul1, sul2, dfrA5 | |||||||||||
| P34 | 1 | B2 | 73 | ONT: H1 | NR |
fim Operon, cnf1, pic, hlyA-var2, sfaDE, vat, iroN, kpsM, kpsFEDUCS, sfa, set, foc, fecIRABCDE, sitABCD |
|||||
| P35 | 2 | B1 | 3,234 | ONT: H8 | AMP | astA, fim Operon, K88ab (F4), fecIRABCDE | |||||
| P37 | 3 | B1 | 446 | O88: H8 | NR | fim Operon, f17, fecIRA | |||||
| P40 | 3 | B1 | 2,521 | ONT: H7 | NR | fim Operon, acfC, fecIRA | |||||
| P42 | 3 | B1 | 58 | ONT: H16 | NR | fim Operon, f17, afaD-VIII, afaE-VIII, fecIRABCDE | |||||
| P44 | 3 | A | 18,186 | O1: H21 | NR | fim Operon, aidA, fecIRABCDE | |||||
| P49 | 1 | B1 | 10 | ONT: H32 | NR |
fim Operon, fecIRABCDE, sitABCD |
1AMC, amoxicillin and clavulanate; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CFZ, cefazolin; CTX, cefotaxime; FOF, fosfomycin; GEN, gentamicin; PIP, piperacillin; SXT, trimethoprim & sulfamethoxazole; TET, tetracycline; TOB, tobramycin; NR, not resistant; 2QRDR: quinolone-resistanc-determining region.
Table 3.
Phenotypic and genotypic characterization of E. coli isolates from bovine mastitis using Microarray technology and PCRs.
| Sample number | Mastitis score | Phylotype | Resistance phenotyp1 | Virulence genes |
|---|---|---|---|---|
| P2 | 3 | NT | NR | astA, fimH1, fimH2, hlyA-var2, fecI, fecE |
| P4 | 3 | D | AMP, TET | fimH1, fimH2, iha, cnf1, hlyA-var2, fecI, fecE |
| P6 | 3 | B1 | NR | fimH1, fimH2, hlyA-var2, fecI, fecE |
| P8 | 3 | A | NR | fimH1, fimH2, astA, fecI, fecE |
| P9 | 3 | B1 | NR | fimH1, fimH2, fecI |
| P11 | 3 | E | NR | fimH1, fimH2, fecI, fecE |
| P13 | 3 | C | AMP, AMC, FQR, TET, CHL, SXT | fimH1, fimH2, astA, iha, papC1, papC2, iucD1, iucD2, fecI, fecE |
| P15 | 3 | C | AMP, AMC, FQR, TET, CHL, SXT | fimH1, fimH2, fecI, fecE |
| P16 | 3 | A | NR | fimH1, fimH2, fecI, fecE |
| P18 | 3 | B1 | NR | fimH1, fecI, fecE |
| P19 | 3 | B1 | NR | fimH1, fecI, fecE |
| P21 | 3 | A | NR | fimH1, fecI, fecE |
| P23 | 3 | B1 | AMP | fimH1, fecI |
| P24 | 3 | B2 | NR | fimH1, fecI, fecE |
| P25 | 3 | B1 | AMP | fimH1, fimH2, astA, hlyA-var2, fecI, fecE |
| P26 | 3 | C | NR | fimH1, fimH2, fecI, fecE |
| P29 | 3 | C | AMP, AMC, FQR, TET, CHL, SXT | fimH1, fimH2, astA, iha, papC1, papC2, iucD1, iucD2, fecI, fecE |
| P30 | 3 | B1 | NR | fimH1, fimH2, fecI |
| P32 | 2 | B1 | NR | fimH1, fimH2, astA, fecI, fecE |
| P33 | 3 | B1 | NR | fimH1, fimH2, f17, fecI, fecE |
| P36 | 2 | B1 | AMP, AMC | fimH1, fimH2, astA, hlyA-var2, fecI, fecE |
| P38 | 2 | A | NR | fimH1, fimH2, fecI, fecE |
| P39 | 2 | B1 | NR | fimH1, fimH2, fecI, fecE |
| P41 | 3 | B1 | NR | fimH1, fimH2, fecI |
| P43 | 3 | B1 | NR | fimH1, fimH2, cnf1, papC1, papC2, hlyA-var2, iucD1, iucD2, fecI, fecE |
| P45 | 3 | B2 | NR | fimH1, fimH2, cnf1, hlyA -var2, fecE |
| P46 | 4 | B1 | FOS | fimH1, fimH2, sfaDE, fecI, fecE |
| P47 | 3 | B1 | AMP, AMC, TET | fimH1, fimH2, cnf1, f17, papC1, papC2, hlyA-var2, iucD1, iucD2, fecI, fecE |
| P48 | 3 | NT | NR | fimH1, fecE |
| P50 | 3 | A | NR | fimH1, fimH2, fecI, fecE |
1AMP, ampicillin; AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CFZ, cefazolin; CTX, cefotaxime; FOF, fosfomycin; GEN, gentamicin; PIP, piperacillin; SXT, trimethoprim-sulfamethoxazole; TET, tetracycline; TOB, tobramycin; NR, not resistant.
3.3. Molecular characterization of E. coli
The predominant phylogenetic group was B1 (n = 25, 50%), followed by A (n = 8, 16%), C (n = 6, 12%), and B2 (n = 4, 8%). Groups D and E each comprised two isolates (4% per group), and group F was represented by only one isolate (2%). Two isolates could not be assigned to any known phylogroup.
Twenty selected isolates underwent WGS, revealing 16 distinct sequence types (STs). ST10 and ST58 were each detected in two isolates (10% each), while the remaining STs (ST2598, ST1125, ST410, ST69, ST1396, ST117, ST744, ST1508, ST88, ST547, ST73, ST3234, ST446, ST2521, and two new ST18186 and ST18185) were singletons. No clustering was observed by cgMLST. WGS-based serotype analysis identified six distinctive O: H serogenotypes. Thirteen strains were O-non-typeable, and 12 distinct H-types were observed. Two multidrug-resistant isolates (P22, P31) belong to serotype O8: H25. Isolate P22 (phylogroup C, ST88) was ESBL-producing (blaCTX-M-3 and blaTEM-1) and resistant to gentamicin, tetracycline, and trimethoprim-sulfamethoxazole, while P31 (phylogroup B1, ST58) contained blaTEM-1 was resistant to was resistant to ampicillin, amoxycillin-clavulanic acid, gentamicin, tobramycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. P5 (ONT: H9, C, ST410) carried blaOXA-1 and mutations in quinolone-resistance-determining region (QRDR) (gyrA D87N, gyrA S83L, parC S80I, parE S458A). P17 (O101: H9, A, ST744) had blaOXA-1 with QRDR mutations (gyrA D87N, gyrA S83L, parC A56T, parC S80I). All four isolates were multidrug-resistant. P10 (phylogroup D, ST69) with the serotype O15: H18 carried the blaTEM-1 gene. P14 (ONT: H4, phylogroup F, ST117) carried blaTEM-1 and gyrA S83L. Phenotypic resistance, which is well reflected by the observation that the isolates carried various AMR genes: tetracycline resistance genes (tet(A), tet(B), trimethoprim-sulfamethoxazole resistance genes (sul1, sul2, dfrA17, dfrA36, dfrA5), chloramphenicol resistance genes (floR, catA1) and aminoglycoside resistance gene (aac (3)-IId, ant(2″)-Ia, aph(3′)-Ia, aadA1, aadA5, aph(3″)-Ib, aph (6)-Id). Biocide resistance genes were found in 4/20 isolates: four carried qacE. Isolates carried distinct virulence-associated genes. The fec operon was detected in 15 of 20 (75%) isolates in this study. Whole-genome sequencing revealed that all strains harbor fecIRABCDE, except for four strains. Strains P37 and P40 carry fecIRA, followed by a plasmid insertion, resulting in the absence of additional fec genes. Isolate P12 possesses fecIRAB, with a subsequent deletion extending to one epimerase gene. Strain P27 lacks all fec genes (Table 2). Among isolates that were whole-genome sequenced, the most prevalent virulence-associated genes were those in the fim operon (95%, 19/20). Less frequent genes included iroN (20%), vat (15%), cdt (10%), ibeA (5%), and kpsM (10%). The adhesins afaD-VIII and afaE-VIII were observed in 3/20 (15%) isolates. One isolate showed the fimbrial adhesin genes foc and sfa. Finally, eight isolates carried sitABCD (iron uptake/transport relevant). The genes for the identification of enteroaggregative E. coli astA and pic were also detected using a DNA microarray (Table 1). Using a DNA microarray, all isolates carried the fimH1 gene; 41 (82%) harbored fimH2. The virulence gene papC was present in 11 isolates (22%), while iucD (siderophore biosynthesis) was detected in 10 isolates (20%). Hemolysin gene hlyA was detected in 20 (40%), and f17 in five (10%). Each of the VAGs acfC, aidA, and K88 was detected in a single isolate. fecI (48/50, 96%) and/or fecE (43/50, 86%)—genes associated with ferric dicitrate uptake (Tables 2, 3).
4. Discussion
The aim of the present study was to characterize phenotypically and genetically mastitis-associated E. coli isolates from clinical and subclinical mastitis cases in Austria using a polyphasic approach that included susceptibility testing, PCR, DNA microarray-based technology, and WGS. The E. coli isolates examined showed genetic heterogeneity, reflected in the distribution of phylogroups and distinct STs, as well as in the variable presence of virulence genes. The virulence genes detected occurred in different combinations, showing a flexible set of pathogenicity factors.
Most isolates originated from severe clinical mastitis cases. The majority of isolates were assigned to phylogroups B1 and A, and lower rates were assigned to phylogroups C, B2, E, and F, consistent with previous studies (6, 9, 30).
The MAEC isolates analyzed in the present study exhibited sequence type (ST) diversity, reflecting substantial genetic variability within the selected population. Due to differences in the molecular methods applied to date (PCR, MLST, DNA microarray, whole-genome sequencing), it is often challenging to compare distinct MAEC virulence and resistance genes and their STs. ST10, ST58, ST88, ST117 and ST1125 found in our study, were also identified in isolates from different farms and with varying scores of mastitis (10, 30),and all these STs have multiple entries (bovine/mastitis) in the Enterobase Escherichia/Shigella Database (https://enterobase.warwick.ac.uk/species/index/ecoli, last accessed 18. November 2025). The affiliations of ST10, ST58, ST410, and ST73 are often associated with pandemic high-risk ExPEC strains that can survive in various ecological niches, including the human gastrointestinal tract and the environments of domestic and farm animals (31). One of the most characteristic genes we detected, which is observed frequently in MAEC and UPEC genomes than in other E. coli strains, is the fec operon (fecIRABCDE), which encodes the ferric dicitrate transport system (10, 32, 33)and represents an important molecular determinant contributing to the pathogenicity of MAEC. Isolate P7, with a new sequence type ST18185, carried previously detected genes in MAEC (papC, hlyA, cnf1, cdt, iucD, iutA), fecIRABCDE, and adhesin gene f17, which transmits adhesion to host cells. f17 are frequently found in ETEC strains and in up to 20% of MAEC isolates, depending on the study (6). The adhesin gene fimH was consistently detected across all isolates. Beyond this gene, different STs harbored distinct arrays of VGs associated with previously reported ExPEC pathotypes (UPEC and MAEC) (6, 9). ExPEC-associated VGs such as hlyA (haemolysin), papC (P-fimbriae), iucD (siderophore synthesis), and cnf1 (cytotoxic necrosis factor 1) were present in 16 to 24% of isolates, as well as astA, a virulence-associated gene associated with enteroaggregative E. coli. Other VAGs, including adhesins (sfaDE, iha) and toxin or siderophore genes (vat, cdt, iroN), were less common, consistent with previous MAEC studies (6, 9, 10).
Specifically, 33 isolates (66%) were phenotypically susceptible to all tested antibiotics, while seven of the fifty isolates (14%) exhibited a multidrug-resistant (MDR) profile. The highest resistance rates were observed for ampicillin (30%), tetracycline (20%), amoxicillin-clavulanate (20%), and trimethoprim-sulfamethoxazole (14%), consistent with data reported for MAEC isolates from Belgium (ampicillin 28.8%), France (amoxycillin/clavulanic acid 22%; tetracycline 20%), and Portugal (trimethoprim-sulfamethoxazole 13.1%) (34).
Genotypic analysis of β-lactamase genes revealed blaTEM-1 in three isolates (ST58, ST69, ST117), blaCTX-M-3/blaTEM-1 in one multidrug-resistant isolate (ST88), and blaOXA-1 in two isolates (ST410, ST744). The prevalence of β-lactamase and ESBL genes (blaCTX-M, blaTEM, and occasionally blaOXA) in MAEC varies considerably across geographical regions (34, 35). The multidrug-resistant isolates also showed phenotypic and genotypic resistance to tetracyclines (tet(A), tet(B)) and trimethoprim-sulfamethoxazole (sul1, sul2, dfrA17, dfrA36, dfrA5), commonly observed in E. coli from farm animals due to the widespread veterinary use of these antibiotics (34, 36).
The strains P5 (ST410), P17 (ST744), P22 (ST88) and P31 (ST58) harbored genotypically the biocide resistance genes qacE (Quaternary Ammonium Compound Efflux Gene) encode efflux pumps that confer tolerance to quaternary ammonium compounds (QACs), and the antibiotic resistance genes sul1 and aadA which are often found together with on class 1 integrons, promoting co-selection (37). Eight isolates carried the operon sitABCD, originally described as a metal ion transporter that contributes to survival in iron-poor environments such as the mammary gland and improves fitness in ExPEC isolates during infection by enhancing iron uptake, as with siderophore systems such as iucD or iroN (38). The co-occurrence with qacE in mastitis-associated strains could indicate adaptation to multiple traits, such as environmental persistence (qacE) or fitness for colonization of the host (sitABCD) (38). The limitation of this study is the lack of phenotypic testing for biocide resistance patterns.
The presence of high-risk pandemic sequence types and multidrug-resistant strains in Austrian MAEC highlights a One Health concern regarding pathogen transmission between livestock, the environment, and humans. Mitigating these strains is both a public health priority and an economic necessity, essential for reducing production losses and ensuring the dairy sector’s financial sustainability.
The number and regional distribution of tested strains were insufficient to determine the nationwide prevalence of resistance and virulence genes. Due to variation in virulence factors assessed worldwide and the high heterogeneity of these strains, it is challenging to establish uniform characteristics for this pathotype. Furthermore, investigation of the entire fec operon was limited by the availability of primers only for fecI and fecE. An additional limitation of this study is the lack of phenotypic testing for biocide resistance patterns. Future studies should incorporate a broader range of isolates from all Austrian localities and provide more comprehensive characterization, such as analyzing all isolates using whole genome sequencing rather than a selected subset.
5. Conclusion
This study is the first in Austria to conduct a genotypic analysis of mastitis-associated E. coli strains. The aim was to provide an overview of potential virulence mechanisms and regional antimicrobial resistance patterns associated with bovine mastitis, a severe disease affecting dairy cows. The isolates showed a high genetic variability. The occurrence of typical ExPEC VAGs such as fimH, fimA, papC, hlyA, cnf1, vat, iroN, and iucD, as well as the fec operon, underscores the importance of characterization of bovine E. coli isolates originating from mastitis. The detection of ESBL-producing isolates in milk represents a significant public health concern, reinforcing the need for integrated One Health surveillance programs. The identification of the fec operon and specific multidrug-resistant sequence types provides actionable targets for the development of regional diagnostic tools and the refinement of targeted antimicrobial therapies to reduce economic losses in Austrian dairy farming.
Funding Statement
The author(s) declared that financial support was received for this work and/or its publication. Institutional funds have been used to cover the costs of the laboratory examinations. Open Access Funding by the University of Veterinary Medicine Vienna.
Footnotes
Edited by: Om P. Dhungyel, The University of Sydney, Australia
Reviewed by: David Erickson, Brigham Young University, United States
Michael Olson, Snow College, United States
Walid Mousa, University of Sadat City, Egypt
Data availability statement
The original contributions presented in the study are publicly available. This data can be found here: NCBI Sequence Read Archive, accession PRJNA1471175.
Ethics statement
Samples were submitted to the Clinical Department for Farm Animals and Food Safety Systems, University of Veterinary Medicine Vienna for routine bacteriological diagnosis of mastitis. Samples were submitted by third parties and thus were exempt from ethical review by the Animal Welfare Commission of the University of Veterinary Medicine Vienna. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.
Author contributions
SP: Writing – review & editing, Writing – original draft. AR: Formal analysis, Writing – review & editing. WR: Validation, Methodology, Writing – review & editing. EM: Methodology, Writing – review & editing, Validation. SB: Methodology, Validation, Writing – review & editing. SM: Writing – review & editing, Methodology, Validation. RE: Validation, Writing – review & editing, Methodology. AW: Writing – review & editing. VU: Writing – review & editing, Formal analysis. TW: Supervision, Writing – review & editing. MS: Writing – review & editing, Data curation. MB: Writing – review & editing, Supervision. IL: Writing – original draft, Project administration, Writing – review & editing, Formal analysis, Supervision.
Conflict of interest
EM, SD, SM, RE were employed by InfectoGnostics Research Campus Jena e.V.
The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The original contributions presented in the study are publicly available. This data can be found here: NCBI Sequence Read Archive, accession PRJNA1471175.