1. Introduction
Antimicrobial resistance (AMR) is a problem of global concern [
1]. The spread of antibiotic-resistant bacteria, as stated by the World Health Organization (WHO), can render many drugs previously essential for treating infections in humans and animals ineffective [
2]. About 50-80% of the total antibiotic use in developed countries has been attributed to livestock [
3], although significant efforts are being made to rationalize antibiotic use. According to a recent report published by the European Food Safety Authority [
4], in Europe antibiotic use has decreased to become lower in food-producing animals than in humans. Nevertheless, the animal industry still plays a crucial role in the occurrence and transmission of AMR [
5].
In recent years, the prevalence of bacterial strains producing extended-spectrum beta-lactamases (ESBLs), i.e., enzymes that confer resistance to beta-lactams, has increased worldwide [
6,
7]. Beta-lactam drugs represent the most widely used group of antimicrobial agents and comprise penicillins and cephalosporins. Resistance to these drugs occurs through several mechanisms, among which the most widely used by Gram-negative bacteria is the production of β-lactamases. These enzymes hydrolyze a specific site in the β-lactam ring structure, causing it to open: the drugs are thus unable to bind to target proteins present on the bacterial wall [
8].
The presence of ESBL-producing
Escherichia coli is a common occurrence in dairy cattle, with the highest incidence observed in calves [
9]. Food-producing animals acquire AMR microorganisms due to several factors such as antibiotic use, forage, soil, water, and interaction with wildlife. Resistant bacteria colonizing the animal gastrointestinal tract are then shed in feces, thus favoring intra-farm spread and maintenance as well as environmental contamination via the farm waste products, including untreated wastewater, sewage sludge, and organic fertilizers such as manure [
3,
10,
11]. Adding to the control of pathogens from outside and inside the farm, biosecurity measures can therefore be also crucial for avoiding the selection, maintenance, and spread of AMR microbes within and outside the farm. Accordingly, although a rational use of antibiotics is key for reducing AMR, adequate farm management practices also play a fundamental role in containing the AMR burden [
4].
With these premises, we investigated the distribution of ESBL-producing E. coli in a medium-large herd in Northern Italy. A detailed questionnaire was administered to the farmer to assess farming practices, and calf feces and cow feces were assessed together with samples from the farm and animal environment to gain a better picture and further information on their prevalence, distribution, and antimicrobial resistance traits.
3. Discussion
This study aimed to assess the presence, distribution, and antimicrobial resistance profiles of ESBL-producing
E. coli in a medium-sized dairy herd in Northern Italy, hosting nearly 1,000 animals, including calves, heifers, and lactating and dry cows. We collected calf and cow feces, waste milk, environmental samples, and water, and we administered a questionnaire to assess the associated risk factors. As a result, most pre-weaned calves, including males and females, carried ESBL
E. coli in their intestines, and ESBL
E. coli were also diffusely present in the environment and farm equipment in contact with them. This result is in agreement with a previous paper [
13] showing that the shedding of AMR
E. coli increased with herd size. The herd management interview enabled the gathering of information on potential risk factors for the distribution of ESBL microorganisms on the farm. Not much is known about the diffusion and transmission of ESBL
E. coli among calves and cows, and how they are affected by environmental factors [
14]. The hierarchical clustering of ESBL
E. coli isolates based on the MIC results suggested that isolates with different resistance characteristics are circulating in the farm and that different sources and routes could be involved in their dissemination and maintenance, facilitated by incorrect or inadequate management, biosecurity, and hygiene practices.
The farmer used waste milk for feeding male calves. According to hierarchical clustering based on the isolate MIC profiles, the largest statistically significant cluster included about 40% of all calves’ fecal isolates and the waste milk isolate. ESBL
E. coli were also detected in the feces of two out of three cows treated for mastitis that contributed to the waste milk. Among the risk factors associated with the spread of ESBL-producing bacteria on cattle farms, the use of waste milk containing antibiotic residues as calf feed appears to play a crucial role [
3,
15]. However, waste milk is still used for this purpose in different countries [
16], although many of them, including Italy, have recently issued guidelines discouraging or forbidding this practice.
Based on hierarchical clustering, the MIC profiles of the isolates from the calf feeding bucket and drinking water were related to the fecal isolates of over 30% of the calves. Incorrect management practices such as shared or improperly cleaned feeding equipment [
3] can favor the diffusion of AMR-carrying bacteria on the farm. Notably, the feeding buckets and calf water buckets were not cleaned with detergents or disinfectants, and sometimes not even rinsed with water between feedings; furthermore, the number of buckets was not adequate for the number of animals on the farm. ESBL
E. coli were isolated from all these pieces of equipment as well as from the calf drinking water. Moreover, the pasteurizer used to reduce bacterial contamination of milk was not cleaned between cycles. Poor cleaning is one of the factors favoring bacterial contamination and multiplication, leading to higher microbial loads [
17]. Indeed, we isolated ESBL
E. coli also from the farm’s pasteurized waste milk.
Shared calf pens and their poor hygiene may also play a role in promoting the diffusion of AMR bacteria. We isolated ESBL
E. coli from both male and female calf pens, and we observed a relationship between the MIC profiles of these isolates and those from calf feces based on hierarchical clustering. As highlighted in an EFSA scientific opinion paper on calf welfare, the level of cleanliness of the areas used for housing calves is a major determinant of their health [
18]. Inaccurate cleaning procedures of the single pens or calf hutches may not adequately remove fecal contamination from the walls, leading them to serve as a reservoir [
14].
The cows underwent blanket dry cow therapy (BDCT) with a β-lactam, specifically amoxicillin/clavulanic acid. Although this practice is not allowed in Italy, some farms are still using it. BDCT has been reported to be linked to a significant increase of ESBL
E. coli in calf feces during the colostral phase [
9].
The ESBL
E. coli isolated from the feces of cows treated for mastitis clustered with the isolates from the cows’ alley floors, suggesting fecal shedding.
E. coli ESBL shedding can vary greatly among individuals [
19], and antibiotic treatment for mastitis could play a role in increasing animal colonization, shedding, and subsequent environmental contamination by AMR-carrying bacteria [
20]. The farm evaluated in this study did not have a sick pen, and this represents a lack of biosecurity.
ESBL
E. coli often carry multiple resistance genes that confer resistance to other antimicrobial drugs than β-lactams, leading to MDR [
21]. All the isolates obtained in our study, both from the calves, their equipment, and the farm environment, were MDR. On the other hand, resistance to colistin, an antibiotic of last resort for humans, was not detected. Many developed countries have prohibited its usage in food-producing animals, and the Antimicrobial Advice Ad Hoc Expert Group (AMEG) [
22] has placed antibiotics in this category as very important in human medicine. All ESBL
E. coli isolates were also carbapenem-sensitive. This is also a positive finding, as carbapenemase-producing Enterobacteriaceae cause serious human infections. The study by Waade et al. conducted in Germany in 2021 reported similar results since the ESBL-producing isolates were 92.9%
E. coli; 60.6% of ESBL-producing isolates were resistant to one or more classes of antibiotics including penicillins and cephalosporins but were sensitive to carbapenems [
23].
As a final consideration, although these bacteria were never isolated from fecal samples, 42.85% of environmental samples were positive for ESBL-producing
A. baumannii.
A. baumannii is reported as a relevant cause of nosocomial infections in humans, and MDR strains can pose significant risks for human health [
24]. MDR
A. baumannii has also been associated with inadequate cleaning of dairy equipment and the dairy cattle environment [
25]. Therefore, improving farm management practices can help control also this relevant AMR microorganism.
Author Contributions
Conceptualization: M.P., R.P, M.F.A.; methodology, M.P., R.P, M.F.A.; farm activities and sample collection: M.P, L.F.P.; formal analysis: M.P., L.F.P., L.M.; resources: R.P., M.F.A.; data curation: M.P., A.G., M.F.A.; writing, original draft: M.P., M.F.A.; writing, review and editing, all authors; supervision: R.P., M.F.A. All authors have read and agreed to the published version of the manuscript.