Colostrum as a source of ESBL-Escherichia coli in feces of newborn calves

The aim of the present study was to determine if colostrum and the equipment for harvesting and feeding colostrum are sources of fecal ESBL/AmpC-producing Escherichia coli (ESBL/AmpC-E. coli) in calves. Therefore, 15 male calves fed with pooled colostrum on a dairy farm and held individually in an experimental barn, the colostrum pool and the equipment for harvesting and feeding colostrum were sampled and analyzed for the occurrence of ESBL/AmpC-E. coli. The ESBL-AmpC-E. coli suspicious isolates were subjected to whole-genome sequence analysis. Forty-three of 45 fecal samples were tested positive for ESBL/AmpC-E. coli. In the colostrum sample and in the milking pot, we also found ESBL/AmpC-E. coli. All 45 E. coli isolates were ESBL-producers, mainly commensal sequence type (ST) 10, but also human-extraintestinal pathogenic E. coli ST131 and ST117 were found. The clonal identity of six fecal isolates with the ESBL-E. coli isolate from the colostrum and of five fecal isolates with the strain from the milking pot demonstrates that the hygiene of colostrum or the colostrum equipment can play a significant role in the spread of ESBL-E. coli. Effective sanitation procedures for colostrum harvesting and feeding equipment are crucial to reduce the ESBL-E. coli shedding of neonatal dairy calves.


Clonal expansion
Phylogeny analysis of ST10 isolates based on WGS data revealed three phylogenetic clusters.The size of the core genome of the ST 10 isolates studied was 5,140,627 bp and within each cluster core genomes of the isolates differed only in 0-4 SNPs or in 0-55 SNPs, respectively (see Fig. 1).The isolate No. 2012 (swab of milking pot) belonged to one cluster, in which only the fecal isolate No. 2009 differed in a single SNP from the other five fecal isolates.All isolates of that milking pot-cluster harbored the CTX-M-15 gene.To the second cluster the isolate No. 996 (colostrum sample) was allocated differing only in 2-4 SNPs to six fecal isolates.Three additional ESBL-E.coli isolates of that cluster exhibited more varying SNPs (52-55) compared to the other seven isolates.CTX-M-1 was detected in all isolates of the colostrum-cluster.The third cluster only contained fecal isolates which differed in 0-4 SNPs from each other.In the feces-cluster CTX-M-1 gene was present.The fecal isolate No. 2005 could not be allocated to one of the clusters.The difference to all other isolates of ST10 was 187-699 SNPs.In contrast to the isolates nearby in the phylogenetic tree, this isolate was CTX-M-15 positive.

Biofilm formation
To test the ability to form biofilms, one representative was selected from each of the three different clusters we identified in the clonal expansion analysis: No. 2012 (milking pot isolate), No. 996 (colostrum isolate), and No. 461 (fecal isolate).The test showed that the ability to form specific biofilms is high in the isolates from milking pot and colostrum and low in the fecal isolate from the feces-cluster (see Fig. 2).Phylotype

Discussion
Today, livestock production is known to be a significant source of AMR.In the past decades, the occurrence of ESBL-carrying pathogens increased worldwide 22 .ESBL/AmpC-E. coli are less prevalent in dairy cattle than other livestock species, with the exception of suckling calves, which have a high ESBL/AmpC-E.coli prevalence, e.g., above 50% in Germany 11,17 .Due to their immature immune system, newborn calves depend on passive immunization with maternal antibodies from the colostrum 23 .As it is common practice in conventional dairy farms to separate calf and dam right after birth, the colostrum is usually administered to the calf via bottle, bucket, or drench.A major advantage of this method compared to natural intake is the controllability of the amount and timing of colostrum intake.
Already on the first day of life, 14 out of 15 calves in our study were tested positive for ESBL-E.coli, although they had neither spatial nor temporal contact with each other and did not get any antibiotics.Before the next calf could enter the experimental barn, it was cleaned, disinfected and unoccupied for at least 1 week to prevent nosocomial transmission.Therefore, horizontal transmission between the calves and from the equipment of the experimental barn seemed unlikely.Moreover, the transport vehicle has also been cleaned and disinfected, so that transmission of ESBL-E.coli during transportation is also unlikely.Moreover, according to previous research, there is little chance that transmission from the dams to the calves is the main reason for ESBL-E.coli occurrence in the calves as the prevalence of the corresponding dams is quite low 11 and the ESBL-E.coli strains of calves differ from that of older animals 10 .Therefore, a sample of the colostrum pool and swabs of the equipment for harvesting and administering colostrum were analyzed for the occurrence of ESBL/AmpC-E.coli.In colostrum and in the milking pot for harvesting colostrum, we could detect ESBL-E.coli belonging to commensal ST10.
WGS revealed that six of 43 fecal isolates were nearly identical to the colostrum strain.Since the number of core genome SNPs was below 17, these isolates fulfil the clone definition of Ludden et al. 24 who developed this SNP cut-off to demonstrate transmission between patients.An even higher degree of core genomic identity was also demonstrated between five fecal isolates and the milking pot strain, demonstrating direct transmission of ESBL E. coli via colostrum or equipment in a total of 11 of the 43 ESBL E. coli isolates (25.6%).Our findings are underlined by two recent studies, which assumed that the feeding of colostrum contaminated with ESBL-E.coli can be responsible for intestinal colonization of calves with ESBL-E.coli, as the same bacterial resistome (i.e., all AMR genes and their precursors) was found in colostrum and in feces of neonatal calves 13,21 .In addition to the suggestion that antibiotic residues in colostrum may lead to increased occurrence of ESBL-E.coli in suckling calves 19 , our results prove that colostrum serves as an early vector for resistant bacteria in calves.
It remains unknown how exactly the ESBL-E.coli contamination of the colostrum occurred.However, based on the detection of an ESBL-E.coli in the milking pot, we assume that the presence of ESBL E. coli in the colostrum is due to poor hygiene of the milking equipment used to collect colostrum, particularly since both strains found in colostrum and milking pot have a high ability to form specific biofilms.The ability of biofilm formation allows ESBL E. coli to withstand standard cleaning procedures 8 and to persist in the milking equipment.In this way, washing out bacteria results in contamination of colostrum and colonization of calves.According to two German studies, hygiene management of the calf 's feeding equipment is associated with the occurrence of ESBL-E.coli in dairy farms.Heinemann and colleagues found ESBL-E.coli in the inner surface of nipples of feeding buckets and concluded that sanitation measures in dairy farms are inadequate, maybe leading to ESBL-E. coli infection of the calves 20 .In addition, the cleaning procedure of feeding buckets was associated with the ESBL-E.coli prevalence of dairy calves 11 .
Acidification or pasteurization of colostrum, which are common to optimize hygiene in milk rations for calves, possible methods to sanitize colostrum 25,26 .However, if the equipment for colostrum administration, particularly the nipple, is contaminated with biofilm forming ESBL-E.coli, transmission of resistant bacteria is still possible.According to the data of Heinemann et al. 20 nipples for calves are regularly contaminated with ESBL-E.coli and other bacteria.www.nature.com/scientificreports/ In addition to ST10, we found other STs of ESBL-producing E. coli, including ST131, a globally distributed clonal lineage known to cause severe extraintestinal infections in humans and animals 22,27 .This single lineage is mainly responsible for the increase in urinary tract and bloodstream infections with ESBL-E.coli worldwide.Strains of ST131 usually carry bla CTX-M-15 on a plasmid 26,28 .Deviating from that, our isolate of ST131 is bla CTX-M-1 -positive.ESBL-E.coli carrying bla CTX-M1 are primarily isolated from livestock in Europe 29,30 .CTX-M15 is predominant in ST131 29,31 , therefore, horizontal gene transfer of a bla CTX-M-1 encoding plasmid seems certainly probable for our isolate.In studies with companion animals, ESBL-E.coli producing CTX-M-15 and other variants (e.g., CTX-M-14, CTX-M-55), which were frequently found in humans, could be detected, indicating a direct transfer of viable bacteria between companion animals and humans 30,32 .Transmission of ESBL-E.coli from animals to humans is often postulated.Regarding livestock production, contaminated slurry and waste water from abattoirs and animal rendering plants can harbor a significant risk for human health 8,33,34 .
Among our study, we also detected ST88, ST117 and ST362, which seem to be associated with calves as they were also found in a farm in Mecklenburg Western Pomerania 10 .ST362 is also known to be an efficient biofilm former 33,35 , therefore, is able to survive at surfaces of milking or feeding equipment or at calf barns/igloos 10,20 .Just like ST131, ST117 is also related to extraintestinal infections in humans and results of five studies indicate that poultry may be the reservoir, but it could also be found in calves 34,36 .With the present study being at least the third detecting ST117 in calves, it cannot be denied that calves may also play a role as a vector for human or avian ST117 colonization.
To our concern, > 50% of all isolates fulfilled the definition of being MDR and 31% of the ESBL-E.coli showed phenotypic quinolone resistance.Fortunately, none of the strains associated with human infections harbored multiple AMR.
In the present study, we were able to detect ESBL E. coli in a large number of different STs, which underlines the fundamental need for improved hygiene in calf husbandry.The transmission modes of the individual STs must be the subject of further investigations.With regard to colostrum as a possible source of ESBL E. coli, we were able to demonstrate the direct transmission of viable ESBL-E.coli via contaminated colostrum.Besides lowering the use of antibiotic agents in dairy herds, excellent sanitation procedures of the equipment for harvesting and feeding colostrum is crucial to reduce the prevalence of ESBL-E.coli in neonatal dairy calves, consequently minimizing the spread of AMR.Therefore, more attention should be paid on the improvement of farm hygiene, as it remains to be a simple and efficient method to prevent the environmental contamination with ESBL-E.coli.

Animals
Fecal, colostrum and equipment samples were collected as part of another study 37 in which calves were initially fed with pooled colostrum at birth and thereafter moved to an experimental barn where they were individually housed and fed with milk replacer within the first eight days of life.Briefly, 15 male Holstein-Friesian calves from a German dairy farm were fed 3 L of colostrum right after birth at the farm.The colostrum was pooled prior to the beginning of the experiments and stored frozen in portions so that all calves received the same colostrum.The calves were then transported to the University of Leipzig within their first 24 h of life.There, they were stalled in one of two separate barns (control and infected with Cryptosporidium parvum, see below) with only one calf at a time.The calves were fed 3 × 2 L of milk replacer (Union A50 S, Arla Foods, Viby J, Denmark) daily and had free access to water in a bucket.Additionally, from day 4 onward they received 2 L of electrolytes (Ursolyt G oral, Serumwerk Bernburg AG, Bernburg, Germany) daily.
Upon arrival at the University, the animals were examined clinically, and blood and fecal samples were collected.Afterwards, one group of calves was infected by oral application of 2 × 10 7 Cryptosporidium parvum oocysts (n = 5) whereas the control group received only water.In the following days, the calves underwent several blood and fecal samplings for study purposes according to the aims of the concurrent study 37 .
The experiments, including all animal sampling, were conducted in accordance with the German legislation on the protection of animals and were approved by the Landesdirektion Leipzig as TVV 19/20.All authors comply with the ARRIVE guidelines.

Sampling, bacteriological examination
Fecal swabs (Sigma Transwab, MWE, United Kingdom) for the determination of ESBL/AmpC-E.coli carriage of the calves were taken at the 1st day (before infection with C. parvum), day 5 and day 8 of life, preserved in Amies medium and stored at 5 °C.A sample of the stored colostrum pool was additionally analyzed, and subsequently the dairy farm was visited and the equipment for harvesting and administering colostrum including liner, tube and milking pot and the nipple of the colostrum bottle was sampled using swabs.
Fecal, colostrum and equipment samples were cultured on CHROM ID agar plates (Mast Group, Reinfeld, Germany) supplemented with 2 µg/mL cefotaxime (Alfa Aesar by Thermo Fisher Scientific, Kandel, Germany) and incubated at 37 °C overnight.Moreover, the pooled colostrum was enriched in LB-broth supplemented with 2 µg/mL cefotaxime before cultivation.According to the manufacturer's protocol, pink-violet colored, shiny colonies represent presumptive ESBL/AmpC-E.coli-positive results.Positive colonies were picked and sub-cultivated until a pure culture was achieved.All isolates were stored at − 80 °C until further use.

Antimicrobial susceptibility testing
AST was carried out using VITEK2 (bioMérieux, Nürtingen, Germany).Testing was performed using software version 9.02 and AST-N428 and AST-XN24 card, according to the manufacturer's instructions.The AST card used for the VITEK2 included an ESBL confirmation test.Second and 3rd generation cephalosporins (ceftazidime, cefotaxime and cefuroxime) were used alone or in combination with tazobactam/avibactam.A reduction of growth in the presence of inhibitor of β-lactamases was considered indicative of ESBL production.
Minimal inhibitory concentration (MIC) breakpoints were set according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint tables for interpretation of MICs and zone diameters (Version 13.1, 2023.http:// www.eucast.org).
Snp-dists v. 0.8.2.(https:// github.com/ tseem ann/ snp-dists, accessed on 05.06.2023) was used to convert the FASTA alignment to a SNP distance matrix.(The distance matrix was depicted as a heat map diagram beside the core SNP phylogeny.)

Biofilm formation
Biofilm formation on polystyrene surfaces was assessed using crystal violet (CV) staining, as previously described 5 .The strength of biofilm formation was determined as specific biofilm formation (SBF), which was calculated using the formula: SBF = (B − NC)/G, where B is the OD570 of the stained bacteria, NC is the OD570 of the stained control wells to account for CV adhering to the polystyrene surface due to non-biological factors, and G is the OD600 representing the cell density in the culture medium.To evaluate biofilm formation, the test was also performed with two strains which are weak (PBIO729 50 ) or strong (W3110) biofilm formers, respectively. https://doi.org/10.1038/s41598-024-60461-4www.nature.com/scientificreports/

Figure 1 .
Figure 1.Core SNP phylogeny of ESBL-producing ST10 isolates originating from calf feces, pooled colostrum, and milking pot.Core genome SNPs were called using snippy v. 4.4.1.SNP distances were calculated and depicted in a heat diagram (SNP distance ranged from 0 to 760 sites).The phylogenetic tree is based on a core SNP alignment and visualized in iTOL.Additionally, presence of CTX-M-1 and CTX-M-15 were depicted in the phylogenetic tree.

Table 1 .
Phenotypic resistance profiles of ESBL-E.coli isolates.R resistant, S sensitive, I intermediate.