Abstract
Objectives: The Enterobacteriaceae family is a group of bacteria that serve as indicators of milk collection hygiene at the farm level. The objective of this study was to detect and identify members of this family in raw cow’s milk and pasteurized milk and to evaluate their antimicrobial susceptibility and the presence of resistance genes.
Materials and Methods: A total of 14 samples of raw bulk milk and 14 samples of pasteurized milk were collected from different dairy plants in Slovakia. Isolates were identified at the family level by PCR and at the species level by MALDI-TOF MS. Ten antibiotics (ampicillin, cefoxitin, ceftizoxime, cefazolin, kanamycin, streptomycin, tetracycline, gentamicin, ofloxacin, and chloramphenicol) were tested using the disc diffusion method. Genes that confer antimicrobial resistance to beta-lactam antibiotics were detected by PCR using gene-specific primers.
Results: Klebsiella oxytoca was the most frequently identified isolate (30.0%), followed by Enterobacter cloacae (13.3%) and Citrobacter gillenii (13.3%). According to the CLSI criteria, 22 of the 30 isolates (73.3%) were classified as cefoxitin-resistant, whereas EUCAST classified 26/30 (86.7%) as cefoxitin-resistant. The presence of resistance genes (at least one of the genes tested) was confirmed in 26 isolates. In 20 isolates, blaTEM was found; in 11, blaCTX–M; and in 10, ampC genes.
Conclusions: Our findings highlight the absence of Enterobacteriaceae in pasteurized milk, discrepancies in evaluation between the CLSI and EUCAST systems, a high level of beta-lactam resistance, and the presence of resistance genes detected only in raw milk samples.
Keywords: Enterobacteriaceae (sensu stricto), pasteurization, resistance genes
1. Introduction
The family Enterobacteriaceae belongs phylogenetically to the order Enterobacterales, the class Gammaproteobacteria, and the phylum Proteobacteria. This group of bacteria was created in 1937 and was initially based on representatives of the genus Enterobacter. Enterobacteriaceae have undergone several major taxonomic revisions. A significant change occurred when some genera originally classified within Enterobacteriaceae were reassigned to separate families. These changes were driven by new data obtained through gene sequencing technologies [1, 2].
In animals, economically important Enterobacteriaceae infections include gastrointestinal tract diseases. The most common causes of mastitis in cattle are Citrobacter koseri, Enterobacter aerogenes, and Klebsiella pneumoniae, belonging to this family. In humans, they are responsible for more than 50% of nosocomial infections. They are responsible for almost 50% of septicemias, 60–70% of intestinal infections, and 70% of urinary tract infections [3, 4].
Milk is a suitable medium for the growth of various microorganisms because it is rich in nutrients, has a high-water content, and maintains a neutral pH. Rapid proliferation of microorganisms, particularly at high ambient temperatures, can alter the physical and chemical composition of milk. As summarized by Chege and Ndungu [5], raw milk can become contaminated at various points along the processing chain and from multiple sources. In general, microorganisms can contaminate raw milk in two primary ways. The first is endogenous contamination, where milk is contaminated via direct transmission from a sick animal (e.g., systemic infections or mastitis). The second is exogenous contamination, which occurs during or after milking through contact with feces, the external parts of the udder, teats, the environment, or equipment [6].
The genera most isolated from the Enterobacteriaceae family in milk and dairy products include Citrobacter, Enterobacter, Escherichia, Klebsiella, and Kluyvera. The genus Enterobacter is the most predominant in raw and pasteurized milk. Species of the genus Escherichia are found in approximately 10–17% of raw milk samples, and in less than 10% of all coliforms in pasteurized milk [7, 8, 9]. The presence of Enterobacteriaceae in raw milk is a reason to use effective heat treatment, as required by current legislation. In accordance with EU Regulation 2073/2005, the acceptance criterion for pasteurized milk is set at m = M = 10 CFU/ml with an allowable number of exceeding samples (c) of zero. Consequently, any detection below this 10 CFU/ml threshold is classified as legally satisfactory, indicating that the manufacturing process is operating within controlled hygiene parameters [10]. Pasteurization should eliminate Enterobacteriaceae in milk for further manufacturing. This is why some studies focus on undesirable bacteria in milk after heat treatment, as their presence after pasteurization may affect, among other things, subsequent processing.
As is well known, antimicrobial resistance (AMR) in Enterobacteriaceae from raw materials of animal origin may be closely related to the use of antibiotics in therapeutic interventions on farms, particularly for the treatment of mastitis in dairy cows. The AMR situation shows specific characteristics depending on therapeutic practices in a given country. This applies to both human and animal populations and their interconnectedness. [11, 12].
The application of antibiotics can affect the food industry, as antibiotic resistance in animals can be transferred to food products. There is also an indirect risk of horizontal transfer of resistance genes to pathogenic microorganisms at different points in the food chain [13]. Multi-resistance is often associated with the presence of Escherichia coli and Salmonella spp., which are considered the most common foodborne pathogens. According to Fakruddin et al. [14], multi-resistant K. pneumoniae bacteria were detected in raw milk samples, and multi-resistant bacteria of the genera Enterobacter, Citrobacter, and Klebsiella were also detected in various food samples, including powdered milk. Currently, the positions on the assessment of antibiotic susceptibility across the two systems, namely CLSI and EUCAST, are a subject of debate due to many differences observed when both are used in parallel. AMR is a global problem, and unifying both systems would be appropriate.
Many researchers have highlighted the potential risk of transmission of ESBL (extended-spectrum beta-lactamases), ampC (aminopenicillin-inactivating cephalosporinases), and CP (carbapenemases) produced by bacteria of the Enterobacteriaceae family to consumers through milk and dairy products sold directly from farms, such as E. coli and K. pneumoniae, that confer resistance to many commonly used antibiotics [15, 16]. Genes encoding ESBLs are most frequently found in transposons or plasmid insertion sequences. Consequently, animal production has become a major concern in recent decades due to its close connection with the food chain and its potential role as a reservoir, vehicle, or transmission route for the dissemination of ESBLs. In the past, these genes were mainly investigated in isolates exhibiting phenotypic resistance. However, it was later discovered that their presence could also be detected in isolates that were not resistant [17].
Based on the above, the aim of our study was to determine the occurrence of Enterobacteriaceae in raw bulk milk and milk after pasteurization, identify them, assess their antimicrobial resistance profiles and the presence of resistance genes, and determine the occurrence of Enterobacteriaceae in raw bulk milk and milk after pasteurization. Isolates were identified and classified according to current taxonomic systems. Additionally, evaluating the two standard systems for assessing AMR may reveal differences between them. The findings would contribute to a better understanding of AMR in foodborne bacteria and underscore important implications for food safety and public health.
2. Materials and Methods
2.1. Ethical approval
Ethical approval was not required for this study because it involved the microbiological analysis of milk samples collected from dairy plants and did not involve human participants or experimental animals.
2.2. Isolation and identification of Enterobacteriaceae
A total of 14 samples of raw bulk milk and 14 samples of pasteurized milk were collected from four dairy plants (west, north, east, and southeast) in Slovakia, which are focused on artisanal cheesemaking. The samples were taken in accordance with ISO 7218 [18]. Dairies receive milk daily from farms in close proximity; the dairy in the southeast also has its own farm with dairy cows on-site. Our visits in diaries were announced in advance to each dairy plant. The samples of pasteurized milk were taken after heat treatment of each raw bulk milk; they matched each other. Each sample (raw or pasteurized milk), with a volume of 500 ml, was transported at 4°C without the addition of preservatives to the laboratory at the University of Veterinary Medicine and Pharmacy in Košice, Slovakia, for analysis. Within 4 h of collection, all samples were analyzed microbiologically.
The samples were diluted decimally according to ISO 6887-5 [19]. Then, 1.0 ml of each diluted sample was placed into sterile Petri dishes, poured with Violet Red Bile Glucose Agar (Merck, Germany) as a selective agar for Enterobacteriaceae, and incubated at 37°C for 24 h. After incubation, the bacterial colonies were counted on each Petri dish. Each milk sample was evaluated by determining the number of colony-forming units (CFU) in 1 ml and then recalculated to log10. Based on their characteristic appearance, five typical colonies per plate were selected, purified on Brain Heart Infusion Agar (Merck, Germany), and stored at –18°C for further investigation.
The DNA was extracted from the isolates according to Hein et al. [20]. PCR was used to identify the Enterobacteriaceae family according to Ke et al. [21]. In our study, the term “Enterobacteriaceae” refers to taxa classified within the family Enterobacteriaceae according to the post-2016 taxonomic revision. The 16S rRNA gene sequences marked as Ent-F 5′-CGT TAC YCG CAG AAG AAG CA-3′ and Ent-R 5′-CTG AGC GTC AGT CTT YGT CC-3′, which covered the identification of 89% of the genus of the family Enterobacteriaceae (sensu stricto) [2], were used. Due to possible overlap of genera that no longer belong to the Enterobacteriaceae family, all collected colonies were identified by PCR and MALDI-TOF MS. Each primer was synthesized in Metabion (Germany), and the size of the product was 259 bp. The PCR protocol was optimized as follows: initial denaturation at 95°C for 13 min, followed by 30 cycles (denaturation at 95°C for 20 sec, annealing at 52°C for 30 sec, and extension at 72°C for 2 min). The final extension was performed at 72°C for 10 min. The HotFirepol® Mastermix (Solis BioDyne, Tart, Estonia) was used in the PCR. The PCR products were visualized on a 1.5% agarose gel stained with GoldviewTM nucleic acid stain (Beijing SBS Genetech Co., Ltd., Beijing).
All isolates examined were streaked on Columbia Blood Agar (Merck, Darmstadt, Germany) to prepare for MALDI-TOF MS examination and incubated at 37°C for 24 h. Individual samples were prepared via an extraction procedure using ethanol and formic acid [22]. Then, the colonies were used for identification by MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA).
2.3. Antimicrobial susceptibility testing
The identified isolates of the family Enterobacteriaceae were tested for antibiotic susceptibility using the disc diffusion method (DDM) according to the procedure described in the CLSI document [23]. Ten antibiotics were tested: ampicillin (AMP, 10 µg), cefoxitin (FOX, 30 µg), ceftizoxime (ZOX, 30 µg), cefazolin (CZ, 30 µg), kanamycin (K, 30 µg), streptomycin (S, 10 µg), tetracycline (TE, 30 µg), gentamicin (CN, 10 µg), ofloxacin (OFX, 5 µg), and chloramphenicol (C, 30 µg).
Antibiotics were selected based on the most commonly used antimicrobial agents in veterinary and human medicine in Slovakia for treatment [24, 25]. Escherichia coli ATCC 25922 was used as a reference strain. The results were evaluated according to two systems, namely the European Committee on Antimicrobial Susceptibility Testing [26] and the Clinical and Laboratory Standards Institute guidelines [23]. According to the CLSI system, it was possible to evaluate all ten tested antibiotics, and according to the EUCAST system, it was possible to assess susceptibility to these antibiotics, namely ampicillin (AMP, 10 µg), cefoxitin (FOX, 30 µg), ofloxacin (OFX, 5 µg), gentamicin (CN, 10 µg), and chloramphenicol (C, 30 µg). The discs used for susceptibility testing were manufactured by HIMEDIA (Mumbai, India). The diameters of the inhibition zones were recorded in millimeters (mm) and interpreted as susceptible, intermediate-susceptible, or resistant.
2.4. Detection of antimicrobial-resistant genes
Enterobacteriaceae isolates for the resistance genes blaTEM (Figure 6A), ampC (Figure 6B), and blaCTX–M (Figure 7), were tested. Genes that can confer AMR to beta-lactam antibiotics were detected in PCR using the gene-specific primers shown in Table 1 [27, 28]. The PCR protocol was optimized as follows: initial denaturation at 95°C for 13 min, followed by 30 cycles of denaturation at 95°C for 20 sec, annealing at different temperatures depending on the gene for 30 sec, and extension at 72°C for 2 min. The final extension was performed at 72°C for 10 min. PCR amplicons were run on 1.5% agarose gel. The expected sizes of the PCR products varied among genes (Table 1).
Figure 6.
A. Agarose gel electrophoresis of PCR product of blaTEM (445 bp). Lane L: size marker; Lanes 1–7: positive samples; Lane 8: positive control; Lane 9: negative control (A). B. Agarose gel electrophoresis of PCR product of ampC (491 bp). Lane L: size marker; Lanes 1–5, 10, 17: positive samples; Lanes 6 – 9, 11–16: negative samples, Lane 18: positive control; Lane 19: negative control (B).
Figure 7.
Agarose gel electrophoresis of PCR product of blaCTX-M; Lane L: size marker; Lanes 3, 11–15: positive samples; Lanes 1, 2, 4–10, 16–23 negative samples; Lane 24: positive control; Lane 25: negative control.
Table 1.
The primers used in this study for the detection of resistance genes in Enterobacteriaceae isolates using a PCR-based method.
| Gene | Primer sequence (5′-3′) | Product size (bp) | Annealing temperature | References |
|---|---|---|---|---|
| bla TEM | TCG CCG CAT ACA CTA TTC TCA GAA TGA | 445 | 61°C | [27] |
| ACG CTC ACC GGC TCC AGA TTT AT | ||||
| ampC | CGC CTC TTG CTC CAC AT | 491 | 51°C | [28] |
| CGC CGA ACA AAC CGA TA | ||||
| bla CTX–M | ATG TGC AGY ACC AGT AAR GTK ATG GC | 593 | 65°C | [27] |
| TGG GTR AAR TAR GTS ACC AGA AYC AGC GG |
bp, basic pairs.
2.5. Sequencing
In isolates with identified genes blaTEM, blaCTX–M, and ampC, the corresponding products of the specified size were sequenced using the Sanger method by SEQme s.r.o. (Dobříš, Czech Republic). The sequences obtained were analyzed for homology with the sequences available in the GenBank-EMBL database using the BLAST program (NCBI, software package 3.40, Bethesda, MD, USA).
2.6. Data analysis
Data were entered, cleaned, and validated in a MicrosoftTM Excel spreadsheet (MS Office Excel® 2021, Wanchai, Hong Kong). The average number of family Enterobacteriaceae in the milk samples was recalculated to log10 transformation. The distribution of each species in the milk samples was determined by calculating the percentage of each species out of the total number of isolated bacteria. The PCR results (positive or negative) were reference variables for descriptive analyses. Univariate analyses were conducted for descriptive statistics, and the data was presented as percentages. Statistical methods of basic statistics such as arithmetic mean, sample standard deviation, median, coefficient of variation, minimum, and maximum were used in the work to evaluate the counts of Enterobacteriaceae in milk samples.
3. Results
3.1. Isolation and enumeration of the family Enterobacteriaceae
Enterobacteriaceae were present in 12 (85.7%) of the 14 raw bulk milk samples tested. The average count in raw cow’s milk samples was 3.27 log10 ± 1.94 CFU/ml (colony-forming units, CFU). For the positive raw milk samples, the average count was 3.81 log10 ± 1.47 CFU/ml (Table 2). In all milk samples tested after batch pasteurization, no Enterobacteriaceae were detected. All dairy plants where the samples were taken use, in accordance with current legislation, a combination of low temperature and a longer pasteurization time to protect the milk proteins before cheesemaking.
Table 2.
Enumeration of Enterobacteriaceae in milk samples from artisanal dairy plants in Slovakia.
| Total samples (raw milk) | Positive samples (raw milk) | Pasteurized milk | |
| Average in log10 | 3.27 CFU/ml | 3.81 CFU/ml | 0.0 CFU/ml |
| Standard deviation | ± 1.94 | ± 1.47 | ± 0.0 |
| Median in log10 | 3.38 CFU/ml | 3.90 CFU/ml | 0.0 CFU/ml |
| Coefficient of variation | 0.59 | 0.39 | 0.0 |
| Minimum in log10 | 0.00 CFU/ml | 1.3 CFU/ml | 0.0 CFU/ml |
| Maximum in log10 | 6.24 CFU/ml | 6.24 CFU/ml | 0.0 CFU/ml |
| Positive samples (number) | 12 (85.7%) | – | 0 |
| Total samples (number) | 14 | – | 14 |
53 of 59 isolates examined were positive after PCR (Figure 1) using primers that should cover 89% of the genus within the family Enterobacteriaceae [2]. All 59 isolates were simultaneously identified using MALDI-TOF MS. Isolates confirmed by MALDI-TOF MS to belong to genera that were originally part of Enterobacteriaceae but have since been reassigned to other families within the order Enterobacterales were excluded from further analysis. The use of this primer set identified all 53 isolates belonging to the Enterobacteriaceae family at the family level, but genera currently outside this family were also identified as positive. Therefore, MALDI-TOF MS identification was appropriate, unambiguously determining that the isolates no longer belong to Enterobacteriaceae. Only isolates with a MALDI-TOF MS score higher than 2.299, indicating high confidence at the species level, were included. Finally, identification by MALDI-TOF MS showed that all 59 isolates belonged to the order Enterobacterales (six isolates had negative PCR results), but only 30 isolates belonged to the Enterobacteriaceae family according to current taxonomy. The complete dataset is available in Supplementary Table S1. In general, the most common genus was Hafnia, belonging to the Hafniaceae family, identified by MALDI-TOF MS. Other isolates confirmed by PCR, but outside the Enterobacteriaceae, were Serratia liquefaciens and Pantoea agglomerans, which belong to the family Yersiniacae and family Erwiniaceae, order Enterobacterales.
Figure 1.
Agarose gel electrophoresis of the PCR product of 16S rRNA gene sequences of the family Enterobacteriaceae. Lane L: size marker; Lanes 1–6, 8–20: positive samples; Lanes 7, 21: negative samples; Lane 22: positive control; Lane 23: negative control.
In Figure 2, a result is obtained after identification at the species level by MALDI-TOF MS. Finally, 30 of 53 isolates belonged to the family Enterobacteriaceae. In the samples taken (Figure 3), experimental analysis confirmed the presence of eleven species of the family Enterobacteriaceae. Overall, the most common species was Klebsiella oxytoca (9, 30.0%), followed by Enterobacter cloacae and Citrobacter gillenii (4, 13.3%). In our study, Enterobacteriaceae refers to the family as amended in 2016 (order Enterobacterales) and includes only genera still within this family.
Figure 2.
MALDI-TOF identification result: A total of 53 confirmed isolates belonging to the order Enterobacterales (including family Hafniaceae, Yersiniaceae, Erwiniaceae) of which 30 isolates are part of the family Enterobacteriaceae.
Figure 3.
MALDI-TOF identification result: Numerical representation of individual species of the Enterobacteriaceae family (30) obtained after their isolation from samples of raw cow’s milk with the highest incidence of Klebsiella oxytoca (30.0%).
3.2. Antimicrobial susceptibility test results for representatives of family Enterobacteriaceae
The antimicrobial susceptibility results were evaluated separately according to the CLSI 2023 and EUCAST 2023 documents due to the expectation differences between these two systems. Evaluation using the CLSI criteria was possible for all selected antibiotics, and evaluation using the EUCAST criteria for the Enterobacteriaceae family was possible for five antibiotics (FOX, AMP, OFX, CN, C). The zone diameter breaking points defined by CLSI are different compared to the values defined by EUCAST. The results according to CLSI and EUCAST for all isolates tested from milk samples are presented in Table 3.
Table 3.
Antimicrobial-resistant phenotypes of Enterobacteriaceae identified by MALDI-TOF MS according to CLSI and EUCAST.
| Resistant | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ZOX | FOX | AMP | K | CZ | OFX | S | CN | C | TE | |
| CLSI | 12 (40.0%) | 22 (73.3%) | 19 (63.3%) | 1 (46.7%) | 14 (46.7%) | 1 (3.3%) | 12 (40.0%) | 5 (16.7%) | 0 (0.0%) | 3 (10.0%) |
| EUCAST | Nda | 26 (86.7%) | 19 (63.3%) | Nda | Nda | 16 (53.3%) | Nda | 17 (56.7%) | 0 (0.0%) | Nda |
Nda, no data available; ZOX, ceftizoxime; FOX, cefoxitin; AMP, ampicillin; K, kanamycin; CZ, cefazolin; OFX, ofloxacin; S, streptomycin; CN, gentamicin; C, chloramphenicol; TE, tetracycline.
Although numerically prevalence differs, in many cases the trend was similar (e.g., cefoxitin and ampicillin are top resistance in both systems, just at different absolute percentages). As already mentioned, it was possible to compare five antibiotics with each other. In particular, the number of resistant isolates was lower under the CLSI criteria than under the EUCAST criteria for the same antibiotics. The most significant differences were observed with ofloxacin and gentamicin; only one isolate was evaluated as resistant to ofloxacin by the CLSI, versus 16 by the EUCAST system. In all isolates investigated, the highest resistance to cephalosporins was observed, according to EUCAST to cefoxitin in 26 (86.7%) isolates and according to CLSI to cefoxitin in 22 (73.3%) followed by ampicillin in 19 (63.3%) isolates according to both systems. A high level of resistance was observed in isolates against gentamicin (56.7%) and ofloxacin (53.3%), but only according to the EUCAST criteria. The number and distribution of antimicrobial-resistant phenotypes of Enterobacteriaceae by species according to CLSI (Figure 4) showed that all nine isolates (100%) of K. oxytoca were resistant to ampicillin and eight (88.9%) of these isolates were resistant to cefoxitin. All five isolates of the genus Enterobacter were resistant to cefoxitin. Resistance to both isolates of Raoultella terrigena was observed simultaneously against ampicillin, streptomycin, and tetracycline. According to these criteria, only one isolation of K. oxytoca was resistant to ofloxacin. No resistance was observed in one isolation of Lelliottia amnigena to all antibiotics tested.
Figure 4.
Distribution of antimicrobial-resistant phenotypes of Enterobacteriaceae identified by MALDI-TOF MS at species level according to the CLSI system. ceftizoxime (ZOX), cefoxitin (FOX), ampicillin (AMP), kanamycin (K), CZ (cefazolin), ofloxacin (OFX), streptomycin (S), gentamicin (CN), chloramphenicol (C), tetracycline (TE).
The number and distribution of antimicrobial-resistant phenotypes of Enterobacteriaceae by species according to the EUCAST system are illustrated in Figure 5. This system could evaluate five antibiotics and showed resistance in all nine (100%) K. oxytoca to cefoxitin, the same result as in the case of the CLSI criteria. On the other hand, the most significant differences from CLSI were found in the assessment of resistance to ofloxacin and gentamicin, regarding the high number of resistant isolates and the greater variability at the species level.
Figure 5.
Distribution of antimicrobial-resistant phenotypes of Enterobacteriaceae identified by MALDI-TOF MS at species level according to the EUCAST system. cefoxitin (FOX), ampicillin (AMP), ofloxacin (OFX), gentamicin (CN), chloramphenicol (C).
3.3. Detection of resistance genes among all isolates of family Enterobacteriaceae
The assessment of Enterobacteriaceae safety through in vitro expression of virulence traits does not always reflect the real hazard in these groups of bacteria due to the presence of silent genes. Bacteria in raw milk can be under antibiotic pressure during the treatment of animals with mastitis, and could potentially be activated by environmental conditions, thus improving the pathogenicity of these bacteria [16]. In our study, the presence of resistance genes (blaTEM, blaCTX–M, ampC) was investigated in all isolates with or without phenotypic expression.
The phenotypic expression of resistance against ceftizoxime, cefoxitin, cefoxitin, ampicillin according to both systems and the presence of resistance genes in the identified isolates are represented in Table 4. As documented in the table, among all isolates identified belonging to the Enterobacteriaceae family, there were 20 (66.7%) isolates with confirmed blaTEM. Most of them were identified as K. oxytoca. Overall, the resistance gene ampC was found in eleven isolates, including seven species of the family. The primers used are designed to detect plasmid ampC resistance genes [28] and do not amplify the innately present chromosomal ampC genes in the several species of Enterobacteriaceae. Nine isolates identified as K. oxytoca and one of C. gillenii harbored the blaCTX-M gene. As shown in Table 4, there are differences in resistance phenotype expression against cefoxitin (FOX1, FOX2) only in Lelliottia spp. and Raoultella spp.
Table 4.
Antimicrobial-resistant phenotypes of Enterobacteriaceae and number of isolates with confirmed resistance genes blaTEM, blaCTX–M, and ampC.
| Isolates | Antibiotics | Genes | Number of genes | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| ZOX | FOX1 | FOX2 | AMP | CZ | bla TEM | ampC | bla CTX–M | 0 | 1 | 2 | |
| Buttiauxella gavinae | – | 1 | 1 | – | 1 | – | – | – | 1 | – | – |
| Citrobacter freundii | – | 1 | 1 | – | 1 | 1 | 1 | – | – | – | 1 |
| Citrobacter gillenii | – | 1 | 1 | 2 | – | 3 | – | 1 | 1 | 2 | 1 |
| Escherichia coli | 2 | 2 | 2 | – | 1 | 1 | 2 | – | – | 1 | 1 |
| Enterobacter bugandensis | – | 1 | 1 | – | – | – | 1 | – | – | 1 | – |
| Enterobacter cloacae | 1 | 4 | 4 | 3 | 3 | 4 | 3 | – | – | 1 | 3 |
| Enterobacter kobei | 1 | 1 | 1 | 1 | 1 | – | 1 | – | – | 1 | – |
| Klebsiella oxytoca | 4 | 8 | 8 | 9 | 4 | 6 | – | 9 | – | 3 | 6 |
| Lelliottia amnigena | 2 | 1 | 3 | – | 1 | 1 | – | – | 2 | 1 | – |
| Raoultella ornithinolytica | 1 | 1 | 2 | 2 | 1 | 2 | 1 | – | – | 1 | 1 |
| Raoultella terrigena | 1 | 1 | 2 | 2 | 1 | 2 | 2 | – | – | – | 2 |
| Total | 12 | 22 | 26 | 19 | 14 | 20 | 11 | 10 | 4 | 11 | 15 |
ceftizoxime (ZOX–CLSI), cefoxitin (FOX1–CLSI), cefoxitin (FOX2–EUCAST), ampicillin (AMP–CLSI, EUCAST), cefazolin (CZ–CLSI).
In general, in 26 isolates identified from the Enterobacteriaceae family, one or two genes were confirmed. Half of all identified isolates had two genes simultaneously. In six isolates confirmed as Klebsiella oxytoca, a combination of the blaTEM and ampC genes was found. Four isolates, namely Buttiauxella gavinae (1), Citrobacter gillenii (1), and Lelliottia amnigena (2), did not have the presence of blaTEM/blaCTX-M/ampC.
4. Discussion
Family Enterobacteriaceae are a group of bacteria that serve as indicators of milk collection hygiene at the farm level. They are also indicators of poor hygiene in milk processing operations at dairies, and consequently, the Enterobacteriaceae can also be detected in final dairy products.
In our study, the presence of Enterobacteriaceae was detected in 12 (85.7%) samples of raw cow’s milk, with an average count of 3.81 log10 ± 1.47 CFU/ml. The number of positive samples ranged from 1.3 to 6.24 log10 CFU/ml. An important finding regarding the effectiveness of heat treatment in dairy plants was that no Enterobacteriaceae were detected after pasteurization, indicating an effective process, but also suggesting that the main findings of our study on resistance could apply only to raw milk bacteria.
In the study by Farhat et al. [29], the presence of Enterobacteriaceae was investigated using selective VRBG agar, and Enterobacteriaceae were detected in all 50 (100%) raw milk samples. The average count reported in their study was significantly higher than our findings, at 7.41 log10 CFU/ml. In another work by Tepeli and Zorba [30], the total number of Enterobacteriaceae ranged from 3.0 to 5.0 log10 CFU/ml in farm milk and from 4.0 to 7.0 log10 CFU/ml in pooled milk samples. A study conducted in Egypt analyzed 100 raw milk samples and found Enterobacteriaceae in 84 samples, with an average of 6.00 log10 ± 5.30 CFU/ml. The highest frequency of positive samples (48.81%) fell within the range of 5.0 to 6.0 log10 CFU/ml [31].
As mentioned in the results section, the most common species detected in our work was Klebsiella oxytoca, followed by Enterobacter cloacae and Citrobacter gillenii. Their presence in bulk tank milk could be due to subclinical mastitis in cows. However, this assertion is limited by the lack of information on the animals’ health status and the antimicrobial use practices on the farms.
A study by Slovak authors published in 2017 reported that enterobacteria in raw milk samples collected in Slovakia ranged from 0.00 to 4.34 log10 CFU/ml [32]. Compared with our study, the maximum Enterobacteriaceae count in that study was lower.
Another study in Slovakia examined the representation of individual bacterial species within the family Enterobacteriaceae, in which the majority of isolates belonged to Citrobacter spp. (21.4%), followed by isolates of Shigella spp. (20.0%), E. coli (14.2%) and Enterobacter spp. (12.8%) [33]. The identification of individual bacterial species in this study was performed similarly to our method using MALDI-TOF MS. Similar results were also presented in the work of Kagkli et al. [34], where the most common genus of the family Enterobacteriaceae in raw milk was Escherichia, Enterobacter, Klebsiella, and Citrobacter, which was consistent with the results of our research. A 2020 study in Egypt also investigated the presence of Enterobacteriaceae in raw milk. This study also included genera that have already been assigned to separate families under the new taxonomic classification. Representatives of the Hafniaceae (17.65%) and Yersiniaceae (Serratia spp., 25.81%) families were among the most frequently isolated from raw milk in this study. Both families currently belong to separate orders within the Enterobacterales. Among the genera of the family Enterobacteriaceae, Klebsiella (15.69%), Enterobacter (7.84%), Escherichia (6.54%), and Citrobacter (3.92%) were recorded, constituting a minority [31].
The situation regarding AMR shows specificities depending on therapeutic practices in human and veterinary medicine in a given country. This applies to both human and animal populations and their mutual connection. Hleba et al. [35] investigated the susceptibility of Enterobacteriaceae to selected antibiotics in milk collected on farms in Slovakia. In this work, antibiotic susceptibility was assessed according to EUCAST criteria. The highest resistance to ampicillin was recorded in milk (57.14%). Compared to our results, these values were comparable. Resistance to streptomycin was 14.28% among milk isolates; according to current EUCAST criteria, no established values are available for this antibiotic. It is interesting to compare resistance to gentamicin: in 2011, it was not detected in raw milk isolates; in contrast, in our study, resistance was detected in milk isolates at almost 60%. It is necessary to account for the significant time gap between the works. The same Slovak authors published another work in 2015, whose objective was to investigate AMR in Enterobacteriaceae isolates from milk and dairy products, and they found results similar to those of their previous work with ampicillin. Similarly, four years later, they did not record resistance to gentamicin. For this reason, it is very important to evaluate AMR in the given country and its antibiotic treatment practices. Intrinsic resistance of some Enterobacteriaceae to beta-lactam antibiotics is due to the presence of inducible chromosomal ampC beta-lactamases, low outer membrane permeability, and active efflux mechanisms. These aspects need to be considered, especially when evaluating the phenotypic manifestation of beta-lactam antibiotic resistance [36].
Raw milk is also a reservoir of antimicrobial resistance genes [36, 37]. In a review of the AMR situation over the past 10 years, which examined 306 studies, it was noted that when investigating resistance and resistance genes in milk, the greatest attention is generally focused on the genus Staphylococcus, and the samples collected primarily represent milk from mastitis dairy cows [38].
Many studies investigating the presence of ESBL genes in Enterobacteriaceae first select isolates based on ESBL phenotype. In a 2024 study [39], the presence of blaTEM, blaSHV, and blaCTX-M resistance genes were investigated in isolates from milk and swab samples collected from the milk collection environment in Ethiopia. The dominant isolates were those in which the presence of the blaTEM (85.0%) and blaCTX–M (78.8%) genes were confirmed. This representation of isolates with confirmed resistance genes was significantly higher than in our work. However, it is necessary to add, for the objectivity of the comparison of the results, that in the study from Ethiopia, resistance genes were investigated only in the so-called ESBL producers. Dey et al. [40] analyzed ESBL-, MBL-, and ampC-producing Enterobacteriaceae in raw milk from farms and dairies. In addition to other ESBL genes, blaTEM and blaCTX–M1 were also examined in ESBL producers. In this work, ESBL genes were detected in 9 isolates, representing 2.13% of all Enterobacteriaceae examined. In our work, there were 15 isolates with detected blaTEM and blaCTX–M genes in raw milk samples, which had a representation of these genes individually or in combination. This study also examined the presence of the ampC genes in milk, which was detected in 28 isolates (6.65%) of the total number, which could not be compared with our data, since the mentioned study investigated several genes responsible for ampC production. In our work, some isolates’ beta-lactam resistance was likely due to intrinsic chromosomal enzymes that were not detected by the chosen primers. It should be noted that our study did not include several other clinically relevant beta-lactamases. For example, K. oxytoca produces the intrinsic OXY beta-lactamase, and in some studies, the presence of Klebsiella spp. has been investigated. blaSHV, blaIMP, blaOXA genes [38, 40]. In our work, the selected PCR targets (blaTEM, blaCTX–M, and plasmid-mediated ampC) cover the major ESBL and ampC plasmid families, but they do not encompass all possible beta-lactamase genes. This represents a limitation, as additional resistance determinants may have remained undetected.
However, our expectation of detecting silent genes in phenotypically susceptible isolates was not confirmed. This suggests that Enterobacteriaceae in raw milk may be selected by prior antimicrobial exposure. As mentioned, the limitation of sample size can have lacked any silent genes, and it is essential to consider the intrinsic resistance of some species of Enterobacteriaceae against some beta-lactam agents. From this perspective, awareness of AMR development at the individual farm level is essential for managing bacterial mastitis and other infections.
5. Conclusions
This study investigated the presence of the family Enterobacteriaceae (sensu stricto) as a potential source of AMR in milk before and after pasteurization. Enterobacteriaceae were present in 12 of all the milk samples tested (raw and pasteurized). The relatively small sample size substantially limits our findings and limits the extent to which the results can be generalized to broader populations. Our effort was to precisely identify isolates of the family Enterobacteriaceae according to current taxonomy and to evaluate phenotypic resistance as objectively as possible, using two independent assessment systems. Overall, the most commonly identified species were Klebsiella oxytoca, followed by Citrobacter gillenii and Enterobacter cloacae. Most isolates showed resistance to cefoxitin and ampicillin. Antimicrobial susceptibility testing was performed using two systems to assess potential discrepancies. Significant differences were observed, particularly between ofloxacin and gentamicin. Resistance to ofloxacin was 53.3% according to EUCAST (2023), compared to only 3.3% based on CLSI (2023). Similarly, resistance to gentamicin was 56.7% according to EUCAST, versus 16.7% according to CLSI. Such discrepancies can significantly affect the final interpretation of resistance data and raise concerns regarding the simultaneous use of both systems in scientific studies. Importantly, no Enterobacteriaceae isolates were detected in any of the pasteurized milk samples. The evaluation of the efficiency of heat treatment in these dairy plants is important for public health, given our results (the presence of Enterobacteriaceae with a high prevalence of beta-lactamase genes in raw milk). Ensuring food safety must be a daily priority. Raw milk serves as the primary input for dairy products, and its microbial quality is vital for food safety. Increased awareness of bacteria that pose a health risk and the potential spread of AMR should be clear evidence to discourage the consumption of raw milk or its products, despite the long-standing tradition in some countries. Our findings highlight that raw milk can serve as a reservoir for multidrug-resistant Enterobacteriaceae with the potential to be transmitted to humans. Implementing strict pasteurization and hygiene measures is therefore essential to reduce this risk.
Acknowledgment
The authors gratefully acknowledge the financial support provided by the Slovak Research and Development Agency under Contract no. APVV-22-0457. Also, this study was supported by the Visegrad Fund project no. 22420065: Non-antibiotic approaches to control mastitis in dairy cows. The project is co-financed by the governments of Czechia, Hungary, Poland, and Slovakia through Visegrad Grants from the International Visegrad Fund. The mission of the fund is to advance ideas for sustainable regional cooperation in Central Europe.
List of abbreviations
AMP, ampicillin; ampC, aminopenicillins inactivated by cephalosporinases; AMR, antimicrobial resistance; BLAST, Basic Local Alignment Search Tool; C, chloramphenicol; CFU, colony forming units; CLSI, Clinical and Laboratory Standards Institute; CN, gentamicin; CP, carbapenemases; CZ, cefazolin; DDM, disc diffusion method; DNA, Deoxyribonucleic Acid; EMBL, European Molecular Biology Laboratory; ESBL, extended-spectrum beta-lactamase; EUCAST, European Committee on Antimicrobial Susceptibility Testing; FOX, cefoxitin; GMP, Good Manufacturing Practice; HACCP, Hazard Analysis and Critical Control Point; ISO, International Organization for Standardization; K, kanamycin; MALDI-TOF MS, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry; MBL, metallo-beta-lactamases; OFX, ofloxacin; PCR, polymerase chain reaction; pH, potential of hydrogen, S, streptomycin; 16S rRNA, 16S ribosomal ribonucleic acid; TE, tetracycline; VRBG, violet red bile glucose; ZOX, ceftizoxime; °C, degree Celsius; gm, gram; h, hour; µg, microgram; ml, milliliter; mm, millimeter; min, minute; sec, second.
Data availability
The data presented in this study are available from the corresponding author upon reasonable request.
Conflicts of interest
The authors declare there is no conflict of interest in this research study.
Author contributions
ZH and ED contributed to the conception and design of the study. ZH, ED, and VL were involved in sample collection and investigation. FZ prepared the initial draft of the manuscript. VL and FZ performed the statistical analyses and interpretation. ZH and ED participated in the preparation and critical review of the manuscript. All authors have read and agreed to the published version of the manuscript.
Supplementary material
Supplementary Table S1.
List of all identified isolates by the PCR and MALDI-TOF MS method and their belonging to the family Enterobacteriaceae.
| No. of isolate | PCR | MALDI-TOF MS | Family Enterobacteriaceae | No. of isolate | PCR | MALDI-TOF MS | Family Enterobacteriaceae |
|---|---|---|---|---|---|---|---|
| 1 | + | Klebsiella oxytoca | + | 31 | + | Hafnia alvei | – |
| 2 | + | Klebsiella oxytoca | + | 32 | + | Enterobacter cloacae | + |
| 3 | + | Lelliottia amnigena | + | 33 | + | Hafnia alvei | – |
| 4 | + | Hafnia alvei | – | 34 | + | Hafnia alvei | – |
| 5 | + | Lelliottia amnigena | + | 35 | + | Klebsiella oxytoca | + |
| 6 | + | Hafnia alvei | – | 36 | + | Serratia liquefaciens | – |
| 7 | + | Klebsiella oxytoca | + | 37 | + | Hafnia alvei | – |
| 8 | + | Lelliottia amnigena | + | 38 | + | Hafnia alvei | – |
| 9 | + | Citrobacter gillenii | + | 39 | + | Hafnia alvei | – |
| 10 | + | Hafnia alvei | – | 40 | + | Enterobacter cloacae | + |
| 11 | + | Enterobacter cloacae | + | 41 | + | Hafnia alvei | – |
| 12 | + | Hafnia alvei | – | 42 | + | Klebsiella oxytoca | + |
| 13 | + | Hafnia alvei | – | 43 | + | Enterobacter cloacae | + |
| 14 | + | Hafnia alvei | – | 44 | – | Hafnia alvei | – |
| 15 | – | Hafnia alvei | – | 45 | + | Hafnia alvei | – |
| 16 | – | Hafnia alvei | – | 46 | + | Citrobacter gillenii | + |
| 17 | + | Klebsiella oxytoca | + | 47 | + | Klebsiella oxytoca | + |
| 18 | + | Hafnia alvei | – | 48 | – | Hafnia alvei | – |
| 19 | + | Citrobacter gillenii | + | 49 | + | Serratia liquefaciens | – |
| 20 | + | Buttiauxella gavinae | + | 50 | + | Enterobacter kobei | + |
| 21 | + | Hafnia alvei | – | 51 | – | Hafnia alvei | – |
| 22 | + | Serratia liquefaciens | – | 52 | – | Hafnia alvei | – |
| 23 | + | Citrobacter gillenii | + | 53 | + | Escherichia coli | + |
| 24 | + | Klebsiella oxytoca | + | 54 | + | Raoultella terrigena | + |
| 25 | + | Hafnia alvei | – | 55 | + | Klebsiella oxytoca | + |
| 26 | + | Raoultella ornithinolytica | + | 56 | + | Raoultella ornithinolytica | + |
| 27 | + | Hafnia alvei | – | 57 | + | Escherichia coli | + |
| 28 | + | Pantoea agglomerans | – | 58 | + | Raoultella terrigena | + |
| 29 | + | Hafnia alvei | – | 59 | + | Citrobacter freundii | + |
| 30 | + | Enterobacter bugandensis | + |
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Associated Data
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Data Availability Statement
The data presented in this study are available from the corresponding author upon reasonable request.