Occurrence of Enterococci in Mastitic Cow's Milk and their Antimicrobial Resistance.

INTRODUCTION: The aim of the study was to evaluate the occurrence of enterococci in inflammatory secretions from mastitic bovine udders and to assess their antimicrobial resistance.
MATERIAL AND METHODS: A total of 2,000 mastitic milk samples from cows were tested in 2014-2017. The isolation of enterococci was performed by precultivation in buffered peptone water, selective multiplication in a broth with sodium azide and cristal violet, and cultivation on Slanetz and Bartley agar. The identification of enterococci was carried out using Api rapid ID 32 strep kits. The antimicrobial susceptibility was evaluated using the MIC technique.
RESULTS: Enterococci were isolated from 426 samples (21.3%). Enterococcus faecalis was the predominant species (360 strains), followed by E. faecium (35 isolates), and small numbers of others. The highest level of resistance was observed to lincomycin, tetracycline, quinupristin/dalfopristin (Synercid), erythromycin, kanamycin, streptomycin, chloramphenicol, and tylosin. Single strains were resistant to vancomycin and ciprofloxacin. All isolates were sensitive to daptomycin. E. faecalis presented a higher level of resistance in comparison to E. faecium, except to nitrofurantoin.
CONCLUSION: The results showed frequent occurrence of enterococci in mastitic cow's milk and confirmed the high rate of their antimicrobial resistance.

Introduction

Udder inflammations (mastitis) are the most frequent and cost-generating illness of dairy cows all over the world (1–3, 9, 17, 20, 21, 25, 26, 28, 29). Many different microorganisms can infect the mammary gland. Due to their different aetiology and epidemiology, contagious and environmental cases of mastitis are distinguished (1, 4, 9, 20, 26, 27). Streptococci are mainly responsible for contagious mastitis. Enterococci are one of the environmental causative agents of mastitis. These opportunistic bacteria are a part of normal physiological gut flora in humans and animals, but over last years they have become one of the main pathogens causing numerous infections in humans, mainly those hospital-acquired, such as bacteraemia and infections of the urinary tract, skin, soft tissue, abdomen and pelvis, and central nervous system. These infections are caused mainly by E. faecalis (about 80.0%) and E. faecium (10.0%–15.0%) (3, 6, 14, 16, 19, 22, 31). The high tolerance of enterococci to disadvantageous conditions allows for their long survival in the environment, including in abattoirs. For this reason, potential infections of the mammary gland are easy and simple. Enterococci are characterised by a high level of resistance to many antibacterial substances, both by intrinsic and acquired mechanisms. Due to their ability to acquire and transfer resistance-determining genes to other bacteria, they are perceived as a good indicator of antimicrobial resistance in the environment (15, 20, 22). Another matter of concern is the possibility of the transmission of enterococci from the inflamed udder to humans. The growing consumption of raw, unpasteurised milk and products produced from this milk seems to indicate that there is a possibility of transfer of potentially pathogenic and antimicrobial-resistant enterococci to humans via the food chain. (1, 3, 6, 8, 13, 15, 22, 24, 28, 31, 32). There are no sufficient data from Poland about the occurrence of Enterococcus spp. in mastitic cow’s milk or about their susceptibility to antimicrobials. For this reason, this investigation was undertaken to evaluate the occurrence of enterococci in milk of cows with clinical and subclinical mastitis and to assess their antimicrobial resistance.

Material and Methods

In total, 2,000 milk samples were taken by physicians from individual cows suspected of mastitis. The samples were collected in different parts of Poland and were sent to our laboratory under temperature-controlled conditions. The samples were frozen until analysis. To isolate enterococci, 1 ml of the sample was incubated in buffered peptone water overnight at 37 ±1°C. Then, 100 μL of the culture was transferred into a broth with sodium azide and crystal violet (azide dextrose broth supplemented with 1.6% bromocresol purple and 1.3 mL/L crystal violet, Merck, Germany). After incubation (24 h at 37°C), one loop (10 μL) of the culture was spread on the surface of Slanetz and Bartley agar (Oxoid, UK) and incubated overnight at 37°C. The suspected colonies were identified using the API rapid ID 32 STREP kit (bioMérieux, France) following the manufacturer’s instructions. The antimicrobial resistance of the confirmed Enterococcus isolates was evaluated using the minimum inhibitory concentration (MIC) technique (in Sensititre CMV3AGPF NARMS Plates, Trek Diagnostic Systems, UK) with decreasing levels of tigecycline (TGC), tetracycline (TET), chloramphenicol (CHL), daptomycin (DAP), streptomycin (STR), tylosin tartrate (TYLT), quinupristin/dalfopristin (SYN), linezolid (LZD), nitrofurantoin (NIT), penicillin (PEN), kanamycin (KAN), erythromycin (ERY), ciprofloxacin (CIP), vancomycin (VAN), lincomycin (LIN), and gentamycin (GEN). All investigations were performed with a Sensititre Autoreader device (Trek, UK). The reference E. faecalis ATCC 29212 strain was used as a control. The results were interpreted automatically, according to CLSI standards. For comparison of the resistance to each substance possessed by E. faecalis and E. faecium, the Z test for two independent proportions was used. For trends evaluation, the mobile means method was applied.

Results

Enterococcus spp. bacteria were isolated from 426 samples (21.3%). In this number, 360 isolates were identified as Enterococcus faecalis and 35 as E. faecium. A small number of isolated specimens were identified as E. hirae, E. casseliflavus, E. durans, and E. avium. They were not included in the result analysis. The results of antimicrobial resistance of the isolated Enterococcus spp., E. faecalis, and E. faecium strains are summarised in Table 1 and Fig. 1. The highest Enterococcus spp. resistance was to lincomycin (82.16%), followed by tetracycline (61.5%), Synercid (60.8%), erythromycin (48.83%), kanamycin (47.42%), streptomycin (46.48%), chloramphenicol (44.83%), and tylosin (42.49%). The lowest rates of resistance were to vancomycin and ciprofloxacin (0.94 and 0.47%, respectively). All strains were sensitive to daptomycin. As shown in Table 1 and Fig. 1, significant differences in resistance between E. faecalis and E. faecium were observed, especially to chloramphenicol, erythromycin, nitrofurantoin, quinupristin/dalfopristin (Synercid), streptomycin, and tetracycline. In total, the isolates of E. faecium presented lower resistance than E. faecalis, except to nitrofurantoin.

Table 1

Antimicrobial resistance of enterococci isolated from mastitic cow’s milk (n = 2,000)

		Number/% of resistant strains
Antimicrobials	Enterococcus spp.	Enterococcus faecalis	Enterococcus faecium
	(n = 426)	(n = 360)	(n = 35)
Chloramphenicol (CHL)	191/44.84	173/49.43	7/20.0
Ciprofloxacin (CIP)	2/0.47	2/0.57	1/2.86
Daptomycin (DAP)	0	0	0
Erythromycin (ERY)	208/48.83	177/50.57	11/31.43
Gentamicin (GEN)	47/11.03	31/8.86	2/5.71
Kanamycin (KAN)	202/47.42	196/56.0	15/42.86
Linkomycin (LIN)	350/82.16	294/84.0	25/71.43
Linezolid (LZD)	15/3.52	14/4.0	1/2.86
Nitrofurantoin (NIT)	27/6.34	17/4.86	13/37.14
Penicillin (PEN)	11/2.58	12/3.43	2/5.71
Qunupristin/dalfopristin (SYN)	259/60.8	296/84.57	15/42.86
Streptomycin (STR)	198/46.48	167/47.71	10/28.57
Tetracycline (TET)	262/61.5	287/82.0	11/31.43
Tigecycline (TGC)	5/1.17	4/1.14	0
Tylosin (TYLT)	181/42.49	170/48.57	11/31.43
Vancomycin (VAN)	4/0.94	3/0.86	1/2.86

Fig. 1

Comparison of the antimicrobial resistance of enterococci isolated from mastitic milk samples. For antibiotic abbreviations see Table 1

The antimicrobial resistance trends of E. faecalis isolated in 2014–2017 are shown in Table 2 and Fig. 2. Because of the low number of E. faecium isolates, the trends for this species were not analysed. The presented data indicate some differences between the years, especially for chloramphenicol, erythromycin, lincomycin, streptomycin, tetracycline, and tylosin, for which a decrease in resistance was observed. On the basis of the results, 53 resistance profiles were established. In total, 193 E. faecalis strains (45.31%) were resistant to at least three substances from three different groups (multiresistant strains).

Table 2

Trends in antimicrobial resistance of Enterococcus faecalis in 2014–2017

		Number/% of resistant strains
Antimicrobials	Total	2014	2015	2016	2017
	(n = 360)	(n = 127)	(n = 64)	(n = 90)	(n = 79)
Chloramphenicol (CHL) Ciprofloxacin (CIP)	173/49.43 2/0.57	89/70.08 0	27/42.19 0	31/34.44 1/1.11	26/32.91 1/1.27
Daptomycin (DAP)	0	0	0	0	0
Erythromycin (ERY)	177/50.57	92/72.44	29/45.31	29/32.22	27/34.18
Gentamicin (GEN)	31/8.86	4/3.15	8/12.5	10/11.11	9/11.39
Kanamycin (KAN)	196/56.0	90/70.87	35/54.69	37/41.11	34/43.04
Linkomycin (LIN)	294/84.0	116/91.34	44/68.75	71/78.89	63/79.75
Linezolid (LZD)	14/4.0	2/1.57	8/12.5	3/3.33	1/1.27
Nitrofurantoin (NIT)	17/4.86	2/1.57	8/12.5	4/4.44	3/3.8
Penicillin (PEN)	12//3.43	6/4.72	1/1.56	2/2.22	3/3.8
Qunupristin/dalfopristin (SYN)	296/84.57	106/83.46	42/65.63	81/90.0	67/84.81
Streptomycin (STR)	167/47.71	89/70.08	19/29.69	31/34.44	28/35.44
Tetracycline (TET)	287/82.0	111/87.4	42/65.63	71/78.89	63/79.75
Tigecycline (TGC)	4/1.14	2/1.57	1/1.56	1/1.11	0
Tylosin (TYLT)	170/48.57	82/64.57	27/42.19	29/32.22	32/40.51
Vancomycin (VAN)	3/0.86	2/1.57	0	1/1.11	0

Fig. 2

Trends in antibacterial resistance of E. faecalis in 2014–2017 For antibiotic abbreviations see Table 2

Discussion

Opinions concerning the occurrence of enterococci in mastitic cow’s milk vary. Some authors indicate a rare occurrence of enterococci in mastitic milk samples. Petersson-Wolfe et al. (28) showed 0.3% to 1.3% prevalence in milk samples from cows with subclinical mastitis in Sweden. Botrel et al. (2) isolated these bacteria in 2.4% of milk samples from cows with clinical mastitis and 3.1% in subclinical cases in France. Similar findings were reported by Gürler et al. (15) who isolated Enterococcus spp. in 3.26% of milk samples from subclinically mastitic cows in Turkey. Concurring results were described by other authors in Sudan (11), Slovakia (18), and Poland (24). In contrast, Kuyucouglu et al. (23) isolated enterococci from 10.9% of mastitic milk samples in Turkey. Our results are much more similar to those reported by three groups: Cameron et al. (5) who recovered enterococci from 15.25% of samples tested in Canada; Cervinkova et al. (7) who isolated these bacteria in 16.1% of samples in the Czech Republic; and Kateete et al. (21) who isolated enterococci in 19.5% of samples in Uganda. According to Hamzah et al. (17), enterococci were present in 60.0% of mastitic milk samples collected in Iraq. The cited data showed enterococci diversity in mastitic milk samples depending on geographical region and animal production specifics in different countries. In our experiments, E. faecalis was the predominant species, similar to the results presented by Cameron et al. (5), Kunyucouglu et al. (23), and Hamzah et al. (17). In studies performed by Kateete et al. (21), E. faecium was most often isolated, whereas Klimiene et al. (22) pointed to E. durans as the predominant strain in Lithuania.

Enterococci exploit several mechanisms of intrinsic and acquired resistance to antimicrobials. The intrinsic resistance concerns β-lactams, cephalosporins, clindamycin, and low concentrations of aminoglycosides, whereas the acquired one reduces susceptibility to tetracyclines, ciprofloxacin, erythromycin, linezolid, daptomycin, quinupristin/dalfopristin, and vancomycin (8, 15). This regularity, described by many authors, seems to be partially confirmed in our results. In general, almost all authors indicated a high level of antimicrobial resistance of enterococci; however, this level differs by species, substance, and country. The enterococci isolated from mastitic milk samples in Poland resisted these therapeutics to a greater extent than those from other countries.

According to the data presented in Table 1 and Fig.1, E. faecalis demonstrates a higher rate of resistance than E. faecium, except as it resists nitrofurantoin (4.86% and 37.14%, respectively). The data presented by other authors are not clear. According to Klimiene et al. (22), E. faecium showed higher resistance to β-lactams than E. faecalis. El Zubeir et al. (11) noted the wide range of resistance of enterococci to antimicrobials, especially to enrofloxacin, kanamycin, gentamicin, lincomycin, and oxacillin. According to Erbas et al. (12), 1.1% of the isolates in Turkey showed resistance to vancomycin, 10.7% to chloramphenicol, 28.7% to erythromycin, and 81.% to tetracycline. Fabianova et al. (13) noted 4.5 % of strains resistant to vancomycin, 2.1% to teicoplanin, 13.0 % to erythromycin, 39.1% to gentamicin, 45.6% to tetracycline, and 93.5% to ampicillin. There are no data to which to compare the trends in antimicrobial resistance of enterococci in the analysed period (2014–2017). In our opinion, the obtained results (Table 2, Fig. 2) should only be treated as tentative because the samples were not taken in the same places each year. The frequent occurrence of antimicrobial-resistant enterococci in mastitic milk samples indicates a potential possibility of transfer of these bacteria to humans, especially when consuming unpasteurised milk because these bacteria are also isolated from healthy cows prior to exhibition of mastitis symptoms (7). Similar markers of pathogenicity and resistance were detected in strains isolated from food of animal origin and from humans (6, 8, 10, 11, 13). Our results confirmed the frequent presence of the resistant enterococci in mastitic milk, which can be considered as a potential risk to humans.