- Email: [email protected]
In vitro and in vivo safety analysis of Enterococcus faecium 2C isolated from human breast milk
Accepted Manuscript In vitro and in vivo safety analysis of Enterococcus faecium 2C isolated from human breast milk Soodabeh Khalkhali, Naheed Mojgani PII:
S0882-4010(17)30148-1
DOI:
10.1016/j.micpath.2018.01.012
Reference:
YMPAT 2729
To appear in:
Microbial Pathogenesis
Received Date: 15 February 2017 Revised Date:
13 November 2017
Accepted Date: 8 January 2018
Please cite this article as: Khalkhali S, Mojgani N, In vitro and in vivo safety analysis of Enterococcus faecium 2C isolated from human breast milk, Microbial Pathogenesis (2018), doi: 10.1016/ j.micpath.2018.01.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
In vitro and in vivo safety analysis of Enterococcus faecium 2C isolated from human breast milk Soodabeh Khalkhali1,2, Naheed Mojgani3*
2
Department of Microbiology, Shiraz Branch, Islamic Azad University, Shiraz, Iran.
RI PT
1
Department of Microbiology, Fars Research and Science Branch, Islamic Azad University, Fars, Iran 3
Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension
SC
Organization, Karaj, IR Iran
M AN U
Corresponding Author: Naheed Mojgani, Razi Vaccine and Serum Research Institute Hessarak, Karaj, IR Iran, P.O. Box 31975/148, Tel: +98 9121646063
EP
TE D
Corresponding Author Email: [email protected]
AC C
Running title: Safety considerations for Enterococcus faecium as a probiotic candidate
1
ACCEPTED MANUSCRIPT
Abstract: Introduction: Safety analysis of probiotic bacteria is an obligatory characteristic to be evaluated prior to application in food or pharmacological products. This study was designed to evaluate in
RI PT
vitro and in vivo safety parameters of Enterococcus faecium 2C strain, a probiotic candidate isolated from human breast milk.
Material and methods: E.faecium 2C was studied for its hemolytic activity and phenotypic
SC
antibiotics resistance profile. In vivo safety of the mentioned Enterococcus strain was studied by determining acute oral toxicity in Wistar Male rats. The animals were randomly divided into two
M AN U
groups of 3 animals each. The test group animals were gavaged daily with bacterial dose of 1 x 1011 CFU/kg of animal body weight for 21 consecutive days. The animals in control group received normal basal diet without any supplementations. Hematological and biochemical parameters, organ weight, body weight and common health features of the animals were
TE D
recorded.
Results: E.faecium 2C appeared non-hemolytic and sensitive to the majority of the tested antibiotics. The Wistar male rats fed orally with the mentioned bacterial suspensions survived the
EP
test period, and showed normal growth and development. No adverse effects on the general health condition, behavior, and growth were seen in the treated animals compared to control
AC C
group. Additionally, no significant changes in the hematological results, blood biochemistry, organ weights and histopathology of the rats in treatment groups were observed. None of the vital organs of the treated animals showed signs of bacteremia or infectivity. Conclusion: E.faecium 2C strain isolated from human breast milk might be considered safe for use in probiotic formulations intended for man and animals. Keywords: Enterococcus faecium; Probiotic; in vivo Safety; Breast milk; Wistar rats
2
ACCEPTED MANUSCRIPT
Introduction
Probiotics are a group of viable bacteria or yeast with huge beneficial effects on host health [1,
RI PT
2]. A number of investigations have demonstrated the role of probiotics in preventing and treating specific disorders in human and animals [3, 4]. Among the most widely studied group of probiotic bacteria, lactic acid bacteria (LAB) including Lactobacillus, Lactococcus, Pediococcus,
SC
and Enterococcus are well known owing to their generally recognized as safe (GRAS) status [5]. The desirable health benefits of Enterococcal strains have previously been reported by several
M AN U
researchers, which led to their approval for use in a number of probiotic formulations for human and animal [5]. Recently, our investigations on human breast milk lead to identification of Enterococcus faecium 2C strain with probiotic properties [6]. During these investigations, we were able to reveal the crucial probiotic characteristics of the mentioned Enterococcus strain including their acid and bile resistance, survival in simulated gastric and intestinal conditions and
TE D
adhesion to Caco2 cell lines. However, Enterococci are well known as opportunistic pathogens and some strains might harbor pathogenic determinants, and thus it is essential to evaluate their
EP
safety [7, 8]. In general, a number of phenotypic and genotypic virulence traits are evaluated in a candidate probiotic bacteria before considering them safe for use. In a recent investigation, we
AC C
were able to show in vitro safety of E.faecium 2C (TA0033) using biochemical and molecular methods [6]. E.faecium 2C lacked the virulence genes including agg, cylA, efaA, hyl, esp, vancomycin resistance (VanA,VanB, VanC) and biogenic amine (hdc1, hdc2, tdc, ldc, odc). The mentioned strain also appeared DNase, gelatinase, hyaluronidase, lecithinase and lipase negative.
Although in vitro evaluations of virulence traits are a pre-requisite of an Enterococcus probiotic candidate strain, in vivo studies in appropriate animal model are essential for confirming their
3
ACCEPTED MANUSCRIPT
safety. For in vivo studies, the animal of choice for acute oral toxicity studies is the rat, as they are shown to be sensitive to toxic effects of a variety of drugs and chemicals [9]. These animals are also considered standard animal model for repeated dose toxicology studies as per the
RI PT
regulatory requirements [9]. Additionally, previous investigations revealed that hematological investigations are valuable diagnostic tools for evaluating health in human and animals [10-14]. In this study, we aimed to ascertain the safety of E.faecium 2C as a probiotic candidate by
SC
assessing their phenotypic antibiotic resistance profile, acute oral toxicity, and bacterial
M AN U
translocation in Albino Wistar rat model.
Materials and methods
Bacterial Strains and Culture Conditions
E.faecium 2C (accession number KX158836) was cultured in MRS (DeMan Rogosa and Sharpe, Merck, USA), KF agar supplemented with 1% TTC (2, 3, 5-Triphenyll-tetrazolium chloride,
TE D
SIGMA, UK) and Kanamycin Aesculin-Azide Agar (KAA, OXOID, UK). The isolate grew well in the respective media at 37 ºC for 24 h in aerobic conditions. Stock cultures were maintained in
Hemolytic Activity
EP
20% glycerol solution at -70 ºC.
AC C
In order to evaluate the hemolytic activity, E.faecium 2C was cultured on blood agar plates (containing 5% defibrinated sheep blood) and incubated at 37 °C for 24 h. The presence of clear, green and no hemolysis zones around the bacterial colonies indicated the presence of βhemolysis, α-hemolysis, and or γ-hemolysis, respectively. Phenotypic Antibiotics resistance profile The phenotypic resistance pattern of E.faecium 2C against a number of antibiotics was tested using disc diffusion assay which is described previously [15]. The antibiotic discs which are used 4
ACCEPTED MANUSCRIPT
in the study were as follow: ampicillin 10 µg (AM), chloramphenicol 30 µg (C), clindamycin 2 µg (CL), erythromycin 15 µg (E), tetracycline 30 µg (TET), gentamicin 10 µg (G), kanamycin 30 µg (K) and vancomycin 30 µg (V). The discs were purchased from Paditan Teb Co. (Tehran,
RI PT
Iran). Fifty µl of the inoculum prepared by adjusting freshly grown overnight cultures to 0.5 McFarland standards (1.5 x 108 CFU/mL of bacteria), were streaked onto the surface of agar plates with sterile swabs. Corresponding antibiotic discs were placed on the agar plates in
SC
duplicates and incubated at 37 ºC for 24 to 48 h. All experiments were performed in triplicate. The plates were evaluated regarding the inhibition zone around the discs and their diameters
M AN U
were measured (millimeters). The phenotypic antibiotics resistance profile was reported as sensitive (S), intermediate (I) and resistant (R) in a standard manner [16]. Minimum Inhibitory Concentration (MIC) for Antibiotic Resistance Based on the ISO 10932/IDF 233 standard protocol, the minimum inhibitory concentrations
TE D
(MICs) of E.faecium 2C were identified using broth microdilution [17]. The antibiotics (Padtan Teb Co., Iran) were reconstituted in appropriate buffer according to manufacturer’s recommendations. Final inoculum size of E.faecium 2C added to respective antibiotic solutions
EP
was 3x105 CFU/mL. After incubating the plates at the earlier mentioned conditions, the MICs values were measured. Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC
AC C
2921 were used as reference strains. Animal ethics
All animals used in the study were handled based on the standard criteria obtained from Guide for Care and Use of Laboratory Animals protocol (NIH US publication 86-23 revised 1985;http://oacu.od.nih.gov/regs/guide/guidex.htm) and the protocol of the experiments was approved by the Ethical Committee of Islamic Azad University, Shiraz Branch..
5
ACCEPTED MANUSCRIPT
Acute toxicity study A total of 6 male albino Wistar rats (220 ± 10 g) obtained from Department of Laboratory Animal Research, Razi Vaccine and Serum Research Institute, Iran were divided randomly into
RI PT
two groups, 3 each. The animals were caged individually in rooms with controlled temperature and humidity (22 ºC with 48% humidity), with 12 h light-dark cycle. Food and water was provided to the animals ad libitum.
SC
Acute oral toxicity study was performed according to the protocol of the "Organization for Economic Cooperation and Development (OECD) Guideline for the Testing of Chemicals No.
M AN U
423; Acute Oral Toxicity–Acute Toxic Class Method, adopted December 17, 2001". Detailed clinical observations were made prior to the exposure of the animals to test culture. The animals in the test group were gavaged with E.faecium 2C at a concentration of 1.1×1011 CFU/kg, daily for a period of 21 days based on the suggestion of other researchers [18, 19]. The
TE D
animals in control group received basal diet without supplementation with the mentioned bacterial suspension. Immediately after receiving the first dose, general behavior signs of toxicity
EP
including changes in the skin, eyes, and also respiratory, digestive, and central nervous systems were recorded methodically.
Observations were also made on behavior patterns, such as
AC C
tremors, convulsions, salivation, stool consistency, lethargy, sleep, and changes in posture and response to handling. The body weight (BW) of the rats was logged on days 0, 7, 14 and 21.
Following an overnight fast, on day 21, the animals in both the groups were sacrificed with ether anesthesia. The final BW of the rats was recorded following overnight fasting before sacrifice and the weight gain recorded systematically [20, 21].
Hematological analysis 6
ACCEPTED MANUSCRIPT
A day prior to the end of test period (day 21), the animals were fasted overnight and blood samples were obtained by cardiac puncture after anaesthetization of the animals. Blood was collected under sterile conditions in ethylene-diamine-tetracetic acid-2K (EDTA-2K) pretreated
RI PT
and non-pretreated tubes for hematological and serum biochemical investigations, respectively. White blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT) and differential leukocyte count were evaluated on the blood samples of E.faecium 2C
SC
treated and non-treated (controls) rats, at Mabna Veterinary Laboratory, Karaj, Iran. Blood Biochemistry
M AN U
The blood urea, creatinine, Alkaline phosphatase (ALP), Asparatate Amino Transferase (AST), Alanine Amino Transferase (ALT), Creatine phosphokinase (CK), Sodium (Na), Potassium (K), Chloride (Cl) and Cholesterol levels were determined using standard kits at Mabna Veterinary
Translocation Assay
TE D
Laboratory, Karaj, Iran.
The vital animal organs (heart, liver, kidney and spleens) were removed under sterile conditions
EP
for gross and microscopic examination [20-23]. The organs were weighed before culturing on appropriate growth media. The dissected organs were homogenized in 0.1% sterile PBS (0.1
AC C
g/mL), serially diluted and 100 microliters of the respective homogenized samples and also blood samples cultured on KF Agar. The plates were incubated aerobically at 37 ºC for a period of 72 h, and observed for the presence or absence of growth during the mentioned period. Statistical analysis
After evaluation of the Gaussian distribution data, student t-test under SPSS software version 18 has been used to analyze the differences of the variables between E.faecium 2C treated in comparison to non-treated rats and a p value less than 0.05 was considered as significant. 7
ACCEPTED MANUSCRIPT
Results Safety of E.faecium 2C, a potentially probiotic strain isolated previously from human breast milk, was assessed by determining the antibiotic resistance and in vivo acute oral toxicity in male
RI PT
albino Wistar rat models. During initial examinations, E.faecium 2C was recorded as nonhemolytic, owing to their inability to lyse sheep red blood cells demonstrated by no zone of clearance around the bacterial colonies on blood agar plates.
SC
Table 1, the antibiotic resistance profile of the test strain is indicated by disc diffusion and microdilution assay. E.faecium 2C was sensitive to the majority of the tested antibiotics during
M AN U
disc diffusion assay, and the determined MIC values for the respective antibiotics were below the standard Cut-off values recommended for probiotic bacteria.
During in vivo investigations, the results demonstrated that oral administration of 1.1×1011 CFU of E.faecium 2C per kg BW, does not affect the survival rate of the tested animals. No mortality
TE D
in the tested albino Wistar rat models in treatment group was seen at the end of test period and all animals not only survived but also appeared healthy, showing normal growth and development pattern. All behavioral signs of the treated animals were comparable to the animals
EP
in the control group. Additionally, no changes in the skin, fur, eyes, mucous membrane, occurrence of secretions or excretions, respiratory, circulatory, autonomic and central nervous
AC C
system in the animals in different treatment groups were seen. As seen in Table 2, no differences in the weights of the treated animals compared to the animals in control group were observed. Overall results indicated the weight gain for the treated animals (220.825 ± 4.84, 260 ± 7.21 and 300 ± 7.94 for day 0, 7 and 21, respectively) was nonsignificantly different (p< 0.1) compared to the controls (222.5 ± 5.21, 224.15 ± 3.85 and 304 ± 8.42 for day 0, 7 and 21, respectively). Table 2 also revealed that the weight of heart, kidney,
8
ACCEPTED MANUSCRIPT
spleen and liver in the Enterococcus faecium treated did not differ when compared to non-treated rats (p< 0.1). Additionally, no substantial effects on the mean feed intake in the treated and the control group
RI PT
animals were recorded and the results were non-significant (p< 0.1).
The hematological analysis shown in Table 3, revealed that the red blood cell count (p= 0.426), platelet count (p= 0.895), white blood cell count (p= 0.465) and hemoglobin (p= 0.543) levels
SC
were similar in both the treatment and control groups, respectively with no significant differences (p≥0.05).
M AN U
During biochemical analysis, E.faecium 2C treatment resulted in decreased serum levels of alanine amino transferase (p= 0.004) and alkaline phosphatase (p= 0.014), while other biochemical
parameters
including
aspartate
amino
transferase
(p=
0.089),
creatine
phosphokinase (p= 0.408), creatinine (p= 0.625), Cholesterol (p= 0.885), sodium (p= 0.544),
TE D
potassium (p= 0.305) and chloride (p= 1.0) levels were not affected (Table 4). Table 5 the variations in organ weight among control and treated rats such as spleen, heart, kidney and liver are recorded. Variation in organ weight among control and treated rats such as
EP
spleen (0.7 to 0.8 g), heart (0.9 to 1 g), kidney (2.1 to 2.4 g), liver (10 to 12 g), respectively, were found to be non-significant (p≥0.05).
AC C
Absence of bacterial growth was observed during culturing of the organs. No pathogenic changes in the organs were seen during gross and microscopic examinations. Discussion
E.faecium is a well-known normal resident of the human gastrointestinal tract and has been successfully used in probiotic preparations [1, 24]. However, some species/strains belonging to genus Enterococcus have been reported as opportunistic pathogens for humans. Hence, safety
9
ACCEPTED MANUSCRIPT
assessment of species and strains belonging to this genus are essential for considering them as a probiotic bacteria and evaluating them for use in different probiotic formulations in the food and feed industry. In our previous study, an E.faecium 2C strain with probiotic potential, from breast
RI PT
milk of healthy young mothers was isolated [6]. In this context, the probiotic potential and in vitro safety parameters of E.faecium 2C including absence of virulence traits and vancomycin resistance were studied by biochemical and molecular analysis.
SC
In this study, the safety of the mentioned strain in vivo conditions was investigated, in order to develop a new probiotic candidate. Acute oral toxicity in male albino rat models was studied by
M AN U
oral administration of a fixed dose of E.faecium 2C via gavage. In the United States, bacteria considered safe for human consumption are assigned ‘Generally Regarded As Safe’ status (GRAS) by the Food and Drug Administration on a case-by-case basis. Similar system is being considered in the European Union (EU), referred to as ‘Qualified Presumption of Safety’ (QPS)
24].
TE D
that aims to harmonize the safety assessment of micro-organisms throughout the food chain [1,
Although enterococci are associated with low pathogenicity, they are considered a cause of
EP
nosocomial infection in immune-compromised patients. It appears that the intrinsic tolerance of the bacteria against several antimicrobial agents is the main reason for the complication [25].
AC C
Based on the definitions of the Qualified Presumption of Safety (QPS), prior to the approval of a bacterial candidate as a potential probiotic, it is necessary to determine the nature of antibiotic resistance including intrinsic or acquired resistance [26]. Hence, antibiotic resistance in these bacteria is an essential criterion that is considered for evaluating the risk of resistance transfer and emergence of antibiotic resistance. In this study, the antibiotic resistance pattern of the selected bacterial strain was initially evaluated by disc diffusion assay, while the MIC (mg/L)
10
ACCEPTED MANUSCRIPT
values were later determined for the antibiotics to which the bacteria were either resistant or mildly sensitive/resistant. All the MIC values for E.faecium 2C were within the standard antibiotic resistance scope proposed by the Scientific Committee on Animal Nutrition (SCAN,
RI PT
European Union) [27].
Acute oral toxicity test is a fundamental test to evaluate the safety of probiotic bacterial strains
SC
[22, 28]. Similar to the results reported by Tompkins and his colleagues [29], E.faecium 2C appeared a safe strain like E.faecium R0026, and the tested rat model treated with E.faecium 2C
M AN U
remained healthy and survived the test dose during 21 days of inoculations. Absence of adverse effects on the body weight and food intake, indicated that the tested candidate strain does not have gross oral toxicity effects on the tested animals’ health status, growth and development. Contrasting results have been reported by Uzokwe et al., who reported that using a commercial E.faecium as supplementary material in food has suspected side effects [30]. Authors have
TE D
reported that E.faecium (C1R1;221) treatment leads to mild centrilobular degeneration of the fatty tissues and contamination of some vital organs such as heart, kidney and liver of the tested
EP
animal model [30].
During our investigation, no changes in urine, hematological examination, and blood
AC C
biochemistry of the tested animal compared to control group were recorded. Previous investigations have reported that serum levels of the liver enzymes are increased by several stressful factors including viral and non-viral hepatitis [31]. However, decreased serum levels of ALT and ALP in the treated animals observed in our studies might suggest a better functioning of the liver in the treated animals than the controls. Increased peripheral blood neutrophils or eosinophils are well known indicators of bacterial infection [32]. In the current study, no difference was detected in the mentioned parameters 11
ACCEPTED MANUSCRIPT
between the animals in treatment and the control group. These results indicate that the rats fed with the Enterococcus strain did not experience infection during the treatment time. Bacterial translocation is a prospective indicator of probiotics toxicity, as it is the first step in the
RI PT
pathogenesis process for many opportunistic indigenous lumen strains [20]. In spite of the high dose administration of E.faecium 2C, the rats showed no signs of bacteremia and/or organ infections. These data imply that high doses of the test probiotic orally administered to rat do not
SC
increase bacterial translocation either to blood or to spleen or liver.
The results of our study confirmed the safety of the tested E.faecium 2C strain, as administration
M AN U
of high doses of the test strain showed no toxic effects on the health of orally fed rats. Similar results have been documented by other investigators, who reported the safety of their tested candidate probiotic strains. For instance, Sudha and his colleagues were able to show that feeding of Bacillus coagulants strain for 2 weeks had no adverse effects on the growth, health
TE D
status, hematology, histology parameters or blood biochemistry of the tested animals [20]. Similarly, Zhou and his colleagues showed that consumption of three probiotics including Bifidobacterium lactics HN019 (DR10), L. rhamnosus HN001 (DR20) and L. acidophilus
EP
HN017 for three weeks did not affect the general health parameters of the treated animals [33]. Based on our results it may be concluded that E.faecium 2C isolated from Iranian human breast
AC C
milk is a safe strain with potential probiotic properties. Conclusion remark
Taken together, the current investigation demonstrated that E.faecium 2C isolated from Iranian human breast milk is a safe probiotic bacterial strain that might be used for producing functional foods.
12
ACCEPTED MANUSCRIPT
Acknowledgment The author would like to thank the Laboratory Animal Department at RVSRI for providing appropriate animal care facilities and cooperating in animal handling including gavage, blood
RI PT
collection and dissection of the animals.
This study was supported by a grant from Islamic Azad University, Shiraz Branch. Conflict of Interest
SC
There is no conflict of interest to declare. References
AC C
EP
TE D
M AN U
[1] Anadon A, Martinez-Larranaga MR, Martinez MA. Probiotics for animal nutrition in the European Union.Regulation and safety assessment. Regulatory Toxicology and Pharmacology. 2006;45:91-5. [2] Salminen S, Wright AV, Morelli L, Marteau P, Brassart D, M.deVos W, et al. Demonstration of safety of probiotics-a review. International Jurnal of Food Microbiology. 1998;44:93-106. [3] Gueimonde M, Salminen S. New methods for selecting and evaluating probiotics. Digestive and Liver Disease. 2006;38:s242-s7. [4] Gaggia F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. International Jurnal of Food Microbiology. 2010;141:S15-S28. [5] Ogier JC, Serror P. Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol. 2008;126:291-301. [6] Khalkhali S, Mojgani N. Bacteriocinogenic Potential and Virulence Traits of Enterococcus faecium and E.faecalis Isolated from Human Milk. Iran J Microbiol. 2017;Article in press. [7] O'Brien J, Crittenden R, C.Ouwehand A, Salminen S. Safety evaluation of probiotics. Trends in Food Science & Technology. 1999;10:418-24. [8] Vankerckhoven V, Huys G, Vancanneyt M, Vael C, Klare I, Marie-Be'ne'dicte R, et al. Biosafety assessment of probiotics used for human consumption:recommendations from the EUPROSAFE project. Trends in Food Science & Technology. 2008;19:102-14. [9] Borriello SP, Hammes WP, Holzapfel W, Marteau P, Schrezenmeir J, Vaara M, et al. Safety of probiotics that contain lactobacilli or bifidobacteria. Clinical Infectious Disease. 2003;36:77580. [10] R.Snydman D. The safety of probiotics. Clinical Infectious Disease. 2008;46:S104-S11. [11] Ishibashi N, Yamazaki S. Probiotics and safety. The American Journal Of Nutrition. 2001;73:465S-70S. [12] Sanders ME, M.A.Akermans L, Haller D, Hammerman C, Heimbach J, Huys G, et al. Safety assessment of probiotics for human use. Gut Microbes. 2010;1:164-85. [13] kaur IK, chopra K, saini A. Probiotics:potential pharmaceutical applications. European journal of pharmaceutical sciences. 2002;15:1-9. [14] Amara AA, Shibl A. Role of probiotics in health improvement,infection control and disease treatment and management. Saudi Pharmceutical Journal. 2013;14:1-8.
13
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[15] Bauer AW, Kirby WMM, Sherris JC, Turk M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology. 1966;45:493-6. [16] Acar JF, Goldstein FW. Disk susceptibility test. Antibiotic in Laboratory Medicine, ed Lorian V. 1991:17-52. [17] ISO E. Milk and milk products: Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococal lactic acid bacteria. ESA2012. p. 109-32. [18] Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM. Characterization of Bacillus probiotics available for human use. Appl Environ Microb. 2004;70:2161-71. [19] Endres JR, Clewell A, Jade KA, Farber T, Hauswirth J, Schauss AG. Safety assessment of a proprietary preparation of a novel Probiotic, Bacillus coagulans, as a food ingredient. Food Chem Toxicol. 2009;47:1231-8. [20] Sudha RM, Sunita M, Sekhar BM. SAFETY STUDIES OF BACILLUS COAGULANS Uniqe IS-2 IN RATS:MORPHOLOGICAL,BIOCHEMICAL AND CLINICAL EVALUATIONS. international Journal of Probiotics and Prebiotics. 2011;6:43-8. [21] Zavisic G, Petricevic1 S, Radulovic Z, Begovic J, Golic N, Topisirovic L, et al. PROBIOTICS FEATURES OF TWO ORAL LACTOBACILLUS ISOLATES. Brazilian Journal of Microbiology. 2012:418-28. [22] Aboderin FI, Oyetayo VO. Haematological Studies of Rats Fed Different Doses of Probiotic, Lactobacillus plantarum, Isolated from Fermenting Corn Slurry. Pakistan Journal of Nutrition 2006;5:102-5. [23] Huang c-c, Nam m-k, Tsai y-t, cheng-chih T. Safety evaluation for multispecies probiotics in a 28-day feeding study in Sprague-Dawley rats. African Journal of Biotechnology Research. 2014;8(7):127-36. [24] Klaenhammer T, Kullen MJ. Selection and design of probiotics. International Jurnal of Food Microbiology. 1999;50:45-57. [25] Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, Rood JI. Gram-positive pathogens: ASM Press; 2000. [26] Rossetti L, Carminati D, Zago M, Giraffa G. A qualified presumption of safety approach for the safety assessment of Grana Padano whey starters. Int J Food Microbiol. 2009;130:70-3. [27] Wright Av. Regulating the safety of probiotics-the European approach. Current pharmaceut des. 2005;11:17-23. [28] Pooja Thakkar P, Modi HA, Prajapati JB. Isolation, characterization and safety assessment of Lactic acid bacterial isolates from fermented food products. International Journal of Current Microbiology and Applied Sciences. 2015;4:713-25. [29] Tompkins TA, Hagen KE, Wallace TD, Fillion-Forte V. Safety evaluation of two bacterial strains used in asian probiotic products. Can J Microbiol. 2008;54:391-400. [30] Uzokwe F, Adegoke C, Ogunbanwo S. Enterococcus species as feed supplement in albino rats. Trakia Journal of Sciences. 2014;12:101. [31] Ebrahim M, Mirzaei V, Bidaki R, Shabani Z, Daneshvar H, Karimi-Googheri M, et al. Are RIG-1 and MDA5 Expressions Associated with Chronic HBV Infection? Viral immunology. 2015;28:504-8. [32] Dahl MV, Greene WH, Quie PG. Infection, dermatitis, increased IgE, and impaired neutrophil chemotaxis: A possible relationship. Arch Dermatol. 1976;112:1387-90.
14
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[33] Zhou JS, Pillidge CJ, Gopal PK, Gill HS. Antibiotic susceptibility profiles of new probiotic lactobacillus and bifidobacterium strains. International Journal of Food Microbiology. 2005;98:211-7.
15
ACCEPTED MANUSCRIPT
Table 1: Antibiotic susceptibility of Enterococcus faecium (KX158835) Abbreviation Zone size
MIC (µg/ml)
Resistant pattern
EFSA breakpoints (mg/L)a
Clindamycin
CC
28
0.25
S
4
Ampicillin
AM
25
0.25
S
24
1
S S
TE
30
2
Erythromycin
E
23
2
Vancomycin
V
27
2
Gentamicin
G
28
Kanamycin
K
30
16
4 4
S
4
1
S
32
8
S
1024
EP
TE D
LAB with MICs higher than the EFSA breakpoints are considered as resistant strains
AC C
a
2
S
M AN U
Tetracycline
SC
Chloramphenicol C
RI PT
Generic name
ACCEPTED MANUSCRIPT
A Sample Control E.faecium
0-day 222.5 ± 5.21 220.825 ± 4.84
7-day 21-day 224.15 ± 3.85 304 ± 8.42 260 ± 7.21 300 ± 7.94
P value
P> 0.1
P> 0.1
SC
P> 0.1
RI PT
Table 2. Body (A) and organ (B) weight of Enterococcus faecium treated and non-treated rats.
Heart 0.914 ± 0.04 1.148 ± 0.14
Kidney 2.426 ± 0.14 2.213 ± 0.21
Spleen 0.745 ± 0.07 0.887 ± 0.08
Liver 10.456 ± 1.75 10.479 ± 1.87
P value
P> 0.1
P> 0.1
P> 0.1
P> 0.1
M AN U
B Sample Control E.faecium
AC C
EP
TE D
The results presented in the table demonstrated that Enterococcus faecium treatment did not affect animals and their organ weights.
ACCEPTED MANUSCRIPT Table 3. Hematological values in Enterococcus faecium treated and non-treated rats.
Groups
RBC (Number*106/µl) 8.415 ± 0.315 8.795 ± 0.135 0.426
Hb (g/dl) 14.5 ± 0.56 14.2 ± 0.14 0.543
PLT (Number*103/µl) 370.00 ± 14.14 371.50 ± 0.70 0.895
RI PT
Control E.faecium P value
WBC (Number/µl) 9900 ± 282 10100 ± 141 0.465
AC C
EP
TE D
M AN U
SC
Table shows that there were not differences between rats treated and non-treatment with Enterococcus faecium regarding WBC, RBC and platelet counts and also Hb value.
ACCEPTED MANUSCRIPT Table 4. Serum levels of AST, ALT, ALP, CK, Cr, Chol, Na, K and Cl in Enterococcus faecium treated in comparison to non-treated rats. Urea (mg/dl) 14.45 ± 0.35
AST ALT ALP CK Cr Chol Na K (U/L) (U/L) (U/L) (U/L) (mg/dl) (mg/dl) (meq/dl) (meq/dl) Control 51.5 73.5 109.5 704.5 0.67 ± 95.0 ± 154.6 ± 6.25 ± ± ± ± ± 0.08 4.24 1.55 0.21 0.70 0.70 0.70 6.36 E.faecium 15.45 ± 48.0 62.5 103.5 694.5 0.71 ± 95.5 ± 153.65 ± 6.60 ± 0.63 ± ± ± ± 0.02 0.70 0.35 0.28 1.41 0.70 0.70 12.02 P value 0.192 0.089 0.004 0.014 0.408 0.625 0.885 0.544 0.305 Table 4 illustrates that treatment with Enterococcus faecium leads to significant decrease in serum levels of ALT and ALP but has no effects on other variables.
SC
RI PT
Sample
AC C
EP
TE D
M AN U
AST: Aspartate amino transferase, ALT: Alanine amino transferase, ALP: Alkaline phosphatase, CK: Creatine phosphokinase, Cr: Creatine, Chol: Cholesterol, Na: Sodium, K: Potassium, Cl: Chloride
Cl (meq/dl) 107 ± 2.82 107 ± 1.41 1.0
ACCEPTED MANUSCRIPT Safety of probiotic bacteria is a key criterion for application as food pharmacological products. Enterococcus faecium isolated from human breast milk show probiotic feature without side effects.
RI PT
The bacteria might be considered safe to use in probiotic formulations intended for man and
AC C
EP
TE D
M AN U
SC
animals.
S0882-4010(17)30148-1
DOI:
10.1016/j.micpath.2018.01.012
Reference:
YMPAT 2729
To appear in:
Microbial Pathogenesis
Received Date: 15 February 2017 Revised Date:
13 November 2017
Accepted Date: 8 January 2018
Please cite this article as: Khalkhali S, Mojgani N, In vitro and in vivo safety analysis of Enterococcus faecium 2C isolated from human breast milk, Microbial Pathogenesis (2018), doi: 10.1016/ j.micpath.2018.01.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
In vitro and in vivo safety analysis of Enterococcus faecium 2C isolated from human breast milk Soodabeh Khalkhali1,2, Naheed Mojgani3*
2
Department of Microbiology, Shiraz Branch, Islamic Azad University, Shiraz, Iran.
RI PT
1
Department of Microbiology, Fars Research and Science Branch, Islamic Azad University, Fars, Iran 3
Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension
SC
Organization, Karaj, IR Iran
M AN U
Corresponding Author: Naheed Mojgani, Razi Vaccine and Serum Research Institute Hessarak, Karaj, IR Iran, P.O. Box 31975/148, Tel: +98 9121646063
EP
TE D
Corresponding Author Email: [email protected]
AC C
Running title: Safety considerations for Enterococcus faecium as a probiotic candidate
1
ACCEPTED MANUSCRIPT
Abstract: Introduction: Safety analysis of probiotic bacteria is an obligatory characteristic to be evaluated prior to application in food or pharmacological products. This study was designed to evaluate in
RI PT
vitro and in vivo safety parameters of Enterococcus faecium 2C strain, a probiotic candidate isolated from human breast milk.
Material and methods: E.faecium 2C was studied for its hemolytic activity and phenotypic
SC
antibiotics resistance profile. In vivo safety of the mentioned Enterococcus strain was studied by determining acute oral toxicity in Wistar Male rats. The animals were randomly divided into two
M AN U
groups of 3 animals each. The test group animals were gavaged daily with bacterial dose of 1 x 1011 CFU/kg of animal body weight for 21 consecutive days. The animals in control group received normal basal diet without any supplementations. Hematological and biochemical parameters, organ weight, body weight and common health features of the animals were
TE D
recorded.
Results: E.faecium 2C appeared non-hemolytic and sensitive to the majority of the tested antibiotics. The Wistar male rats fed orally with the mentioned bacterial suspensions survived the
EP
test period, and showed normal growth and development. No adverse effects on the general health condition, behavior, and growth were seen in the treated animals compared to control
AC C
group. Additionally, no significant changes in the hematological results, blood biochemistry, organ weights and histopathology of the rats in treatment groups were observed. None of the vital organs of the treated animals showed signs of bacteremia or infectivity. Conclusion: E.faecium 2C strain isolated from human breast milk might be considered safe for use in probiotic formulations intended for man and animals. Keywords: Enterococcus faecium; Probiotic; in vivo Safety; Breast milk; Wistar rats
2
ACCEPTED MANUSCRIPT
Introduction
Probiotics are a group of viable bacteria or yeast with huge beneficial effects on host health [1,
RI PT
2]. A number of investigations have demonstrated the role of probiotics in preventing and treating specific disorders in human and animals [3, 4]. Among the most widely studied group of probiotic bacteria, lactic acid bacteria (LAB) including Lactobacillus, Lactococcus, Pediococcus,
SC
and Enterococcus are well known owing to their generally recognized as safe (GRAS) status [5]. The desirable health benefits of Enterococcal strains have previously been reported by several
M AN U
researchers, which led to their approval for use in a number of probiotic formulations for human and animal [5]. Recently, our investigations on human breast milk lead to identification of Enterococcus faecium 2C strain with probiotic properties [6]. During these investigations, we were able to reveal the crucial probiotic characteristics of the mentioned Enterococcus strain including their acid and bile resistance, survival in simulated gastric and intestinal conditions and
TE D
adhesion to Caco2 cell lines. However, Enterococci are well known as opportunistic pathogens and some strains might harbor pathogenic determinants, and thus it is essential to evaluate their
EP
safety [7, 8]. In general, a number of phenotypic and genotypic virulence traits are evaluated in a candidate probiotic bacteria before considering them safe for use. In a recent investigation, we
AC C
were able to show in vitro safety of E.faecium 2C (TA0033) using biochemical and molecular methods [6]. E.faecium 2C lacked the virulence genes including agg, cylA, efaA, hyl, esp, vancomycin resistance (VanA,VanB, VanC) and biogenic amine (hdc1, hdc2, tdc, ldc, odc). The mentioned strain also appeared DNase, gelatinase, hyaluronidase, lecithinase and lipase negative.
Although in vitro evaluations of virulence traits are a pre-requisite of an Enterococcus probiotic candidate strain, in vivo studies in appropriate animal model are essential for confirming their
3
ACCEPTED MANUSCRIPT
safety. For in vivo studies, the animal of choice for acute oral toxicity studies is the rat, as they are shown to be sensitive to toxic effects of a variety of drugs and chemicals [9]. These animals are also considered standard animal model for repeated dose toxicology studies as per the
RI PT
regulatory requirements [9]. Additionally, previous investigations revealed that hematological investigations are valuable diagnostic tools for evaluating health in human and animals [10-14]. In this study, we aimed to ascertain the safety of E.faecium 2C as a probiotic candidate by
SC
assessing their phenotypic antibiotic resistance profile, acute oral toxicity, and bacterial
M AN U
translocation in Albino Wistar rat model.
Materials and methods
Bacterial Strains and Culture Conditions
E.faecium 2C (accession number KX158836) was cultured in MRS (DeMan Rogosa and Sharpe, Merck, USA), KF agar supplemented with 1% TTC (2, 3, 5-Triphenyll-tetrazolium chloride,
TE D
SIGMA, UK) and Kanamycin Aesculin-Azide Agar (KAA, OXOID, UK). The isolate grew well in the respective media at 37 ºC for 24 h in aerobic conditions. Stock cultures were maintained in
Hemolytic Activity
EP
20% glycerol solution at -70 ºC.
AC C
In order to evaluate the hemolytic activity, E.faecium 2C was cultured on blood agar plates (containing 5% defibrinated sheep blood) and incubated at 37 °C for 24 h. The presence of clear, green and no hemolysis zones around the bacterial colonies indicated the presence of βhemolysis, α-hemolysis, and or γ-hemolysis, respectively. Phenotypic Antibiotics resistance profile The phenotypic resistance pattern of E.faecium 2C against a number of antibiotics was tested using disc diffusion assay which is described previously [15]. The antibiotic discs which are used 4
ACCEPTED MANUSCRIPT
in the study were as follow: ampicillin 10 µg (AM), chloramphenicol 30 µg (C), clindamycin 2 µg (CL), erythromycin 15 µg (E), tetracycline 30 µg (TET), gentamicin 10 µg (G), kanamycin 30 µg (K) and vancomycin 30 µg (V). The discs were purchased from Paditan Teb Co. (Tehran,
RI PT
Iran). Fifty µl of the inoculum prepared by adjusting freshly grown overnight cultures to 0.5 McFarland standards (1.5 x 108 CFU/mL of bacteria), were streaked onto the surface of agar plates with sterile swabs. Corresponding antibiotic discs were placed on the agar plates in
SC
duplicates and incubated at 37 ºC for 24 to 48 h. All experiments were performed in triplicate. The plates were evaluated regarding the inhibition zone around the discs and their diameters
M AN U
were measured (millimeters). The phenotypic antibiotics resistance profile was reported as sensitive (S), intermediate (I) and resistant (R) in a standard manner [16]. Minimum Inhibitory Concentration (MIC) for Antibiotic Resistance Based on the ISO 10932/IDF 233 standard protocol, the minimum inhibitory concentrations
TE D
(MICs) of E.faecium 2C were identified using broth microdilution [17]. The antibiotics (Padtan Teb Co., Iran) were reconstituted in appropriate buffer according to manufacturer’s recommendations. Final inoculum size of E.faecium 2C added to respective antibiotic solutions
EP
was 3x105 CFU/mL. After incubating the plates at the earlier mentioned conditions, the MICs values were measured. Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC
AC C
2921 were used as reference strains. Animal ethics
All animals used in the study were handled based on the standard criteria obtained from Guide for Care and Use of Laboratory Animals protocol (NIH US publication 86-23 revised 1985;http://oacu.od.nih.gov/regs/guide/guidex.htm) and the protocol of the experiments was approved by the Ethical Committee of Islamic Azad University, Shiraz Branch..
5
ACCEPTED MANUSCRIPT
Acute toxicity study A total of 6 male albino Wistar rats (220 ± 10 g) obtained from Department of Laboratory Animal Research, Razi Vaccine and Serum Research Institute, Iran were divided randomly into
RI PT
two groups, 3 each. The animals were caged individually in rooms with controlled temperature and humidity (22 ºC with 48% humidity), with 12 h light-dark cycle. Food and water was provided to the animals ad libitum.
SC
Acute oral toxicity study was performed according to the protocol of the "Organization for Economic Cooperation and Development (OECD) Guideline for the Testing of Chemicals No.
M AN U
423; Acute Oral Toxicity–Acute Toxic Class Method, adopted December 17, 2001". Detailed clinical observations were made prior to the exposure of the animals to test culture. The animals in the test group were gavaged with E.faecium 2C at a concentration of 1.1×1011 CFU/kg, daily for a period of 21 days based on the suggestion of other researchers [18, 19]. The
TE D
animals in control group received basal diet without supplementation with the mentioned bacterial suspension. Immediately after receiving the first dose, general behavior signs of toxicity
EP
including changes in the skin, eyes, and also respiratory, digestive, and central nervous systems were recorded methodically.
Observations were also made on behavior patterns, such as
AC C
tremors, convulsions, salivation, stool consistency, lethargy, sleep, and changes in posture and response to handling. The body weight (BW) of the rats was logged on days 0, 7, 14 and 21.
Following an overnight fast, on day 21, the animals in both the groups were sacrificed with ether anesthesia. The final BW of the rats was recorded following overnight fasting before sacrifice and the weight gain recorded systematically [20, 21].
Hematological analysis 6
ACCEPTED MANUSCRIPT
A day prior to the end of test period (day 21), the animals were fasted overnight and blood samples were obtained by cardiac puncture after anaesthetization of the animals. Blood was collected under sterile conditions in ethylene-diamine-tetracetic acid-2K (EDTA-2K) pretreated
RI PT
and non-pretreated tubes for hematological and serum biochemical investigations, respectively. White blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT) and differential leukocyte count were evaluated on the blood samples of E.faecium 2C
SC
treated and non-treated (controls) rats, at Mabna Veterinary Laboratory, Karaj, Iran. Blood Biochemistry
M AN U
The blood urea, creatinine, Alkaline phosphatase (ALP), Asparatate Amino Transferase (AST), Alanine Amino Transferase (ALT), Creatine phosphokinase (CK), Sodium (Na), Potassium (K), Chloride (Cl) and Cholesterol levels were determined using standard kits at Mabna Veterinary
Translocation Assay
TE D
Laboratory, Karaj, Iran.
The vital animal organs (heart, liver, kidney and spleens) were removed under sterile conditions
EP
for gross and microscopic examination [20-23]. The organs were weighed before culturing on appropriate growth media. The dissected organs were homogenized in 0.1% sterile PBS (0.1
AC C
g/mL), serially diluted and 100 microliters of the respective homogenized samples and also blood samples cultured on KF Agar. The plates were incubated aerobically at 37 ºC for a period of 72 h, and observed for the presence or absence of growth during the mentioned period. Statistical analysis
After evaluation of the Gaussian distribution data, student t-test under SPSS software version 18 has been used to analyze the differences of the variables between E.faecium 2C treated in comparison to non-treated rats and a p value less than 0.05 was considered as significant. 7
ACCEPTED MANUSCRIPT
Results Safety of E.faecium 2C, a potentially probiotic strain isolated previously from human breast milk, was assessed by determining the antibiotic resistance and in vivo acute oral toxicity in male
RI PT
albino Wistar rat models. During initial examinations, E.faecium 2C was recorded as nonhemolytic, owing to their inability to lyse sheep red blood cells demonstrated by no zone of clearance around the bacterial colonies on blood agar plates.
SC
Table 1, the antibiotic resistance profile of the test strain is indicated by disc diffusion and microdilution assay. E.faecium 2C was sensitive to the majority of the tested antibiotics during
M AN U
disc diffusion assay, and the determined MIC values for the respective antibiotics were below the standard Cut-off values recommended for probiotic bacteria.
During in vivo investigations, the results demonstrated that oral administration of 1.1×1011 CFU of E.faecium 2C per kg BW, does not affect the survival rate of the tested animals. No mortality
TE D
in the tested albino Wistar rat models in treatment group was seen at the end of test period and all animals not only survived but also appeared healthy, showing normal growth and development pattern. All behavioral signs of the treated animals were comparable to the animals
EP
in the control group. Additionally, no changes in the skin, fur, eyes, mucous membrane, occurrence of secretions or excretions, respiratory, circulatory, autonomic and central nervous
AC C
system in the animals in different treatment groups were seen. As seen in Table 2, no differences in the weights of the treated animals compared to the animals in control group were observed. Overall results indicated the weight gain for the treated animals (220.825 ± 4.84, 260 ± 7.21 and 300 ± 7.94 for day 0, 7 and 21, respectively) was nonsignificantly different (p< 0.1) compared to the controls (222.5 ± 5.21, 224.15 ± 3.85 and 304 ± 8.42 for day 0, 7 and 21, respectively). Table 2 also revealed that the weight of heart, kidney,
8
ACCEPTED MANUSCRIPT
spleen and liver in the Enterococcus faecium treated did not differ when compared to non-treated rats (p< 0.1). Additionally, no substantial effects on the mean feed intake in the treated and the control group
RI PT
animals were recorded and the results were non-significant (p< 0.1).
The hematological analysis shown in Table 3, revealed that the red blood cell count (p= 0.426), platelet count (p= 0.895), white blood cell count (p= 0.465) and hemoglobin (p= 0.543) levels
SC
were similar in both the treatment and control groups, respectively with no significant differences (p≥0.05).
M AN U
During biochemical analysis, E.faecium 2C treatment resulted in decreased serum levels of alanine amino transferase (p= 0.004) and alkaline phosphatase (p= 0.014), while other biochemical
parameters
including
aspartate
amino
transferase
(p=
0.089),
creatine
phosphokinase (p= 0.408), creatinine (p= 0.625), Cholesterol (p= 0.885), sodium (p= 0.544),
TE D
potassium (p= 0.305) and chloride (p= 1.0) levels were not affected (Table 4). Table 5 the variations in organ weight among control and treated rats such as spleen, heart, kidney and liver are recorded. Variation in organ weight among control and treated rats such as
EP
spleen (0.7 to 0.8 g), heart (0.9 to 1 g), kidney (2.1 to 2.4 g), liver (10 to 12 g), respectively, were found to be non-significant (p≥0.05).
AC C
Absence of bacterial growth was observed during culturing of the organs. No pathogenic changes in the organs were seen during gross and microscopic examinations. Discussion
E.faecium is a well-known normal resident of the human gastrointestinal tract and has been successfully used in probiotic preparations [1, 24]. However, some species/strains belonging to genus Enterococcus have been reported as opportunistic pathogens for humans. Hence, safety
9
ACCEPTED MANUSCRIPT
assessment of species and strains belonging to this genus are essential for considering them as a probiotic bacteria and evaluating them for use in different probiotic formulations in the food and feed industry. In our previous study, an E.faecium 2C strain with probiotic potential, from breast
RI PT
milk of healthy young mothers was isolated [6]. In this context, the probiotic potential and in vitro safety parameters of E.faecium 2C including absence of virulence traits and vancomycin resistance were studied by biochemical and molecular analysis.
SC
In this study, the safety of the mentioned strain in vivo conditions was investigated, in order to develop a new probiotic candidate. Acute oral toxicity in male albino rat models was studied by
M AN U
oral administration of a fixed dose of E.faecium 2C via gavage. In the United States, bacteria considered safe for human consumption are assigned ‘Generally Regarded As Safe’ status (GRAS) by the Food and Drug Administration on a case-by-case basis. Similar system is being considered in the European Union (EU), referred to as ‘Qualified Presumption of Safety’ (QPS)
24].
TE D
that aims to harmonize the safety assessment of micro-organisms throughout the food chain [1,
Although enterococci are associated with low pathogenicity, they are considered a cause of
EP
nosocomial infection in immune-compromised patients. It appears that the intrinsic tolerance of the bacteria against several antimicrobial agents is the main reason for the complication [25].
AC C
Based on the definitions of the Qualified Presumption of Safety (QPS), prior to the approval of a bacterial candidate as a potential probiotic, it is necessary to determine the nature of antibiotic resistance including intrinsic or acquired resistance [26]. Hence, antibiotic resistance in these bacteria is an essential criterion that is considered for evaluating the risk of resistance transfer and emergence of antibiotic resistance. In this study, the antibiotic resistance pattern of the selected bacterial strain was initially evaluated by disc diffusion assay, while the MIC (mg/L)
10
ACCEPTED MANUSCRIPT
values were later determined for the antibiotics to which the bacteria were either resistant or mildly sensitive/resistant. All the MIC values for E.faecium 2C were within the standard antibiotic resistance scope proposed by the Scientific Committee on Animal Nutrition (SCAN,
RI PT
European Union) [27].
Acute oral toxicity test is a fundamental test to evaluate the safety of probiotic bacterial strains
SC
[22, 28]. Similar to the results reported by Tompkins and his colleagues [29], E.faecium 2C appeared a safe strain like E.faecium R0026, and the tested rat model treated with E.faecium 2C
M AN U
remained healthy and survived the test dose during 21 days of inoculations. Absence of adverse effects on the body weight and food intake, indicated that the tested candidate strain does not have gross oral toxicity effects on the tested animals’ health status, growth and development. Contrasting results have been reported by Uzokwe et al., who reported that using a commercial E.faecium as supplementary material in food has suspected side effects [30]. Authors have
TE D
reported that E.faecium (C1R1;221) treatment leads to mild centrilobular degeneration of the fatty tissues and contamination of some vital organs such as heart, kidney and liver of the tested
EP
animal model [30].
During our investigation, no changes in urine, hematological examination, and blood
AC C
biochemistry of the tested animal compared to control group were recorded. Previous investigations have reported that serum levels of the liver enzymes are increased by several stressful factors including viral and non-viral hepatitis [31]. However, decreased serum levels of ALT and ALP in the treated animals observed in our studies might suggest a better functioning of the liver in the treated animals than the controls. Increased peripheral blood neutrophils or eosinophils are well known indicators of bacterial infection [32]. In the current study, no difference was detected in the mentioned parameters 11
ACCEPTED MANUSCRIPT
between the animals in treatment and the control group. These results indicate that the rats fed with the Enterococcus strain did not experience infection during the treatment time. Bacterial translocation is a prospective indicator of probiotics toxicity, as it is the first step in the
RI PT
pathogenesis process for many opportunistic indigenous lumen strains [20]. In spite of the high dose administration of E.faecium 2C, the rats showed no signs of bacteremia and/or organ infections. These data imply that high doses of the test probiotic orally administered to rat do not
SC
increase bacterial translocation either to blood or to spleen or liver.
The results of our study confirmed the safety of the tested E.faecium 2C strain, as administration
M AN U
of high doses of the test strain showed no toxic effects on the health of orally fed rats. Similar results have been documented by other investigators, who reported the safety of their tested candidate probiotic strains. For instance, Sudha and his colleagues were able to show that feeding of Bacillus coagulants strain for 2 weeks had no adverse effects on the growth, health
TE D
status, hematology, histology parameters or blood biochemistry of the tested animals [20]. Similarly, Zhou and his colleagues showed that consumption of three probiotics including Bifidobacterium lactics HN019 (DR10), L. rhamnosus HN001 (DR20) and L. acidophilus
EP
HN017 for three weeks did not affect the general health parameters of the treated animals [33]. Based on our results it may be concluded that E.faecium 2C isolated from Iranian human breast
AC C
milk is a safe strain with potential probiotic properties. Conclusion remark
Taken together, the current investigation demonstrated that E.faecium 2C isolated from Iranian human breast milk is a safe probiotic bacterial strain that might be used for producing functional foods.
12
ACCEPTED MANUSCRIPT
Acknowledgment The author would like to thank the Laboratory Animal Department at RVSRI for providing appropriate animal care facilities and cooperating in animal handling including gavage, blood
RI PT
collection and dissection of the animals.
This study was supported by a grant from Islamic Azad University, Shiraz Branch. Conflict of Interest
SC
There is no conflict of interest to declare. References
AC C
EP
TE D
M AN U
[1] Anadon A, Martinez-Larranaga MR, Martinez MA. Probiotics for animal nutrition in the European Union.Regulation and safety assessment. Regulatory Toxicology and Pharmacology. 2006;45:91-5. [2] Salminen S, Wright AV, Morelli L, Marteau P, Brassart D, M.deVos W, et al. Demonstration of safety of probiotics-a review. International Jurnal of Food Microbiology. 1998;44:93-106. [3] Gueimonde M, Salminen S. New methods for selecting and evaluating probiotics. Digestive and Liver Disease. 2006;38:s242-s7. [4] Gaggia F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. International Jurnal of Food Microbiology. 2010;141:S15-S28. [5] Ogier JC, Serror P. Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol. 2008;126:291-301. [6] Khalkhali S, Mojgani N. Bacteriocinogenic Potential and Virulence Traits of Enterococcus faecium and E.faecalis Isolated from Human Milk. Iran J Microbiol. 2017;Article in press. [7] O'Brien J, Crittenden R, C.Ouwehand A, Salminen S. Safety evaluation of probiotics. Trends in Food Science & Technology. 1999;10:418-24. [8] Vankerckhoven V, Huys G, Vancanneyt M, Vael C, Klare I, Marie-Be'ne'dicte R, et al. Biosafety assessment of probiotics used for human consumption:recommendations from the EUPROSAFE project. Trends in Food Science & Technology. 2008;19:102-14. [9] Borriello SP, Hammes WP, Holzapfel W, Marteau P, Schrezenmeir J, Vaara M, et al. Safety of probiotics that contain lactobacilli or bifidobacteria. Clinical Infectious Disease. 2003;36:77580. [10] R.Snydman D. The safety of probiotics. Clinical Infectious Disease. 2008;46:S104-S11. [11] Ishibashi N, Yamazaki S. Probiotics and safety. The American Journal Of Nutrition. 2001;73:465S-70S. [12] Sanders ME, M.A.Akermans L, Haller D, Hammerman C, Heimbach J, Huys G, et al. Safety assessment of probiotics for human use. Gut Microbes. 2010;1:164-85. [13] kaur IK, chopra K, saini A. Probiotics:potential pharmaceutical applications. European journal of pharmaceutical sciences. 2002;15:1-9. [14] Amara AA, Shibl A. Role of probiotics in health improvement,infection control and disease treatment and management. Saudi Pharmceutical Journal. 2013;14:1-8.
13
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[15] Bauer AW, Kirby WMM, Sherris JC, Turk M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology. 1966;45:493-6. [16] Acar JF, Goldstein FW. Disk susceptibility test. Antibiotic in Laboratory Medicine, ed Lorian V. 1991:17-52. [17] ISO E. Milk and milk products: Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococal lactic acid bacteria. ESA2012. p. 109-32. [18] Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM. Characterization of Bacillus probiotics available for human use. Appl Environ Microb. 2004;70:2161-71. [19] Endres JR, Clewell A, Jade KA, Farber T, Hauswirth J, Schauss AG. Safety assessment of a proprietary preparation of a novel Probiotic, Bacillus coagulans, as a food ingredient. Food Chem Toxicol. 2009;47:1231-8. [20] Sudha RM, Sunita M, Sekhar BM. SAFETY STUDIES OF BACILLUS COAGULANS Uniqe IS-2 IN RATS:MORPHOLOGICAL,BIOCHEMICAL AND CLINICAL EVALUATIONS. international Journal of Probiotics and Prebiotics. 2011;6:43-8. [21] Zavisic G, Petricevic1 S, Radulovic Z, Begovic J, Golic N, Topisirovic L, et al. PROBIOTICS FEATURES OF TWO ORAL LACTOBACILLUS ISOLATES. Brazilian Journal of Microbiology. 2012:418-28. [22] Aboderin FI, Oyetayo VO. Haematological Studies of Rats Fed Different Doses of Probiotic, Lactobacillus plantarum, Isolated from Fermenting Corn Slurry. Pakistan Journal of Nutrition 2006;5:102-5. [23] Huang c-c, Nam m-k, Tsai y-t, cheng-chih T. Safety evaluation for multispecies probiotics in a 28-day feeding study in Sprague-Dawley rats. African Journal of Biotechnology Research. 2014;8(7):127-36. [24] Klaenhammer T, Kullen MJ. Selection and design of probiotics. International Jurnal of Food Microbiology. 1999;50:45-57. [25] Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, Rood JI. Gram-positive pathogens: ASM Press; 2000. [26] Rossetti L, Carminati D, Zago M, Giraffa G. A qualified presumption of safety approach for the safety assessment of Grana Padano whey starters. Int J Food Microbiol. 2009;130:70-3. [27] Wright Av. Regulating the safety of probiotics-the European approach. Current pharmaceut des. 2005;11:17-23. [28] Pooja Thakkar P, Modi HA, Prajapati JB. Isolation, characterization and safety assessment of Lactic acid bacterial isolates from fermented food products. International Journal of Current Microbiology and Applied Sciences. 2015;4:713-25. [29] Tompkins TA, Hagen KE, Wallace TD, Fillion-Forte V. Safety evaluation of two bacterial strains used in asian probiotic products. Can J Microbiol. 2008;54:391-400. [30] Uzokwe F, Adegoke C, Ogunbanwo S. Enterococcus species as feed supplement in albino rats. Trakia Journal of Sciences. 2014;12:101. [31] Ebrahim M, Mirzaei V, Bidaki R, Shabani Z, Daneshvar H, Karimi-Googheri M, et al. Are RIG-1 and MDA5 Expressions Associated with Chronic HBV Infection? Viral immunology. 2015;28:504-8. [32] Dahl MV, Greene WH, Quie PG. Infection, dermatitis, increased IgE, and impaired neutrophil chemotaxis: A possible relationship. Arch Dermatol. 1976;112:1387-90.
14
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[33] Zhou JS, Pillidge CJ, Gopal PK, Gill HS. Antibiotic susceptibility profiles of new probiotic lactobacillus and bifidobacterium strains. International Journal of Food Microbiology. 2005;98:211-7.
15
ACCEPTED MANUSCRIPT
Table 1: Antibiotic susceptibility of Enterococcus faecium (KX158835) Abbreviation Zone size
MIC (µg/ml)
Resistant pattern
EFSA breakpoints (mg/L)a
Clindamycin
CC
28
0.25
S
4
Ampicillin
AM
25
0.25
S
24
1
S S
TE
30
2
Erythromycin
E
23
2
Vancomycin
V
27
2
Gentamicin
G
28
Kanamycin
K
30
16
4 4
S
4
1
S
32
8
S
1024
EP
TE D
LAB with MICs higher than the EFSA breakpoints are considered as resistant strains
AC C
a
2
S
M AN U
Tetracycline
SC
Chloramphenicol C
RI PT
Generic name
ACCEPTED MANUSCRIPT
A Sample Control E.faecium
0-day 222.5 ± 5.21 220.825 ± 4.84
7-day 21-day 224.15 ± 3.85 304 ± 8.42 260 ± 7.21 300 ± 7.94
P value
P> 0.1
P> 0.1
SC
P> 0.1
RI PT
Table 2. Body (A) and organ (B) weight of Enterococcus faecium treated and non-treated rats.
Heart 0.914 ± 0.04 1.148 ± 0.14
Kidney 2.426 ± 0.14 2.213 ± 0.21
Spleen 0.745 ± 0.07 0.887 ± 0.08
Liver 10.456 ± 1.75 10.479 ± 1.87
P value
P> 0.1
P> 0.1
P> 0.1
P> 0.1
M AN U
B Sample Control E.faecium
AC C
EP
TE D
The results presented in the table demonstrated that Enterococcus faecium treatment did not affect animals and their organ weights.
ACCEPTED MANUSCRIPT Table 3. Hematological values in Enterococcus faecium treated and non-treated rats.
Groups
RBC (Number*106/µl) 8.415 ± 0.315 8.795 ± 0.135 0.426
Hb (g/dl) 14.5 ± 0.56 14.2 ± 0.14 0.543
PLT (Number*103/µl) 370.00 ± 14.14 371.50 ± 0.70 0.895
RI PT
Control E.faecium P value
WBC (Number/µl) 9900 ± 282 10100 ± 141 0.465
AC C
EP
TE D
M AN U
SC
Table shows that there were not differences between rats treated and non-treatment with Enterococcus faecium regarding WBC, RBC and platelet counts and also Hb value.
ACCEPTED MANUSCRIPT Table 4. Serum levels of AST, ALT, ALP, CK, Cr, Chol, Na, K and Cl in Enterococcus faecium treated in comparison to non-treated rats. Urea (mg/dl) 14.45 ± 0.35
AST ALT ALP CK Cr Chol Na K (U/L) (U/L) (U/L) (U/L) (mg/dl) (mg/dl) (meq/dl) (meq/dl) Control 51.5 73.5 109.5 704.5 0.67 ± 95.0 ± 154.6 ± 6.25 ± ± ± ± ± 0.08 4.24 1.55 0.21 0.70 0.70 0.70 6.36 E.faecium 15.45 ± 48.0 62.5 103.5 694.5 0.71 ± 95.5 ± 153.65 ± 6.60 ± 0.63 ± ± ± ± 0.02 0.70 0.35 0.28 1.41 0.70 0.70 12.02 P value 0.192 0.089 0.004 0.014 0.408 0.625 0.885 0.544 0.305 Table 4 illustrates that treatment with Enterococcus faecium leads to significant decrease in serum levels of ALT and ALP but has no effects on other variables.
SC
RI PT
Sample
AC C
EP
TE D
M AN U
AST: Aspartate amino transferase, ALT: Alanine amino transferase, ALP: Alkaline phosphatase, CK: Creatine phosphokinase, Cr: Creatine, Chol: Cholesterol, Na: Sodium, K: Potassium, Cl: Chloride
Cl (meq/dl) 107 ± 2.82 107 ± 1.41 1.0
ACCEPTED MANUSCRIPT Safety of probiotic bacteria is a key criterion for application as food pharmacological products. Enterococcus faecium isolated from human breast milk show probiotic feature without side effects.
RI PT
The bacteria might be considered safe to use in probiotic formulations intended for man and
AC C
EP
TE D
M AN U
SC
animals.