International Journal of Contemporary Pediatrics
Srinivas A. Int J Contemp Pediatr. 2020 May;7(5):980-984
http://www.ijpediatrics.com pISSN 2349-3283 | eISSN 2349-3291
DOI: http://dx.doi.org/10.18203/2349-3291.ijcp20201511
Original Research Article
Anti-microbial susceptibility pattern of spores used in Bacillus clausii suspension:
an in vitro study
A. Srinivas*
Department of Probiotics R and D, Virchow Biotech Pvt, Ltd, Medchal II, Qutubullapur, Gagillapur, Hyderabad,
Telangana, India
Received: 18 February 2020
Accepted: 21 March 2020
*Correspondence:
Dr. A. Srinivas,
E-mail: srinivasarewar@gmail.com
Copyright: © the author(s), publisher and licensee Medip Academy. This is an open-access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial
use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Background: Bacillus species have been used as probiotics as they have high stability to gastrointestinal conditions
and impart health benefit on the host. Primarily used in their spore form the diversity of Bacillus species being used
and their applications are remarkable. Here, we present the results of the antimicrobial susceptibility testing of the B.
clausii spore suspension (Benegut®).
Methods: Bacillus clausii spore suspension (Benegut®), used in oral bacteriotherapy was tested for the susceptibility
to therapeutically useful antibiotics. Twelve commercially prepared, paper antibiotic discs of different drugs having
fixed concentrations were used. The antimicrobial susceptibility test was performed by the disc diffusion technique,
using soybean casein digestive agar (SCDA) as media and the results were categorized as susceptible, intermediate, or
resistant; using the criteria published by the Clinical and Laboratory Standards Institute.
Results: B. clausii was found to be resistant to broad-spectrum antibiotic chloramphenicol, antimycobacterial
rifampicin, beta-lactamase inhibitor amoxiclav, first-generation antibiotic cefaloridine, penicillin ampicillin, and
tetracycline. B. clausii was resistant to both the aminoglycoside antibiotics (streptomycin and kanamycin) studied. Off
first-generation fluoroquinolones studied, B. clausii was resistant to ciprofloxacin but partially sensitive to
norfloxacin, ofloxacin and to macrolide azithromycin.
Conclusions: Antimicrobial resistance is an important feature of probiotics. The results of our study indicate that the
antimicrobial and immunomodulatory activity B. clausii spore suspension (Benegut®) can profess beneficial effects
and can be a useful source of improving the intestinal imbalance of microbial flora and is safe for human
consumption.
Keywords: B. clausii, Probiotics, Resistance, Susceptibility
INTRODUCTION processed foods and drinks.3 The potential benefits of
probiotics include improved nutrition and growth and
Probiotics are live microorganisms which when prevention of various gastrointestinal disorders. 4
administered in adequate quantities confer health benefits Probiotics can also be used as an adjunct to antibiotic
to the host.1 Microorganisms have applications in every therapy, as the microorganisms present in them are
area of human life and have been explored for human resistant to antibiotics and therefore prevent the intestinal
benefits in the food and pharmaceutical industry.2 In microbial flora imbalance.5,6
pharmaceutical industries various antibiotics and
vaccines are produced from fungi, viruses and bacteria. 2 However, the use of probiotics for health benefits is
Food and beverage industries also use microbes such as giving rise to concerns regarding resistance transfer from
lactobacillus, Saccharomyces cerevisiae and the probiotic microorganisms to the disease-causing
Propionibacterium sharmanii for manufacturing pathogenic bacteria.5-7 The antimicrobial resistance to
International Journal of Contemporary Pediatrics | May 2020 | Vol 7 | Issue 5 Page 980
Srinivas A. Int J Contemp Pediatr. 2020 May;7(5):980-984
antibiotics can be classified as intrinsic which is non- the antimicrobial susceptibility testing of B. clausii was
transferrable and acquired resistance which is SCDA. The composition of this media is mentioned in
transferrable.7 The resistance transfer, acquired in nature, Table 1. One hundred mL of enriched broth of soybean
can take place either by a mutation in a gene or by the casein digestive medium (SCDM) was prepared (Table 2)
acquisition of foreign DNA through horizontal gene and one vial of B. clausii spore suspension (Benegut®)
transfer which includes transduction, transformation, and was inoculated on it. After that 500µL of this prepared
conjugation.8 media was spread on the SCDA in aseptic conditions and
the broth was incubated at 37ºC±2ºC for 18-24 hrs.
The most common group of bacteria used as probiotics
belongs to the group of lactic acid bacteria, which Table 2: Composition of soybean casein
involves Lactobacillus and Enterococcus.9 Due to digestive medium.
horizontal gene transfer, concerns are still raised,
particularly in lactic acid bacteria strains that carry Chemicals %
mobile genetic elements such as plasmids.10 Tryptone 1.7
Enterococcus, on the other hand, has member strains that Soya peptone 0.3
are opportunistic pathogens and are sometimes the Sodium Chloride 0.5
etiologic agents of some human nosocomial infections, Dextrose 0.25
such as bacteremia and infective endocarditis.9 Therefore, Dipotassium hydrogen phosphate 0.25
bacterial spore formers, mostly of the genus Bacillus
pH - 7.3±0.2
which is a gram-positive species constitute a major
probiotic product in use today.5 Primarily used in their
spore form, these products have been shown to prevent Table 3: Antibiotic discs and their concentration.
gastrointestinal disorders, also the diversity of Bacillus
species being used and their applications are astonishing.5 Disc
Antibiotic name Disc source
It has been suggested that the spore-bearing bacilli have concentration
some potential advantages over other non-spore formers Streptomycin 10 mcg HiMedia
such as Lactobacillus spp.6 Bacillus spores are heat- Chloramphenicol 10 mcg HiMedia
stable, capable of surviving the low pH of the gastric Tetracycline 30 mcg HiMedia
barrier, additionally products made on them can be stored Rifampicin 30 mcg HiMedia
at room temperature without any deleterious effect on Azithromycin 15 mcg HiMedia
viability.6 Amoxyclav 30 mcg HiMedia
Cefaloridine 10 mcg HiMedia
Benegut® is a Bacillus clausii spore suspension, used in Ciprofloxacin 5 mcg HiMedia
the treatment of alterations in the intestinal microbial Norfloxacin 10 mcg HiMedia
flora. In this article, we present the results of the Ofloxacin 2 mcg HiMedia
antimicrobial susceptibility testing of the B. clausii spore
Ampicillin 10 mcg HiMedia
suspension (Benegut®) using Soybean Casein Digestive
Kanamycin 30 mcg HiMedia
Agar (SCDA).
METHODS Table 3 contains the concentration and source of the
antibiotic discs used. Twelve commercially prepared,
The antimicrobial susceptibility test was performed by paper antibiotic discs of different drugs having fixed
the disc diffusion technique to assess the susceptibility of concentrations were placed in the middle of the culture
the strains of B. clausii against the antibiotics. inoculated agar plates. These agar plates were incubated
at 37±2°C for 24 to 48 hours and observed. The zones of
growth inhibition around each antibiotic disks were
Table 1: Composition of soybean casein
measured to the nearest millimeter. The diameter of the
digestive agar.
zone gives the measure of the susceptibility of the isolate
and the amount of drug diffused through the agar
Chemicals %
medium. The zone diameters of each drug are interpreted
Tryptone 1.5
using the criteria published by the Clinical and
Peptone 0.5 Laboratory Standards Institute (CLSI, formerly the
NaCl 0.5 National Committee for Clinical Laboratory Standards or
Agar 2 NCCLS) or those included in the US Food and Drug
pH - 8.0±0.2 Administration (FDA)-approved product inserts for the
disks. Positive, medium and LAFU control were also
The advantages of the disk method are simplicity of incubated at 37°C for 24-48 hrs. Growth observed in the
testing not requiring any special equipment, flexibility in plate was regarded as positive and inhibition of zone
the selection of disks for testing and the results that can around antibiotic disc was regarded as negative.
be easily interpreted by the clinicians. The media used for
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Srinivas A. Int J Contemp Pediatr. 2020 May;7(5):980-984
RESULTS is necessary to access the pattern and stability of
resistance spectra in probiotics, in order to better manage
The antimicrobial susceptibility of B. clausii spore the advantage of antibiotic-resistant probiotics during
suspension (Benegut®) to antibiotics was accessed by the antibiotic therapy.6 Antibiotic resistance is a common
disc diffusion technique and the results were categorized phenomenon in gram-positive bacteria.13 It is
as susceptible, intermediate or resistant. A bacterial strain accomplished by genes acquired either horizontally
is said to be susceptible to a given antibiotic when it is through plasmids, or foreign DNA recombination, or
inhibited in vitro by the antibiotic in therapeutic dose mutations at different chromosomal loci in the bacterial
range; intermediate if it is inhibited by a concentration genome.13
that is associated with an uncertain therapeutic effect and
resistant when it is inhibited by a concentration that is Members of the genus Bacillus are aerobic or facultative
associated with a high likelihood of therapeutic failure. 11 aerobic, endospore-forming and rod-shaped gram-
B. clausii was found to be resistant to broad-spectrum positive bacteria, which inhabit a wide range of habitats,
antibiotic chloramphenicol, antimycobacterial rifampicin, mostly soil, and sediments.12 These bacteria do not
beta-lactamase inhibitor amoxiclav, first-generation belong to the commensal microbiota of the
antibiotic cefaloridine, penicillin ampicillin, and gastrointestinal tract, but some strains of the genus are
tetracycline. B. clausii was resistant to both the included in food supplements and used in human
aminoglycoside antibiotics (streptomycin and kanamycin) nutrition as probiotics, notably B. clausii.12
studied. Off first-generation fluoroquinolones studied, B.
clausii was resistant to ciprofloxacin but partially The B. clausii probiotic strains are resistant to clinically
sensitive to norfloxacin, ofloxacin and to macrolide important antibiotics, including macrolides and
azithromycin. The test results are summarized in Table aminoglycosides.14,15 Resistance to aminoglycosides is
4. Positive and medium control was also maintained and reported to be due to the synthesis of an aminoglycoside-
growth was observed in positive control and not in inactivating enzyme encoded by an aadD2 chromosomal
medium control. gene, and to macrolides due to the presence of an erm(34)
gene.14 The resistance of B. clausii to rifampicin is
Table 4: Results of the antibiotic susceptibility test. reported to be due to a chromosomal mutation.7 A study
by Galopin et al, showed that the resistance to
Name of the Benegut® chloramphenicol in probiotic containing B. clausii was
Antibiotic Resistance Sensitivity due to the production of CAT encoded by the catBcl
Streptomycin ✔ ❌ gene.16 There are domains found in B. clausii which
Chloramphenicol ✔ ❌ confer resistance to different drugs, such as streptomycin
(Pfam) and tetracycline.13 Moreover, all the Bacillus
Tetracycline ✔ ❌
probiotics contain different classes of beta-lactamases
Rifampicin ✔ ❌ rendering them resistant to penicillins such as ampicillin
Partially Partially and amoxiclav.13 These findings are consistent with the
Azithromycin
resistance sensitive fact that the genes found in B. clausii have not been
Amoxiclav ✔ ❌ detected in pathogenic bacteria despite the long term use.7
Cefaloridine ✔ ❌
Ciprofloxacin ✔ ❌ The present study evaluated the antibiotic susceptibility
Partially Partially profile of B. clausii spore suspension (Benegut®). The
Norfloxacin antimicrobial susceptibility test of B. clausii spore
resistance sensitive
Partially Partially suspension (Benegut®) was performed against 12
Ofloxacin antibiotics using the disc diffusion technique. In this
resistance sensitive
Ampicillin ✔ ❌ study, we found that the Benegut® was resistant to
streptomycin, chloramphenicol, tetracycline, rifampicin,
Kanamycin ✔ ❌
amoxiclav, cefaloridine, ciprofloxacin, ampicillin, and
Positive control Growth was observed kanamycin; partially resistant to azithromycin,
Medium control No growth was observed norfloxacin, ofloxacin. In the case of positive and
LAFU control No growth was observed medium controls, growth was observed in positive
✔=Resistant control and not in medium control.
❌=Zone of inhibition more than 1.0 cm
Partially resistant= Zone of inhibition less than 1.0 cm
A couple of studies have reported similar susceptibility of
B. clausii to antibiotics as reported in our study. A study
DISCUSSION
conducted by Gueimonde et al, accessing the antibiotic
resistance patterns of the Lactobacillus and Bacillus
Commercially available probiotics contain
strains, also showed that the strains of Bacillus had genes
microorganisms with antibiotic resistance, due to which
resistant to macrolides, tetracycline, aminoglycosides,
they are used in association with antibiotic therapies in
chloramphenicol, and beta-lactamases.12 Another study
order to prevent microbial flora imbalance.12 Therefore, it
testing Enterogermina (containing four strains of Bacillus
International Journal of Contemporary Pediatrics | May 2020 | Vol 7 | Issue 5 Page 982
Srinivas A. Int J Contemp Pediatr. 2020 May;7(5):980-984
species [O/C, N/R, T, and SIN]) found that different Marseglia et al, performed a pilot study to assess the
strains had varied susceptibility towards different efficacy and the safety of B. clausii in the prevention of
antibiotics. O/C strain was found to be resistant to recurrent respiratory infections in children.22 Results
chloramphenicol, N/R was resistant to rifampicin, T showed that B. clausii treatment was significantly
showed resistance towards tetracycline and SIN showed effective (p=0.037) in reducing the duration of respiratory
resistance to streptomycin.17 infections in children with recurrent disease. Moreover,
the duration of respiratory infections episodes during the
Gueimonde et al. examined five commercial preparations follow-up period was also significantly shortened
of Bacillus strains by disc diffusion technique.12 The (p=0.049).22
authors reported that B. subtilis had a basal level of
resistance to streptomycin. Among the five probiotics The use of probiotics in the treatment and prevention of
examined, Bactisubtil was resistant to both disease, particularly gastrointestinal disease, has yielded
chloramphenicol and tetracycline, Domuvar, which is many successful results.24 As regard to B. clausii, its
closely related to Enterogermina was found to be resistant professed beneficial effect has been credited to
to chloramphenicol.12 According to another study testing antimicrobial and immunomodulatory activity.25 Bacillus
probiotics containing Bacillus strains, all the strains of probiotics have an overall excellent health-promoting
Bacillus were resistant to streptomycin but susceptible to record, especially in preventing and curing of diarrhea,
chloramphenicol, kanamycin, and tetracycline.18 The B. gingivitis, H. pylori infection and maintaining
clausii strain (ATCC 9799) was reported to be resistant to homeostasis of the intestine.26 B. clausii survives transit
cephalosporins in the study by Hong et al, and Girlich et in the gut and maintains a considerable intestinal titre for
al, and sensitive in a study by Abbrescia et al.5,6,19 In this up to 12 days after a single oral administration.25 B.
study out of three fluoroquinolones (ciprofloxacin, clausii strains show different ability to survive and
norfloxacin, and ofloxacin) tested, B. clausii probiotic persist, suggesting a strain dependent adaptation to this
strains were reported to be resistant to ciprofloxacin but environment.25 Analysis of the whole genome sequence
partially resistant to norfloxacin and ofloxacin. However, of B. clausii has revealed that the antibiotic resistance
in a study by Abbrescia et al., B. strains were reported to genes are present in chromosomal DNA which is intrinsic
be sensitive to fluoroquinolones (ciprofloxacin, and not transferable.27 Toxin genes were also found to be
levofloxacin, norfloxacin, and moxifloxacin).6 absent. These results suggest consumption of B. clausii is
safe for humans.27
In the era of antibiotics, probiotic use is increasingly
employed in association with antibiotics therapy for In conclusion, resistant traits found in B. clausii spore
preserving the intestinal microbial flora, owing to suspension (Benegut®) in our study were similar to those
antimicrobial resistance.6 Antimicrobial resistance is a reported from other commercial preparations of Bacillus
desirable feature in probiotics. Although, microbes used strains. Benegut® can be a useful source of improving
as probiotics are not exempted from the natural processes the intestinal imbalance of microbial flora; and effective
governing antibiotic resistance.20 treatment of diarrhea and other intestinal malfunctions.
Therefore, it is imperative to screen microbes effectively ACKNOWLEDGEMENTS
for antibiotic resistance genes before using them as
probiotics.20 Extensive research has been conducted on Authors would like to thank MIS team at medONE
the antimicrobial resistance patterns of probiotic strains Pharma Solutions, Gurugram, Delhi NCR, for assistance
and it was found that probiotic strains possess both in the preparation of this manuscript.
acquired and intrinsic resistance.9,12,20
Funding: No funding sources
B. clausii, probiotic in the spore form appears to have an Conflict of interest: None declared
important role to play management of acute diarrhea and Ethical approval: The study was approved by the
recurrent respiratory infections in children, with a good Institutional Ethics Committee
safety profile.21,22 A systematic review and a meta-
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Microbiol Lett. 2009;296:185-9. clausii suspension: an in vitro study. Int J Contemp
17. Senesi S, Celandroni F, Tavanti A, Ghelardi E. Pediatr 2020;7:980-4.
Molecular Characterization and Identification of
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