[Downloaded free from http://www.indianjotol.org on Monday, August 18, 2014, IP: 218.241.189.21] || Click here to download free Android application for this journal
ORIGINAL ARTICLE
A study of antibacterial and antifungal
properties of human cerumen
Sumit Gupta, Rohit Singh, Kranthi Kosaraju, Indira Bairy, Balakrishnan Ramaswamy
Department of ENT-HNS and Department of Microbiology, Kasturba Medical College, Manipal University,
Karnataka, India
ABSTRACT
Objective: To investigate the effect of human cerumen on the growth of Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa and Candida albicans. Study Design: Prospective study Setting: The study was
conducted in the Department of Otolaryngology-Head and neck Surgery and Department of Microbiology, at
a tertiary care teaching hospital in southern India. Materials and Methods: Cerumen samples were collected
from 120 healthy cases by using sterile Jobson Horn Probe. Sterile samples were taken and further tested.
The bacterial strains were cultured on nutrient agar and Candida was grown on SDA. Serial 10 fold dilutions
of the test organisms were made using normal saline acting as control and using 3.5% cerumen suspension as
test and were incubated at 37°C for 12 hours. Subcultures were performed from test as well as control tubes
to assess the inhibitory activity of human cerumen. Results: Of 120 samples, only sterile samples were tested.
At dilutions of 1 in 103 there was complete inhibition of Escherichia coli and Pseudomonas aeruginosa in all
samples, complete inhibition of Staphylococcus aureus in 83.3% samples and Candida albicans in 80% samples.
The order of inhibition demonstrated by cerumen in the present study was Escherichia coliPseudomonas
aeruginosaStaphylococcus aureusCandida albicans. Conclusion: Human cerumen has antibacterial and
antifungal properties against the commonest bacterial and fungal pathogens. Other than the physical barrier,
wax acts as protective coating over the external auditory canal. Hence, routine wax removal/ear cleaning
is not mandatory unless impacted wax is leading to earache or conductive hearing loss.
KEYWORDS: Antibacterial, Antifungal, Cerumen
INTRODUCTION
Cerumen is produced in the outer third of the cartilaginous
portion of the human ear canal. It is composed of desquamated
sheets of corneocytes, originating from the deep and superficial
external auditory canal, mixed with glandular secretions. It’s
a mixture of secretions from sebaceous glands and modified
apocrine sweat glands.[1] Sebaceous and cerumenous glands
in the auditory canal secrete lipids and peptides, respectively.
Hairs in the external third of the canal also produce glandular
secretions that contribute to cerumen’s composition.[2] The
balance of secretions from the sebaceous and cerumenous
glands varies between ethnic groups, which might partly explain
the phenotypic differences in cerumen observed in different
ethnic groups.[3] However, whether these phenotypic variations
translate into clinically significant differences in the prevalence
of impacted cerumen or the treatment outcome is unknown.
There is some evidence of genetic polymorphisms in cerumen
Address for correspondence: Dr. Rohit Singh,
Department of ENT-HNS, Kasturba Medical College, Manipal University,
Karnataka, India. E-mail: [email protected]
Indian Journal of Otology | October 2012 | Vol 18 | Issue 4 |
phenotypes. Current evidence stratifies cerumen into two
phenotypes: wet and dry. Wet cerumen, which is light or
dark brown and sticky, is characterized by a relatively high
concentration of lipid and pigment granules. Dry cerumen,
which is grey or tan and brittle, tends to express lower levels
of these components.[4] For example, dry wax contains around
20% lipid, compared to approximately 50% in wet cerumen.
Besides this, the two forms show few other biochemical
differences. These two forms are associated with race and are
controlled by two autosomal alleles.[5]
Cerumen production seems to show neither discordance
between sexes, nor marked differences over the years.
However, the lack of marked differences over the year might
Access this article online
Quick Response Code:
Website:
www.indianjotol.org
DOI:
10.4103/0971-7749.104796
189
[Downloaded free from http://www.indianjotol.org on Monday, August 18, 2014, IP: 218.241.189.21] || Click here to download free Android application for this journal
Gupta, et al.: Antimicrobial properties of ear wax
offer one strand of circumstantial evidence against cerumen
playing a clinically or biologically significant antibacterial
role.[6]
Although, the role of human cerumen is believed to protect the
external ear canal against infections there are still controversies
on this topic. Some authors have suggested that cerumen is
unable to prevent infections and that the rich nutrients of
cerumen support abundant growth of bacteria and fungi.
Besides providing a physical barrier against infection, it
is believed that cerumen has antibacterial and antifungal
properties.[7] Our study aims at evaluating the effect of
human cerumen on the growth of Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Candida albicans.
MATERIALS AND METHODS
Study design and setting
This was a prospective study that was conducted at the
Department of Otolaryngology-Head and neck Surgery
and Department of Microbiology, at a tertiary care teaching
hospital, in southern India.
Inclusion criteria
Healthy subjects, of all age groups, without any external or
middle ear pathology, who attended the ENT outpatient clinic
of the hospital during five months of the study period.
After Institutional ethics committee clearance (IEC 016/2010/KH,
Manipal) cerumen samples were collected from 120 normal,
healthy individuals using a sterile Jobson Horn Probe, then
emulsified in a solution containing 30 percent glycerol with
5 percent sodium bicarbonate, producing a cerumen suspension
of 3.5 percent (weight/volume). The mixture of cerumen and
buffer was then emulsified by pumping it back and forth using
a sterile syringe. The cerumen suspension was stored at minus
20°C until microbiological testing.
Microbiological testing
The cerumen suspensions were cultured on nutrient agar plate
and incubated at 37°C overnight. Samples that showed any
microbiological growth, even the commensals of the external
auditory canal, were treated as unsterile and were excluded
from further testing. The samples which did not show any
growth were preserved for further testing.
Bacterial and fungal strains
The bacterial strains that were tested are Staphylococcus aureus
(American Type Culture Collection (ATCC) 25923),
Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 27853) and the fungi Candida albicans.
Antibacterial and antifungal assays
The three bacterial strains and the fungal strain were cultured on
190
nutrient agar and Sabouroud’s dextrose agar (SDA) respectively
and incubated at 37°C for 24 hours to obtain fresh cultures.
A single isolated colony of each culture was transferred to 10ml
nutrient broth and incubated for minimum of 6 hours at 37°C
and inoculum was adjusted to 0.5McF (i.e. 1.5108 cfu/ml).
In the next step, 10-fold serial dilution ranging from 1 in 101 to 1
in 105 was prepared by adding 100 l of the prepared inoculum to
tubes containing 900 l of 3.5% cerumen suspension. The tubes
containing various dilutions of the inoculums were incubated
at 37°C for 16-18 hrs. After overnight incubation, 10 l of
suspension from each dilution was transferred onto a blood agar
plate, streaked and incubated at 37°C for 24 hours to observe for
colonies. Growth in the form of colony formation was observed
following incubation and the results were recorded.
The growth control for each of the strains was also included
to ensure adequate growth of the test bacteria and fungus.
Negative control was also included to ensure sterility of all the
samples and media used in the test procedure.
RESULTS
1. Total number of cerumen samples collected 120 (labeled
from 1 to 120)
2. Total number of sterile samples obtained 60 (considered
for further proceedings in the study)
3. Total number of unsterile samples obtained 60 (rejected,
hence not considered for the study)
4. The growth control showed adequate growth of the
test strains and the negative control showed no growth
indicating the validity of the test conditions.
5. Table 1 shows the number of samples in which the growth
was inhibited.
From the data obtained, it was seen that sterile human cerumen
samples could inhibit,
1. Escherichia coli
2. Pseudomonas aeruginosa
3. Staphylococcus aureus
4. Candida albicans in the decreasing order of their list.
DISCUSSION
Cerumen forms an acidic coat that aids in the prevention of
Table 1: Depicts the number of samples in which the
growth was inhibited
Concentration
1 in 101 1 in 102 1 in 103 1 in 104 1 in 105
(%)
(%)
(%)
(%)
(%)
Staphylococcus
35 (58.3) 45 (75) 50 (83.3) 58 (96.6) 60 (100)
aureus
Escherichia coli
55 (91.6) 60 (100) 60 (100) 60 (100) 60 (100)
Pseudomonas
45 (75) 58 (96.6) 60 (100) 60 (100) 60 (100)
aeruginosa
Candida albicans 20 (33.3) 40 (66.6) 48 (80) 50 (83.3) 53 (88.3)
Indian Journal of Otology | October 2012 | Vol 18 | Issue 4 |
[Downloaded free from http://www.indianjotol.org on Monday, August 18, 2014, IP: 218.241.189.21] || Click here to download free Android application for this journal
Gupta, et al.: Antimicrobial properties of ear wax
external auditory canal infection.[8,9] Absence of cerumen may
lead to infection, such that cerumen serves an antimicrobial
role by physically protecting the external auditory canal skin,
establishing a low pH; thus an inhospitable environment for
pathogens and producing antimicrobial compounds such as
lysozyme, so that its absence leaves the canal vulnerable to
infection.
The traditional view holds that cerumen also protects
the middle ear from bacterial and fungal infection. For
example, some authorities suggest retaining the cerumen
barrier to bolster host defenses against ear infections.[10]
However, the evidence that cerumen plays a biologically or
clinically significant role in host defence seems relatively
weak. It might be expected, for example, that if cerumen
played an important role bolstering host defense systems,
its composition would alter in response to an infection.
Perhaps exposure to bacteria would induce up-regulation
of antibacterial components of cerumen. However, the
cerumen of patients with otitis externa does not seem to
contain more antibacterial polyunsaturated fatty acids than
those without.[11]
In our study the cerumen inhibited the growth of bacteria and
fungi at different concentrations. The study demonstrated that
cerumen has antibacterial and antifungal properties, which
plays a role in the protection of the external auditory canal.
Interestingly, Sololov et al., proved that the cerumen of some
mammals possess antistaphylococcal, antimicrococcal and
antiherpes activities.[12]
Our findings on the bactericidal effect of cerumen on E. coli
are consistent with Stone and Fulghum, Chai and Chai
and Bauman et al.,[13-15] However, Lum et al., and Campos
et al., found insignificant bactericidal effect and stated that
Escherichia coli is not a normal commensal of the ear canal,
and thus may not be recognized by the immune system of
the ear canal.[7,16]
Campos et al., showed that an increase in growth occurred
much more frequently than a decrease in growth in almost
every microorganism tested with wet cerumen, with
the mean increase percentage being much higher than
the mean decrease percentage, except in the case of S.
aureus.[16] Burtenshaw reported inconsistent bactericidal
activity of cerumen against S. aureus.[17] Megarry et al., Stone
and Fulghum reported a significant bactericidal activity
similar to our study.[13,18]
Our results agree with Lum et al., and Chai and Chai who
demonstrated the bactericidal effect of cerumen on two strains
of P. aeruginosa.[7,14] However other studies have reported a
lack of bactericidal effect of cerumen on P. aeruginosa.[15,16,19]
study shows that the human cerumen has more antibacterial
property as compared to antifungal property. Probably some
protective mechanism in the fungus led to less inhibition of
their growth as compared to the bacteria.
In our study, the sterile samples were stored at 20°C and then
processed after some time. This storage might have affected
the antibacterial and antifungal property, which might have
otherwise been higher if the tests were conducted immediately.
Indeed, immunohistochemical studies suggest that
antibody-mediated immune reactions, rather than cerumen,
protect the external auditory canal from infection. The
epidermis and dermis surrounding the sebaceous and
cerumenous glands, as well as the piliary follicles, express cells
capable of activating and sustaining local immune reactions,
including IgA and IgG.[3] However, there is a need for further
studies to characterize the nature of host defence in this unique
anatomical site.
The conflicting results reported in the literature may be
explained by differences in individuals, culture media,
micro-organism virulence and methodology. Staphylococcus
aureus, P. aeruginosa and C. albicans are common pathogens
which cause otitis externa, and the presence of cerumen in
the ear canal may reduce the likelihood of infection by such
micro-organisms.[7]
Our study tries to put forward that apart from being a
physical barrier, cerumen acts as protective coating over the
external auditory canal due to its antibacterial and antifungal
properties. Hence, routine wax removal/ear cleaning is not
mandatory unless impacted wax is leading to earache or
conductive hearing loss.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
Megarry et al., and Lum et al., studied the mycocidal effect
of cerumen consistent with our study.[7, 9] Interestingly, our
Indian Journal of Otology | October 2012 | Vol 18 | Issue 4 |
Alvord LS, Farmer BL. Anatomy and orientation of the human
external ear. J Am Acad Audiol 1997;8:383-90.
Burkhart CN, Kruge MA, Burkhart CG, Black C. Cerumen
composition by flash pyrolysis-gas chromatography/mass
spectrometry. Otol Neurotol 2001;22:715-22.
Sirigu P, Perra MT, Ferreli C, Maxia C, Turno F. Local immune
response in the skin of the external auditory meatus: An
immunohistochemical study. Microsc Res Tech 1997;38:
329-34.
Tomita H, Yamada K, Ghadami M, Ogura T, Yanai Y, Nakatomi K,
et al. Mapping of the wet/dry earwax locus to the pericentromeric
region of chromosome 16. Lancet 2002;359:2000-2.
Petrakis NL, Pingle U, Petrakis SJ, Petrakis SL. Evidence for a
genetic cline in earwax types in the Middle East and Southeast
Asia. Am J Phys Anthropol 1971;35:141-4.
Cipriani C, Taborelli G, Gaddia G, Melagrana A,
Rebora A. Production rate and composition of cerumen: Influence
of sex and season. Laryngoscope 1990;100:275-6.
Lum CL, Jeyanthi S, Prepageran N, Vadivelu J, Raman R.
Antibacterial and antifungal properties of human cerumen.
J Laryngol Otol 2009;123:375-8.
191
[Downloaded free from http://www.indianjotol.org on Monday, August 18, 2014, IP: 218.241.189.21] || Click here to download free Android application for this journal
Gupta, et al.: Antimicrobial properties of ear wax
8.
9.
10.
11.
12.
13.
14.
15.
Campos A, Arias A, Betancor L, Rodríguez C, Hernández AM,
López Aguado D, et al. Study of common aerobic flora of human
cerumen. J Laryngol Otol 1998;112:613-6.
Bojrab DI, Bruderly T, Abdulrazzak Y. Otitis externa. Otolaryngol
Clin North Am 1996;29:761-82.
Lindsey D. It’s time to stop washing out ears! Am J Emerg Med
1991;9:297.
Osborne JE, Baty JD. Do patients with otitis externa produce
biochemically different cerumen? Clin Otolaryngol Allied Sci
1990;15:59-61.
Sokolov VE, Ushakova NA, Chernova OF, Shubkina AB,
Alimbarova LM, Barinskiĭ IF. The anti-infective properties
of mammalian earwax. Izv Akad Nauk Ser Biol 1995;5:
579-85.
Stone M, Fulghum RS. Bactericidal activity of wet cerumen. Ann
Otol Rhinol Laryngol 1984;93:183-6.
Chai TJ, Chai TC. Bactericidal activity of cerumen. Antimicrob
Agents Chemother 1980;18:638-41.
Baumann ES, Carr CD, Senturia BH. Studies of factors considered
responsible for diseases of the external auditory canal. III.
16.
17.
18.
19.
A comparison of lipids in normal and infection-susceptible ears.
Ann Otol Rhinol Laryngol 1961;70:1055-61.
Campos A, Betancor L, Arias A, Rodríguez C, Hernández AM,
López Aguado D, et al. Influence of human wet cerumen on the
growth of common and pathogenic bacteria of the ear. J Laryngol
Otol 2000;114:925-9.
Burtenshaw JM. The mechanism of self-disinfection of the human
skin and its appendages. J Hyg 1942;42:184-210.
Megarry S, Pett A, Scarlett A, Teh W, Zeigler E, Canter RJ. The
activity against yeasts of human cerumen. J Laryngol Otol
1988;102:671-2.
Nichols AC, Perry ET. Studies on the growth of bacteria in the
human ear canal. J Invest Dermatol 1956;27:165-70.
How to cite this article: Gupta S, Singh R, Kosaraju K, Bairy I,
Ramaswamy B. A study of antibacterial and antifungal properties
of human cerumen. Indian J Otol 2012;18:189-92.
Source of Support: Indian Council of Medical Research, New Delhi,
India. (STS2010). Conflict of Interest: None declared.
Author Help: Reference checking facility
The manuscript system (www.journalonweb.com) allows the authors to check and verify the accuracy and style of references. The tool checks
the references with PubMed as per a predefined style. Authors are encouraged to use this facility, before submitting articles to the journal.
• The style as well as bibliographic elements should be 100% accurate, to help get the references verified from the system. Even a
single spelling error or addition of issue number/month of publication will lead to an error when verifying the reference.
• Example of a correct style
Sheahan P, O’leary G, Lee G, Fitzgibbon J. Cystic cervical metastases: Incidence and diagnosis using fine needle aspiration biopsy.
Otolaryngol Head Neck Surg 2002;127:294-8.
• Only the references from journals indexed in PubMed will be checked.
• Enter each reference in new line, without a serial number.
• Add up to a maximum of 15 references at a time.
• If the reference is correct for its bibliographic elements and punctuations, it will be shown as CORRECT and a link to the correct
article in PubMed will be given.
• If any of the bibliographic elements are missing, incorrect or extra (such as issue number), it will be shown as INCORRECT and link to
possible articles in PubMed will be given.
192
Indian Journal of Otology | October 2012 | Vol 18 | Issue 4 |