Curr Microbiol (2012) 64:588–591 DOI 10.1007/s00284-012-0113-8
Lipolytic, Proteolytic, and Cholesterol-Degrading Bacteria from the Human Cerumen Yoram Gerchman • Rinat Patichov Tal Zeltzer
•
Received: 4 January 2012 / Accepted: 9 March 2012 / Published online: 29 March 2012 Ó Springer Science+Business Media, LLC 2012
Abstract Cerumen, also known as ear wax, is a yellowish waxy substance secreted from specialized glands in the ear canal of mammals. Human cerumen is rich in protein (mainly keratin), lipids (long-chain fatty acids), alcohols, squalene, and cholesterol. To-date the role of cerumen is not totally clear but it is believed to have antimicrobial properties. Here we describe the isolation of multiple bacterial species from human cerumen (among them many Staphylococcus spp. and, interestingly, multiple Bacillus spp.) showing that many of these bacteria harbor biochemical traits enabling them to utilize different cerumen components for their growth. We also suggest the existence of microbial consortia.
Introduction Cerumen, also known as ear wax, is a yellowish waxy substance secreted from specialized glands in the ear canal of mammals. Human cerumen is a mixture of dead corneocytes and at least two types of glandular secretions [1, 12]. As such, it is rich in protein, mainly keratin (up to 60 % w/w) although this varies depending on the secretion’s freshness [18]. Cerumen was also found to be rich in Y. Gerchman (&) R. Patichov Department of Biology and Environment, University of Haifa in Oranim, Haifa, Israel e-mail:
[email protected] Y. Gerchman Department of Biology and Environment, Oranim College, Tivon, Israel T. Zeltzer Department of Evolutionary and Environmental Biology, University of Haifa, Haifa, Israel
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different lipids, composed of saturated and unsaturated long-chain fatty acids, and in less hydrophobic chemicals, such as alcohols, squalene and cholesterol (12, 20 and 6–9 % of the wax weight, respectively; [6, 7]). The biological role of cerumen is not totally clear. It was suggested many times to have bactericidal properties and indeed good activity was demonstrated against known human pathogens, such as Escherichia coli K-12, Haemophilus influenza and Serratia marcescens, and a more moderate activity against E. coli K-1, Staphylococcus aureus and Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Propionibacterium acnes and Corynebacterium spp. [5, 23]. Some anti-fungal activity was shown against Candida albicans and C. parapsilosis [15, 16], common ear fungal pathogens [17]. Nevertheless, these results were challenged by the finding that fresh cerumen hosts many bacteria and fungi [2, 20, 24], with bacterial counts as high as 107 g-1 cerumen [3], and recently by the observation that human cerumen is more likely to increase than to decrease the growth of many bacteria [4], putting the antimicrobial properties of cerumen under further doubt. Here we demonstrate that it could host, and its components (proteins, lipids, and cholesterol) could serve as food sources for multiple bacterial species.
Methods Cerumen samples were collected from a 46 years old male, Caucasian, with history of ‘wet cerumen’ accumulation. Samples were collected with a sterile Q-tip, and cerumen was plated immediately by striking on solid LB media plates. The plates were incubated at 37 °C for up to 3 days until colonies appeared. Representative colonies were selected based on morphology and color. Selected colonies
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were isolated by transferring five times on LB plates. The isolates (12 in total) were tested for different biochemical traits. Extracellular proteolytic activity was assayed using modified milk agar (1 % fat-free milk powder, 0.5 % yeast extract, and 1.5 % bacto-agar). Proteolytic activity was determined by appearance of a clear hallow around the colony. Lipolytic activity was assayed using the Rhodamin B—olive oil assay [14] and plates were examined under UV light (365 nm) for fluorescence. Growth on cholesterol was tested by plating the isolates on solid media containing cholesterol as a single carbon source (based on [27] but supplemented with 0.01 % yeast extract). Growth on palmitic acid was tested on the same media, with cholesterol replaced by palmitic acid. To identify the bacteria, a single colony of each isolate was suspended in PCR-grade water until first sign of turbidity and subjected to 16S PCR amplification using universal primers modified from [10]. Eight microliters of each suspension were transferred to a thin-walled PCR tube. One microliter of each primer (11F 50 -GGA TCCAGA CTT TGA TYM TGG CTC AG-30 ; 1512R, 50 -GTG AAG CTT ACG GYT AGC TTG TTA CGA CTT-30 ) were added to give a final concentration of 1 micromolar, and 10 microliters of PCR Reddymix (ABGene, USA) was added. Tubes were placed in a thermocycler (BioER), and the following program was applied: initial denaturation for 3 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 55 °C for 50 s and 72 °C for 2 min, ending with a 10 min final elongation step at 72 °C. Sequences were compared to the NCBI database by using EZ-Taxon server v2.1 for identification [8].
Results Eleven different morphotypes were isolated and tested for biochemical traits. Representative images of the proteolytic and lipolytic plate assay are presented in Fig. 1. Summary of the isolate identities and their biochemical traits are reported in Table 1. Out of the 11 isolates, six were identified as Staphylococcus auricularis or S. haemolyticus. S. auricularis is a known inhabitant of the healthy external human ear [11, 13] while S. haemolyticus is a common skin inhabitant. Of the other five isolates, one was identified as Brevibacterium epidermidis, a skin inhabitant associated with foot odor [25], and the other five were identified as Bacillus spp. (B. safensis, B. pumilus and B. stratosphericus). None of these Bacillus spp. are known common human skin inhabitants although at least one, B. pumilus, was previously reported as a rare cause for cutaneous infection [26]. The presence of the two other Bacillus spp. was more surprising, as they
Fig. 1 Plate assay for biochemical traits of cerumen isolates. a Lipolytic assay on Rhodamin B—olive oil agar, notice the fluorescence around active colonies. b Proteolytic assay on milk agar, notice the transparent halo around active colonies. Numbers are isolate IDs
were originally isolated from high radiation environments (B. safensis from spacecraft assembly-facility surfaces [21]; B. stratosphericus from air samples collected at the stratosphere, [22]), and indeed their 16S gene show high similarity to each other and to that of B. pumilus, questioning species determination. Nevertheless these Bacillus spp. biochemical traits show similar distinction, as only B. safensis was able to grow on cholesterol as a single carbon source. It should also be mentioned that Bacillus spp. were previously isolated from cerumen remains on auriscopes [19], supporting our findings. Interestingly, recent report on the bacterial communities of the human body [9] did not report any members of the Bacillus genus. This seemingly discrepancy is in good agreement with the finding the ear canal microbial communities tend to be highly diverse [9].
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Table 1 16S rDNA based identification and biochemical traits of the different cerumen isolates No.
GeneBank Accession number
Identity
Pairwise Similarity and Diff/Total nt
Proteolytic activity
Lipolytic activity
Growth on cholesterol
Growth on Palmitic acid
1
JQ624774
Staphylococcus auricularis ATCC 33753
99.786 3/1405
-
-
-
-
2
JQ624773
Staphylococcus auricularis ATCC 33753
99.716 4/1407
-
-
-
-
3
JQ624772
Staphylococcus haemolyticus ATCC 29970
99.921 1/1409
-
???
-
-
4
JQ624771
Staphylococcus haemolyticus ATCC 29970
100.000 0/1408
-
?
-
-
5
JQ624770
Staphylococcus auricularis ATCC 33753
99.716 4/1407
-
-
??
-
6
JQ624769
Staphylococcus haemolyticus ATCC 29970
100.00 0/1409
-
-
???
-
12
JQ624775
Bacillus safensis FO-036b
99.058 5/531
???
?
?
??
13
JQ624768
Bacillus pumilus ATCC 7061
99.787 3/1409
???
?
-
??
16
JQ624767
Brevibacterium epidermidis NCDO 2286
99.349 9/1382
-
-
???
-
17
JQ624766
Bacillus safensis FO-036b
99.857 2/1401
???
???
??
??
27B
JQ624765
Bacillus stratosphericus 41KF2a
99.252 7/936
???
?
-
??
‘‘-’’ no growth/no activity, ‘‘?’’ to ‘‘???’’ low to high growth/activity, respectively
Discussion
References
As mentioned above, the antimicrobial role of human cerumen is under continuous debate. Here we demonstrate that its components (proteins, lipids, and cholesterol) could serve as food source for multiple bacterial species, hence, it could naturally host ‘‘cerumen-bacteria’’. Furthermore, the results also bring up the possibility of bacteria consortia. Consortia of different bacteria with different biochemical trait is a well established in many environments, and indeed such a consortium was suggested between two human skin inhabitants, a bacterium, Brevibacterium epidermidis and a fungus Trichophyton interdigitale (previously known as Trichophyton mentagrophytes), where the bacteria utilize amino acids released by the proteolytic activity of the fungus [25]. In the cerumen, cholesteroldegrading bacteria and lipid and protein hydrolyzing bacteria could complement each-other, creating similar consortia. Such process could explain why two of the three isolates identified as S. auricularis, a common resident of the external ear canal, could not grow on any of cerumen related carbon sources—they could scavenge on other bacteria’s enzymatic products. A better understanding of these possible bacterial consortia could broaden our understanding of ear infections process and the process leading to them.
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