Advances in Environmental Biology Staphylococcus Profiles

Advances in Environmental Biology, 7(14) December 2013, Pages: 4562-4566
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Advances in Environmental Biology
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Development of a New Media for Identification of Staphylococcus Species by Fatty Acid
Profiles
1
Bassam Aloklah, 2Anwar Alhajali and 2Sabah Yaziji
1
Department of Industrial and Food Technique, National Commission for Biotechnology, Syria.
Department of Food Science, Faculty of Agriculture, Damascus University, Syria.
2
ARTICLE INFO
Article history:
Received 22 October 2013
Received in revised form 14
January 2014
Accepted 20 January 2014
Available online 25 February 2014
Key words:
Identification, Staphylococcus
Species, Fatty Acids,
Gas Chromatography-Mass
Spectrometry (GC-MS),
Modified TSA Medium
ABSTRACT
22 isolates Staphylococcus species were identified using ID 32 Staph from milk
(6) Staphylococcus epidermidis,(5) samples which included (4) Staphylococcus aureus,
(3) Staphylococcus (4) Staphylococcus hominis and haemolyticus, Staphylococcus.
lugdunensis. Fatty acids were analyzed by means of Gas Chromatography- Mass
Spectrometry for Staphylococcus species. The results revealed that 21 fatty acids were
occurred in tested Staphylococcus species and most of them were from 13 to 20 carbon
atoms. The seven main fatty acids were iso-tridecanoic acid (Ci14:0), iso-tetradecanoic
acid (Ci15:0), antiso-tetradecanoic acid (Ca15:0), hexadecanoic acid (C16:0), antisohexadecanoic acid (Ca17:0), octadecanoic acid (C18:0) and eicosanoic acid (C20:0).
The differences of these fatty acids of Staphylococcus species were tested in TSA and
modified TSA medium. Results showed that the composition of fatty acids profile could
be use to identify Staphylococcus species using modified TSA medium as a complete
and helpful method.
© 2013 AENSI Publisher All rights reserved.
To Cite This Article: Bassam Aloklah, Anwar Alhajali and Sabah Yaziji., Development of a new media for identification of
Staphylococcus species by fatty acid profiles. Adv. Environ. Biol., 7(14), 4562-4566, 2013
INTRODUCTION
Staphylococcus species are one of the most common mastitis causing pathogens [10,12], In 1878, Robert
Koch was the first to describe Staphylococcus spp. in human pus. Later, Pasteur cultivated Staphylococcus spp.
in a liquid medium In 1880 [14]. Then, the name of Staphylococcus was introduced by Ogston in 1883 for
micrococcus group causing inflammation and suppuration. In 1886, the genus Staphylococcus was separated
from Micrococcus by Flügge in 1886 [3].
Staphylococcus spp. are non-motile, non-spore forming, Gram-positive and spherical bacteria about 1
micrometer in diameter that are members of bacterial family called Staphylococcaceae. Staphylococcus spps.
are capable of growth both aerobically and anaerobically media . All species can grow in the presence of bile
salts and are catalase positive [14].
Staphylococcus spps. are normal inhabitants of the skin and mucous membranes of humans and other
organisms [11]. So far, more than 40 species and subspecies in the genus Staphylococcus spp. have been
identified [16].
The species that most frequently cause diseases in humans are Staphylococcus aureus, Staphylococcus
epidermidis, and Staphylococcus haemolyticus. Other significant opportunistic pathogens include
Staphylococcus hominis, and Staphylococcus lugdunensis [8].
The classification of species and subspecies of staphylococci can be based on a variety of phenotypic
character analyses and DNA-DNA (genomic) relationships. In addition, rRNA analysis and ribotyping may be
used to describe the relationship of reference and new species. DNA similarity (>70%) is the criterion that has
been used to determine species boundaries in the formal classification of staphylococcal species and most
subspecies [3]. PCR analysis of the 16S–23S rRNA intergenic spacer region has preliminarily shown successful
results in discriminating among 31 Staphylococcus species and PCR analysis allows for the identification of
pure culture staphylococci within 24–48 h [9]. Conventional methods for the determination of phenotypic
characters at the cellular and population levels were developed first and then examined for their correlation to
DNA relatedness [6]. Now, Key characters are used for species and subspecies identification include colony
morphology, oxygen requirements, coagulase, clumping factor, heat-stable nuclease, hemolysins, catalase,
Corresponding Author: Bassam Aloklah, Department of Industrial and Food Technique, National Commission for
Biotechnology, Syria, B.O.Box 31902.
Fax: +963-11-5130104; E-mail: Bassam78alk @ Gmail.com (B. Aloklah)
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Bassam Aloklah et al, 2013
Advances in Environmental Biology, 7(14) December 2013, Pages: 4562-4566
oxidase, alkaline phosphatase, urease, ornithine decarboxylase, pyrrolidonyl arylamidase, β- galactosidase,
acetoin production, nitrate reduction, esculin hydrolysis and aerobic acid production from a variety of
carbohydrates including D-trehalose, Dmannitol, D-mannose, D-turanose, D-xylose, D-cellobiose, L-arabinose,
maltose, -lactose, sucrose, and raffinose, and intrinsic resistance to novobiocin and polymyxin B [7]. Some
conventional methods may require up to three to five days before a final result can be obtained. Rapid
Identification System (RIS) to facilitate identification in routine analysis or clinical laboratory has been
developed for Staphylococcus species identification such as kits or automated systems requiring only a few
hours to one day for the completion of tests [7]. Now, some identification systems are available such as API
Staph (bioMe´rieux Vitek, Inc., Hazelwood, Mo.) which is a fully automated microbiology system that uses a
Gram-positive identification (GPI) card (bioMe´rieux), MSPI Micro-Scan Positive Identification panel, Crystal
Gram-Positive Identification System, Crystal Rapid Gram-Positive Identification System (Becton Dickinson
Microbiology Systems, Cockeysville, MD, USA) and the MIDI Sherlock® Microbial Identification System
(Microbial ID Inc., Newark, Del.). The Sherlock System identifies microorganisms is based on gas
chromatographic (GC) analysis of extracted microbial fatty acid methyl esters (FAMEs) [3].
FAME has been used to identify staphylococci to the subspecies level [5]. Various studies showed that fatty
acid compositions were useful in distinguishing between species of Staphylococcus. Paradis et al. [10] could
distinguish five species of Staphylococcus by gas chromatography, Babiker and Boehnel [1] found gas
chromatography was good method in identification staphylococcus species and Stoakes et al. [15] studied the
cellular fatty acid contents of staphylococcus species.
The objective of this study was to evaluate the sensitivity of cellular fatty acids analysis for identification
of some isolated staphylococcus species form milk samples which collected from various locations in Syrian
market using Modified-Tryptic Soy Agar medium.
MATERIALS AND METHODS
Isolation and identification of Staphylococcus:
A total number of 22 Staphylococcus strains were isolated from milk samples which collected from various
localities of Syrian market. The Staphylococcus strains were grown on medium Manitol Salt Agar (MSA)
consisting of Proteose peptone 5g\L, Sodium chloride 75g\L, and Mannitol 10g\L, Phenol red 0.025 g\L and
Agar 15g. The isolates were incubated on Manitol Salt Agar for 24 h at 37 C◦, and identified by using API ID32
Staph which consists of 26 assimilation tests (URE, ADH, ODC, ESC, GLU, FRU, MNE, MAL, LAC, TRE,
MAN, RAF, NET, VP, BGAL, ARGA, PAL, PYRA, NOVO, SAC, NAG, TUR, ARA, BGUR, RIB, CEL).
Incubation of isolated Staphylococcus in a new Modified medium:
The isolates were incubated on two mediums. The first one was on Tryptic Soy Agar (TSA) consisting of
Casein 17g\L, Soy protein 3g\L,Sodium chloride 5g\L, Diputassium phosphate 2.5g\L,Glucose 2.5g\L and 15
Agar and the second new one was on Modified Tryptic Soy Agar (M-TSA) consisting of Casein 17g\L, Soy
protein 3g\L,Sodium chloride 5g\L, Diputassium phosphate 2.5g\ L, Glucose 2.5g\ L and 15 Agar with the
addition of Mannitol 5g\L, Mannose 5g\L and Arginen 0.1g\L, for 24 h at 37 C◦.
Fatty acid extraction and preparation of esters:
Cell mass of 0.4 g was harvested for each isolate species. Weighted cell mass was placed into a screw-cap
tube and fatty acids were saponified by addition of a solution of NaOH in aqueous methanol and heated at 100C◦
in a water bath. The fatty acids were converted to fatty acid methyl esters by addition of a solution of HCl in
aqueous methanol and heated at 80C◦. Fatty acid methyl esters were extracted from the aqueous phase with a
hexane–methyl-tert-butyl ether reagent. A dilute solution of NaOH in distilled water was used to remove
residual reagents from the organic extracts, The upper solvent phase was placed in gas-liquid chromatography
vials [13].
Fatty acids analysis was performed on a GC-MS model Agilent with a capillary column DB-1(30m x
0.25mm). The temperature program was applied from 125 C
◦ for 2 min with a rate of 5C◦ /min and up to 250
C◦ with a rate of 4C◦/min with a carrier gas of helium. The output from the chromatograph is fed to an
integrator for calculation of peak retention times and areas. Fatty acids were identified by standard fatty acid
(FAME) from American company( Supleco) and the results were compared with NIST library equipped with
GC-MS.
RESULTS AND DISCUSSION
Isolation and identification of Staphylococcus species from milk samples:
In recent years, several identification methods have been proposed as alternatives ways to compare some
classical Staphylococcus species identification techniques. Among these methods were commercial miniaturized
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Advances in Environmental Biology, 7(14) December 2013, Pages: 4562-4566
systems such as, Staph API ID32C bioMèrieux [7] which is very effective way to identifies Staphylococcus. In
this study, some strains of Staphylococcus were isolated from various milk collected from Syrian market and
identified by using Staph. API ID32 (26 biochemical reaction). They were 22 isolates as follow: Staphylococcus
aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis and Staphylococcus
lugdunensis with a total number of 4, 6, 5, 4 and 3 isolates respectively. Table (1) illustrate the physiological
diagnostic results of Staphylococcus species by ID 32 Staph system.
Table 1: physiological diagnostic results of Staphylococcus species by ID 32 Staph system
Test
Staph .aureus
Staph. epidermidis
Staph. haemolyticus
Staph. hominis
URE
+
+
+
ADH
+
+
ODC
ESC
GLU
+
+
+
+
FRU
+
+
+
+
MNE
+
MAL
+
+
+
+
LAC
+
+
+
TRE
+
+
+
MAN
+
+
RAF
NIT
+
+
+
+
VP
+
+
BGAL
ARGA
PAL
+
+
PYRA
+
NOVO
+
SAC
+
+
+
+
NAG
+
+
+
TUR
+
+
ARA
BGUR
RIB
CEL
-
Staph. lugdunensis
+
+
+
+
+
+
+
+
+
+
+
+
-
Fatty acid profiles of Staphylococcus species by a new modified media:
TSA and TSA-M media were applied for growing Staphylococcus species to study their effects on fatty
acids composition. TSA-M medium were prepared by adding manitol, mannose and arginine. Staphylococcus
species vary in their ability to assimilate last substrates (Staph. aureus can assimilate manitol, mannose and
arginine, while Staph. epidermidis can assimilate mannose and arginine; Staph. haemolyticus can assimilate
manitol and arginine; Staph. lugdunensis assimilate mannose only while Staph. hominis can not assimilate any
one of them, which in turn affects the fatty acids composition.
Tables (2 and 3) show fatty acids composition of Staphylococcus species grown on TSA and TSA-M.
Table 2: Fatty acids composition of Isolated Staphylococcus species in TSA Medium▲
Fatty acid
Staph.aureus
Staph.epidermidis
Staph.haemolyticus
Ci13:0
0.49±0.07
0.18±0.02
0.23±0.03
Ca13:0
0.45±0.03
0.12±0.03
0.05±0.01
Ci14:0
3.88±0.33
1.58±0.23
4.01±0.34
C14:0
0.86±0.1
0.35±0.01
0.34±0.03
Ci15:0
9.05±0.77
6.34±0.83
5.85±0.64
Ca15:0
44.70±2.02
45.01±2.18
41.3±1.88
C15:0
0.17±0.01
0.06±0.01
0.03±0.01
Ci16:0
1.4±0.07
0.83±0.12
2.3±0.62
C16:1
0.15±0.04
0.17±0.03
0.11±0.02
C16:0
3.2±0.12
2.99±0.19
2.4±0.17
Ci17:0
1.7±0.24
2.35±0.12
3.11±0.22
Ca17:0
6.76±0.44
11.4±0.66
12.75±0.89
C17:0
0.3±0.01
0.19±0.02
0.25±0.04
Ci18:0
0.27±0.06
0.51±0.09
1.42±0.07
C18:1
1.61±0.12
1.92±0.22
1.86±0.12
C18:0
11.7±0.51
12.74±1.04
14.76±1.1
Ci19:0
0.73±0.11
0.9±0.08
0.76±0.14
Ca19:0
0.94±0.13
2.51±0.36
2.06±0.37
C19:0
0.97±0.08
0.49±0.02
0.62±0.21
C20:1
0.28±0.05
0.1±0.01
0.16±0.01
C20:0
10.1±1.01
9.82±0.7
6.1±0.3
Staph.hominis
0.48±0.05
0.39±0.02
4.53±0.34
0.3±0.01
6.54±0.45
43.73±1.88
0.2±0.02
1.55±0.13
0.1±0.01
2.14±0.28
1.7±0.21
6.7±0.59
0.13±0.01
1.26±0.05
1.94±0.27
13.76±1.31
1.84±0.12
3.95±0.21
0.34±0.08
0.07±0.02
9.84±0.91
Staph.lugdunensis
0.61±0.1
0.6±0.04
14.05±0.72
3.17±0.4
4.23±0.22
37.36±1.55
0.58±0.02
2.82±0.16
0.25±0.03
6.04±0.42
0.28±0.02
2.26±0.34
0.63±0.03
0.19±0.02
1.75±0.15
14.4±0.78
0.09±0.02
0.07±0.01
1.35±0.05
0.43±0.02
10.5±0.37
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Advances in Environmental Biology, 7(14) December 2013, Pages: 4562-4566
▲Average number of each isolates, I=iso , a=antiso.
Staphylococcus species contained 21 fatty acids in this study which included straight-chain saturated,
brunched (iso and antiso) and unsaturated fatty acids. The main 7 fatty acids were: C18:0, Ca17:0, C16:0,
Ci15:0, Ci14:0 and C20:0, for this reason Durham and Kloss [4] differentiated Staphylococcus species
depending on their fatty acids profile. Table 2 shows that Staph. aureus contained the highest amount of Ci15:0
(9.05%), while Staph. lugdunesis contained the highest amounts of Ci14:0 and C16:0 with 14.05% and 6.04%,
respectively. Staph. epidermidis and Staph. haemolyticus contained the highest amounts of Ca17:0 with 11.4%
and 12.05% respectively. Staph. haemolyticus can be differentiated from other species by the low amount of
C20:0 (6.1%). These results were similar to that obtained by Behme et al., [2] in their study on fatty acids
profiles in different Staphylococcus species. They found that Staph. haemolyticus contained the highest amount
of Ca17:0 compared with the same species studied in this research and with the results obtained by Kotelainen
et al. [5] whom found that Ca15:0 was the highest amounts in Staph. epidermidis, Staph. haemolyticus and
Staph. hominis with 40.59%, 45.41% and 45.50% respectively.
Results shown in table 3 demonstrated the differences in fatty acids percent according to growth modified
TSA medium. The results revealed that Staph. aureus characterized with the low percent of Ci14:0 (2.65%)
compared with other species, also Staph. haemolyticus and Staph. hominis characterized with the high percent
of Ca15:0 with 50.61% and 52.43%, respectively. The high percent of C20:0 in Staph. epidermidis was 14.69%
and Ca17:0 in Staph. haemolyticus was 13.24% compared with other species, while Staph. lugdunesis contained
the lowest amounts of Ci15:0 with 3.95%. The different composition of media effects the fatty acids percentage.
This results were similar to that recorded by Wauthoz et al. [17] in Staph. epidermidis, who found that the fatty
acids percent varied according to media composition.
Table 3: Fatty acids composition of Isolated Staphylococcus species in modified TSA medium▲
Fatty acids
Staph .aureus
Staph epidermidis
Staph. haemolyticus
Staph. hominis
Ci13:0
0.4±0.02
0.85±0.27
0.39±0.03
0.12±0.01
Ca13:0
0.33±0.03
0.32±0.16
0.44±0.1
0.23±0.03
Ci14:0
2.65±0.14
8.6±0.73
4.73±0.47
4.01±0.23
C14:0
0.78±0.21
0.76±0.32
0.29±0.02
0.08±0.01
Ci15:0
8.51±0.65
6.8±0.53
6.83±0.23
6.17±0.29
Ca15:0
36.68±1.92
34.72±1.68
50.61±2.13
52.43±2.25
C15:0
0.22±0.04
0.28±0.06
0.28±0.03
0.1±0.01
Ci16:0
0.98±0.26
1.8±0.09
2.36±0.24
1.5±0.06
C16:1
0.02±0.01
0.11±0.02
0.38±0.04
0.26±0.03
C16:0
5.1±0.16
4.33±0.26
2.4±0.13
1.47±0.13
Ci17:0
2.3±0.25
1.5±0.17
2.66±0.21
1.59±0.15
Ca17:0
5.43±0.36
4.78±0.31
13.24±1.14
7.42±0.45
C17:0
0.52±0.06
0.34±0.01
0.14±0.02 0.39±0.07
Ci18:0
0.48±0.02
0.75±0.06
0.7±0.02
0.9±0.02
C18:1
2.42±0.48
0.26±0.02
0.38±0.02
0.41±0.06
C18:0
13.97±0.77
12.84±1.03
10.67±0.81
12.59±0.64
Ci19:0
1.28±0.13
1.52±0.29
0.38±0.02
1.42±0.21
Ca19:0
1.33±0.17
1.89±0.24
1.18±0.2
3.7±0.12
C19:0
1.37±0.14
1.4±0.1
0.32±0.01
0.23±0.03
C20:1
0.14±0.03
0.18±0.06
0.12±0.01
0.07±0.01
C20:0
11.62±0.82
14.69±0.62
3.18±0.49
6.7±0.34
▲Average number of each isolates, I=iso , a=antiso
Staph. lugdunensis
0.25±0.03
0.21±0.06
6.58±0.25
0.87±0.1
3.95±0.41
39.91±1.76
0.32±0.02
3.05±0.56
1.05±0.23
5.89±0.68
1.14±0.21
6.98±0.34
0.48±0.08
1.03±0.13
2.49±0.47
12.7±0.73
0.38±0.04
1.4±0.21
1.28±0.17
0.06±0.01
9.27±0.35
In conclusion Staphylococcus species can be identified by fatty acids profile to the species level using GCMS applied on cultures grown on TSA and the modified TSA-M media.
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