Introduction opportunistic agents [2].
1. Introduction opportunistic agents [2].
The genus Brevibacterium belongs to family Brevibacterium spp. is gram-positive irregular, Brevibacteriacea, order Actinomycetales which slender, rod-shaped bacteria that display a marked comprises more than 20 species [3]. rod- to-coccus cycle, non-acid-fast. The peptidoglycan Microbes produce an extraordinary array of contains meso-DAP as the principal diamino acid [1]. microbial defense systems. These include They are obligately aerobic and oxidative in their broad-spectrum classical antibiotics, metabolic metabolism of carbohydrates. They are non-motile byproducts, such as the lactic acids produced by and salt-tolerant > 6.5% NaCl. They also produce lactobacilli, lytic agents such as lysozymes, numerous catalase and proteinases and characteristically produce types of protein exotoxins, and bacteriocins, which are methanethiol CH 3 SH from L-methionine [2]. loosely defined as biologically active protein moieties The habitat of the Brevibacterium is primarily milk with a bactericidal mode of action. This biological products, in which the bacteria contribute to the aroma arsenal is striking not only in its diversity, but also in its and the color of surface-ripened cheeses. They are natural abundance. Bacteriocins are found in almost also found on human skin surfaces under moist, every bacterial species examined to date, and within a macerating conditions between the toes and other species. Tens or even hundreds of different kinds of intertriginous areas and are believed to contribute to bacteriocins are present and 99% of all bacteria may body odor, but are rare causes of human infections as make at least one kind of bacteriocin [4].
Corresponding author: Essra Gh. Al-Sammak, Ph.D., The last decade has witnessed the description and assistant professor, research fields: microbiology, classification
characterization of a variety of bacteriocins from of bacteria. E-mail: [email protected].
Numerical Classification of Brevibacterium and Related Genera Using Linocin M18 Bacteriocin
gram-positive bacteria. Most of these are produced by morphology, colonial morphology, pigmentation and lactic acid bacteria with activity spectrums usually
rod-coccus cycle, the production of many enzymes, restricted to closely related strains [5]. The the ability to grow at different temperatures and extraordinary interest in bacteriocins is based concentration of NaCl in addition to their ability to primarily on their potential to inhibit food-borne
oxidizing different CHO [2, 9, 10] and sensitivity to pathogens such as Listeria monocytogenes [6].
antibiotics [11]. Other tests were also carried out. Isolation and characterization of non-lanthionine-
2.2 Bacteriocin Assay
containing linocin M18 from red smear cheese bacteria B. linens M18 were carriedout by Valde’s-
Bacteriocin activity was carried out according to Stauber, et al. [7]. This is the only bacteriocin isolated
Ogunbanuwo [12] by culturing the isolated species in so far from coryneform bacteria. The bacteriocin
a straight line in the center of the media supplement consisted of a single protein subunit with a molecular
with modified yeast extract agar with 2% NaCl. They mass of 31 kDa by SDS-PAGE, but, in its native state,
were incubated at 37 C or 25 C depending on the formed aggregates of extremely high molecular
species for 2 days and then cultured 8 of the test masses (> 2000 kDa).
bacteria and yeast in a right angle 90 to the first line Usually, bacteriocins inhibit only closely related
from each side and incubated in 37 C to 24 h [13]. bacteria, but linocin M18 exhibited an extraordinarily
2.3 Numerical Comparison
broad activity spectrum with activity against species In order to facilitate data handling, the phenotypes
of the genera Bacillus, Arthrobacter, Corynebactrium, were coded in a binary form and were compared using
Micrococcus and Listeria. Oligonucleotide probes simple matching coefficient (Ssm) and clustering
based on the N-terminal amino acid sequence have method of unweighted average linkage between
been used to locate the gene coding for linocin M18 groups by SPSS program. This numerical analysis led
which is a single copy of the gene lin, and was located to the construction of a hierarchical tree.
on chromosomal DNA [8]. So the aims of the study are as follows:
2.4 DNA Isolation
(1) Phenetic classification of Brevibacterium and Selection of species from some clusters was carried related genera using numerical method; out to detect the bacteriocin producing lin gene. The B. (2) Detection of Brevibacterium and related genera linens and other coryneform actinobacteria were ability to produce linocin M18 bacteriocin against grown in modified yeast extract agar. Total DNA some gram positive bacteria and yeast; isolation was done by genomic DNA purification from (3) Detection of the lin gene that is responsible for
Promega Company.
the production of linocin M18 bacteriocin using Gene amplification by PCR was carried out using conventional PCR technique. Promega standard kit as follows: D.W: 10 µL; Green
2. Materials and Methods
master mix: 5 µL (1×); Primer: 5 µL (25 pmol); DNA template: 5 µL (250 ng).
2.1 Sample Collection The sequencing of primer is
5 ′-CGACGACAGCCTCGGCATC-3′ upstream and product, skin and blood from cancer and kidney
Fifty bacterial strains were isolated from dairy
5 ′-GGCGGAGAAGCTGTCCTGG-3′ downstream, failure dialysis patients. The strains were identified
DNA thermocycler was programmed as follows: 1 and classified numerically using a set of 52 phenetic
C (5 min), 68 o C characteristics such as gram staining, cellular o (45 sec), 72
cycle: 94 o
C (5 min); 35 cycle: 94 o
C (1 min).
Numerical Classification of Brevibacterium and Related Genera Using Linocin M18 Bacteriocin
Amplification products were detected by 2% descriptions identified the isolates to nine genera: agarose gel electrophoresis for 120 min. The size of
Corynebacterium , Arthrobacter , the amplified gene was determined by using 100 bp
Brevibacterium ,
Actinomyces , Exiguobacterium, Kocuria, Micrococcus, DNA ladder from Promega Company.
Rothia , Rhodococcus & 22 species which aggregate in seven main clusters and 29 sub-clusters as in the