المعززات الحيوية كمواد بديلة مضادة للبكتيريا ومضادة للأغشية الحيوية لبعض مسببات الأمراض البكتيرية
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Date
2024-06-11
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Summary
Since many beneficial bacteria fall within the lactic acid bacteria (LAB)
group, the focus has been on the isolation of LAB from various sources and
assessing their probiotic qualities in vitro. By using 16S rRNA gene sequencing,
the strains were identified. Following this, whole genome sequencing was used
to identify the strain level, investigate possible therapeutic applications, and
evaluate safety.
150 samples were collected of traditional yogurt, cheese, and infant feses.
The De Man Rogosa and Sharpe (MRS) selective medium was used to separate
the bacteria, and then they were purified. 161 isolates were obtained, divided
into 47 from infant feces, 51 from yogurt, and 63 from cheeses. Initially,
identification was accomplished by morphological and biochemical
investigations using colony and cells characteristics Gram staining, oxidase,
and catalase.
Multiple phases were used to assess the probiotic properties of the
bacterial isolates. First, as a precaution, a hemolysis test was done. The majority
of the isolates exhibited no blood hemolysis. The efficiency of the agar
diffusion, agar spot, and broth microdilution methods was then evaluated
against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella
pneumoniae, Enterococcus faecalis, and Escherichia coli. Only 10 of the total
isolates tested exhibited inhibitory activity against the indicator bacteria. n the
diffusion test against E. faecalis, the largest mean diameter of the inhibition
zone was 21 mm, whereas the spot test against K. pneumoniae had the largest
mean inhibition zone of 25 mm. The microdilution assay yielded a minimum
inhibitory concentration of 1/16. Furthermore, the strains were evaluated for
resistance to simulated stomach and intestinal environments. The logarithm of
IV
live cells decreased somewhat when exposed to the enzyme pepsin and pH3.
However, there were no significant differences in the logarithm of viable cells
when Oxgall was present.
In both the auto-aggregation and co-aggregation ability tests, isolate HR3
had higher percentages of 65.95% and 59.32%, respectively. Anti-biofilm
activities were evaluated. Cell-free supernatants (CFSs) have been shown to
inhibit P. aeruginosa and S. lugdunensis biofilm formation. During the
antibiotic susceptibility test, the probiotic isolates showed consistent resistance
patterns against ciprofloxacin, cloxacillin, gentamicin, kanamycin,
streptomycin, and vancomycin, as well as resistance to tetracycline. These
isolates, however, were susceptible to penicillin, imipenem, erythromycin,
clindamycin, amikacin, and azithromycin.
By using the 16S rRNA gene sequence the isolates HR1 and HR2 were
identified as Lacticaseibacillus sp. and Lacticaseibacillus paracasei while the
other isolates were identified as L. plantarum.
Whole genome resequencing was performed, and sequencing data was
converted into raw data by Illumina SBS technology. After sequencing, overall
reads' quality, total bases, total reads, GC (%) and basic statistics were
calculated. The filtered reads were mapped to the reference genome. After the
genome was assembled, it was annotated. The variants were classified by each
chromosome.
Pathosystems Resource Integration Center (PATRIC) and Rapid
Annotation using Subsystem Technology (RAST) were used for assembly and
annotation. The National Center for Biotechnology Information (NCBI) was
used to register sequences of strains HA3(Isolate HR3) and HA9(Isolate HQM),
under accession numbers JAUTDK000000000.1 and JAUTDL000000000.1.
The genomes measured a single circular 3,314,449 and 3,312,723 base
pairs in size. Their chromosomes contained 44.38% and 44.40% G+C content.
Isolate HA3 comprised 3,253 protein-coding sequences, 3 ribosomal RNA
(rRNA) genes, and 62 transfer RNA (tRNA) genes, while strain HA9 contained
3,298 protein-coding sequences, 2 rRNA genes, and 59 tRNA genes. Both HA3
and HA9 strains were equipped with multiple antibiotic resistance genes across
different groups and stress response factors. The HA3 strain also can produce a
total of 14 bacteriocins, and eight of them exhibit immunogenicity. The HA3
and HA9 strains could synthesize water-soluble B vitamins.
The protein sequences inside PATRIC cross-genus families (PGFams)
were aligned by multiple sequence comparison by log-expectation (MUSCLE)
tool, and the nucleotides that matched each sequence were assigned to a protein.
These sequences revealed that HA3 was 100% comparable to the reference
strain L. plantarum WCFS1, whereas HA9 was 80%.