Molecular Characterisation of Bacteriocinogenic Lactobacillus Plantarum Isolated from Malaysian Fermented Food

Molecular approaches were used in this study to characterize six bacteriocinogenic Lactobacillus plantarum strains isolated from Malaysian foods since biochemical approaches could not differentiate them distinctively. The Lb. plantarum strains were initially identified as Lb. plantarum I with 99.9% similarity by the analysis of carbohydrate fermentation pattern using API CHL50 identification kit. The biochemical identification result was further confirmed by analyzing partial sequence of 16S rDNA that showed 99-100% similarity to Lb. plantarum. Identification up to genus level was also achieved when Amplified Ribosomal DNA Restriction Analaysis (ARDRA) was applied with Lactococcus lactis MG1363, Lb. plantarum ATCC 11305, Lb. johnsonii, Streptococcus thermophilus BAA 250 and Pediococcus acidilactici 446 as reference strains. Furthermore, the studied Lb. plantarum strains were characterized using genotypic methods: plasmid profiling, randomly amplified polymorphic DNA (RAPD), polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP), repetitive extragenic palindromes (Rep)-PCR as well as 16S-23S rDNA (ITS1) and 23S-5S rDNA (ITS2) spacer regions analyses. The strain RG14 was successfully differentiated from others by plasmid profiling. Results from RAPD study in which 6 arbitrary primers were tested, revealed slight differences in the genome of six Lb. plantarum strains. Moreover, sequence analysis of ITS1 revealed a four base pair variable region from which the strains could be divided into four groups. Comparative analysis of ITS1 with 17 Lb. plantarum strains available in GenBank confirmed the variability of this region and showed that the genotype of the studied strains are not present in the strains used for comparative analysis. As for PCR-RFLP study, the studied strains were initially screened for the presence of structural bacteriocin genes. It was found that all studied strains harboured the novel combination of plantaricin EF (Pln EF) and plantaricin W (Pln W), which had not been reported elsewhere. However, the PCR-RFLP technique was not discriminative when the Pln EF genes were digested with restriction enzymes HindIII, MboI and PstI. Although rep-PCR showed strong typing ability, the banding pattern was not discriminative. The ITS2 region showed an extra 5S rDNA sequence downstream of the ribosomal DNA region. The ITS2 region, however, was highly conserved among the strains and encodes rRNA that form secondary structure with the predicted free energy of -11.5 Kcal/mol. In conclusion, the studied strains are novel bacteriocinogenic Lb. plantarum, which were successfully discriminated in a polyphasic approaches using plasmid profiling, RAPD and ITS1 analysis with the RAPD technique showing the highest discriminatory power

Novi bakteriocinogeni sojevi Lactobacillus plantarum i njihova diferencijacija analizom slijeda 16S rDNA, 16S-23S i 23S-5S međugenskih razmaknica, te analizom nasumično umnožene polimorfne DNA

Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11) isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin-producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains.Ispitana je prisutnost strukturalnih bakteriocinskih gena u šest sojeva Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 i RI11), izoliranih iz malezijske hrane. Nova kombinacija strukturalnih gena za plantaricin EF i plantaricin W uspješno je umnožena iz svih ispitanih sojeva, što upućuje na to da se radi o novim sojevima L. plantarum koji proizvode bakteriocine. Usporednom analizom sa 17 sojeva L. plantarum pohranjenih u GenBank otkrivena je varijabilna regija od četiri para baza u kratkim 16S-23S međugenskim razmaknicama tih sojeva, potvrđujući da se radi o novim genotipovima. Ispitani sojevi L. plantarum nakon toga su razvrstani u četiri skupine, na temelju uočene varijabilne regije od četiri para baza kratke 16S-23S međugenske razmaknice. Daljnjom analizom DNA slijeda 23S-5S međugenske razmaknice otkriven je samo jedan tip te razmaknice, pokazujući da je konzerviran u ispitanim sojevima L. plantarum. Trima pokusima nasumičnog umnožavanja polimorfne DNA, koristeći tri različite kombinacije proizvoljno odabranih početnica, uspješno su diferencirani ispitani sojevi L. plantarum, što potvrđuje da se radi o različitim sojevima. Dakle, može se zaključiti da novi sojevi L. plantarum proizvode bakteriocin i da se mogu uspješno razlikovati analizom nasumično umnožene polimorfne DNA i varijabilne regije kratke 16S-23S međugenske razmaknice

Novi bakteriocinogeni sojevi Lactobacillus plantarum i njihova diferencijacija analizom slijeda 16S rDNA, 16S-23S i 23S-5S međugenskih razmaknica, te analizom nasumično umnožene polimorfne DNA

Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11) isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin-producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains.Ispitana je prisutnost strukturalnih bakteriocinskih gena u šest sojeva Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 i RI11), izoliranih iz malezijske hrane. Nova kombinacija strukturalnih gena za plantaricin EF i plantaricin W uspješno je umnožena iz svih ispitanih sojeva, što upućuje na to da se radi o novim sojevima L. plantarum koji proizvode bakteriocine. Usporednom analizom sa 17 sojeva L. plantarum pohranjenih u GenBank otkrivena je varijabilna regija od četiri para baza u kratkim 16S-23S međugenskim razmaknicama tih sojeva, potvrđujući da se radi o novim genotipovima. Ispitani sojevi L. plantarum nakon toga su razvrstani u četiri skupine, na temelju uočene varijabilne regije od četiri para baza kratke 16S-23S međugenske razmaknice. Daljnjom analizom DNA slijeda 23S-5S međugenske razmaknice otkriven je samo jedan tip te razmaknice, pokazujući da je konzerviran u ispitanim sojevima L. plantarum. Trima pokusima nasumičnog umnožavanja polimorfne DNA, koristeći tri različite kombinacije proizvoljno odabranih početnica, uspješno su diferencirani ispitani sojevi L. plantarum, što potvrđuje da se radi o različitim sojevima. Dakle, može se zaključiti da novi sojevi L. plantarum proizvode bakteriocin i da se mogu uspješno razlikovati analizom nasumično umnožene polimorfne DNA i varijabilne regije kratke 16S-23S međugenske razmaknice

Novel bacteriocinogenic Lactobacillus plantarum strains and their differentiation by sequence analysis of 16S rDNA, 16S-23S and 23S-5S intergenic spacer regions and randomly amplified polymorphic DNA

Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11) isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin- producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains

Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity

Moghadam MS, Albersmeier A, Winkler A, et al. Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity. BMC Genomics. 2016;17(1): 117

Novel Bacteriocinogenic Lactobacillus plantarum Strains and Their Differentiation by Sequence Analysis of 16S rDNA, 16S-23S and 23S-5S Intergenic Spacer Regions and Randomly Amplified Polymorphic DNA Analysis

Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11) isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin-producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains

PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively