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Fulltext Estimation of 16S rRNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken

Lee CM, Sieo CC, Abdullah N, Ho YW

FEMS Microbiol Lett, 2008 Oct;287(1):136-41.
PMID: 18707622 DOI: 10.1111/j.1574-6968.2008.01305.x

The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.

Matched MeSH terms: Lactobacillus/isolation & purification
Identification of lactic acid bacteria constituting the predominating microflora in an acid-fermented condiment (tempoyak) popular in Malaysia

Leisner JJ, Vancanneyt M, Rusul G, Pot B, Lefebvre K, Fresi A, et al.

Int J Food Microbiol, 2001 Jan 22;63(1-2):149-57.
PMID: 11205946

Tempoyak is a traditional Malaysian fermented condiment made from the pulp of the durian fruit (Durio zibethinus). Salt is sometime added to proceed fermentation at ambient temperature. In various samples obtained from night markets, lactic acid bacteria (LAB) were the predominant microorganisms, ranging from log 8.4 to log 9.2 cfu g(-1). No other microorganisms were present to such a level. These samples contained reduced amount of saccharose, glucose and fructose but increased amount of D- and L-lactic acid and acetic acid compared with samples of non-fermented durian fruit. Sixty-four isolates of LAB were divided into five groups by use of a few phenotypic tests. A total of 38 strains of LAB were selected for comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their whole cell protein patterns with a SDS-PAGE database of LAB. These strains were also examined for their carbohydrate fermentation patterns by use of API 50 CH. Isolates belonging to the Lactobacillus plantarum group were shown to be the predominant members of the LAB flora. In addition, isolates belonging to the Lactobacillus brevis group, Leuconostoc mesenteroides, Lactobacillus mali, Lactobacilus fermentum and an unidentified Lactobacillus sp. were also observed. A high degree of diversity among isolates belonging to the Lb. plantarum group was demonstrated by analysis of their plasmid profiles.

Matched MeSH terms: Lactobacillus/isolation & purification*
Fulltext Isolation and Characterization of Lactobacillus spp. from Kefir Samples in Malaysia

Talib N, Mohamad NE, Yeap SK, Hussin Y, Aziz MNM, Masarudin MJ, et al.

Molecules, 2019 Jul 17;24(14).
PMID: 31319614 DOI: 10.3390/molecules24142606

Kefir is a homemade, natural fermented product comprised of a probiotic bacteria and yeast complex. Kefir consumption has been associated with many advantageous properties to general health, including as an antioxidative, anti-obesity, anti-inflammatory, anti-microbial, and anti-tumor moiety. This beverage is commonly found and consumed by people in the United States of America, China, France, Brazil, and Japan. Recently, the consumption of kefir has been popularized in other countries including Malaysia. The microflora in kefir from different countries differs due to variations in culture conditions and the starter media. Thus, this study was aimed at isolating and characterizing the lactic acid bacteria that are predominant in Malaysian kefir grains via macroscopic examination and 16S ribosomal RNA gene sequencing. The results revealed that the Malaysian kefir grains are dominated by three different strains of Lactobacillus strains, which are Lactobacillus harbinensis, Lactobacillusparacasei, and Lactobacillus plantarum. The probiotic properties of these strains, such as acid and bile salt tolerances, adherence ability to the intestinal mucosa, antibiotic resistance, and hemolytic test, were subsequently conducted and extensively studied. The isolated Lactobacillus spp. from kefir H maintained its survival rate within 3 h of incubation at pH 3 and pH 4 at 98.0 ± 3.3% and 96.1 ± 1.7% of bacteria growth and exhibited the highest survival at bile salt condition at 0.3% and 0.5%. The same isolate also showed high adherence ability to intestinal cells at 96.3 ± 0.01%, has antibiotic resistance towards ampicillin, penicillin, and tetracycline, and showed no hemolytic activity. In addition, the results of antioxidant activity tests demonstrated that isolated Lactobacillus spp. from kefir G possessed high antioxidant activities for total phenolic content (TPC), total flavonoid content (TFC), ferric reducing ability of plasma (FRAP), and 1,1-diphenyl-2-picryl-hydrazine (DPPH) assay compared to other isolates. From these data, all Lactobacillus spp. isolated from Malaysian kefir serve as promising candidates for probiotics foods and beverage since they exhibit potential probiotic properties and antioxidant activities.

Matched MeSH terms: Lactobacillus/isolation & purification*
Acid and bile tolerance of Lactobacillus isolated from chicken intestine

Jin LZ, Ho YW, Abdullah N, Jalaludin S

Lett Appl Microbiol, 1998 Sep;27(3):183-5.
PMID: 9750324

Twelve Lactobacillus strains isolated from chicken intestine were used to investigate acid and bile tolerance in vitro. Ten out of the 12 strains were slightly affected by 0.3% bile salts, showing a delay of growth (d) of 0.6-37.2 min compared with growth in control cultures. Two strains were not affected by the bile salts. Of the 12 strains, seven could be arbitrarily classified as resistant (d < 15 min) and five as tolerant (15 min < d < or = 40 min). Lactobacillus strains from the caecum showed better tolerance to acid than those from the ileum. Generally, the survival of the ileal strains was very low at pH 1.0 and 2.0, and moderate at pH 3.0. In contrast, caecal Lactobacillus strains could survive at pH 1.0 for up to 2 h of incubation; growth was moderate at pH 2.0 and good at pH 3.0 and 4.0.

Matched MeSH terms: Lactobacillus/isolation & purification
Adhesion of Lactobacillus isolates to intestinal epithelial cells of chicken

Jin LZ, Ho YW, Ali MA, Abdullah N, Ong KB, Jalaludin S

Lett Appl Microbiol, 1996 Mar;22(3):229-32.
PMID: 8852352

A total of 46 Lactobacillus isolates obtained from chicken intestine were assessed on their ability to adhere to the chicken ileal epithelial cell (IEC) in vitro. Twelve out of the 46 isolates showed moderate to good ability to adhere to the IEC. Temperature (between 4 degrees C and 42 degrees C) did not affect attachment. Incubation (contact) time of 30 min was found to be insufficient for the attachment of bacteria to the IEC, but contact time beyond 1 h did not increase this ability. The pH values (4-7) of the suspending buffer did not have any significant effect on the attachment of bacteria to the IEC, but at pH 8 it was reduced significantly (P < 0.05).

Matched MeSH terms: Lactobacillus/isolation & purification
Fulltext Salivary cariogenic bacteria counts are associated with obesity in student women at a Malaysian university

Yeo WZ, Lim SP, Say YH

Asia Pac J Clin Nutr, 2017 12 10;27(1):99-106.
PMID: 29222886 DOI: 10.6133/apjcn.032017.14

BACKGROUND AND OBJECTIVES: The counts of cariogenic bacteria lactobacilli and mutans streptococci have been studied and correlated with sugar intake. This study was to investigate the association between salivary lactobacilli and mutans streptococci counts with sweet food eating behavior and sweet sensitivity among 120 Malaysian women (101 ethnic Chinese, 19 ethnic Indians), while taking into account anthropometric and menstruation variables.
METHODS AND STUDY DESIGN: Demographics, anthropometric measurements and menstrual history were taken. Hedonic preference, intake frequency of a list of sweet foods, intensity perception and pleasantness ratings of sweet stimuli were assessed. Saliva was collected for lactobacilli and mutans streptococci culture.
RESULTS: We found that centrally obese subjects (high waist circumference and waist-hip ratio) had significantly higher salivary lactobacilli and mutans streptococci counts (all p<0.05), while overweight and high total body fat subjects had significantly higher salivary mutans streptococci counts (p<0.001). The sweetness intensity perception of chocolate malt drinks was significantly lower in women who were in their pre-menstrual (post-ovulation) phase. However, menstruation variables (menstrual phases, regularity and pre-menstrual syndromes) did not play a role in determining compulsive eating, sweets/chocolate craving and salivary lactobacilli and mutans streptococci counts.
CONCLUSIONS: Taken together, salivary lactobacilli and mutans streptococci counts of the Malaysian women are associated with central obesity, but not sweet food eating behaviour, sweet sensitivity and menstruation variables. Salivary microbiome analysis could be useful as a potential diagnostic indicator of diseases such as obesity.

Matched MeSH terms: Lactobacillus/isolation & purification*
Identification of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum from honey stomach of honeybee

Tajabadi N, Mardan M, Saari N, Mustafa S, Bahreini R, Manap MY

Braz J Microbiol, 2013;44(3):717-22.
PMID: 24516438

This study aimed to isolate and identify Lactobacillus in the honey stomach of honeybee Apis dorsata. Samples of honeybee were collected from A. dorsata colonies in different bee trees and Lactobacillus bacteria isolated from honey stomachs. Ninety two isolates were Gram-stained and tested for catalase reaction. By using bacterial universal primers, the 16S rDNA gene from DNA of bacterial colonies amplified with polymerase chain reaction (PCR). Forty-nine bacterial 16S rDNA gene were sequenced and entrusted in GenBank. Phylogenetic analysis showed they were different phylotypes of Lactobacillus. Two of them were most closely relevant to the previously described species Lactobacillus plantarum. Other two phylotypes were identified to be closely related to Lactobacillus pentosus. However, only one phylotype was found to be distantly linked to the Lactobacillus fermentum. The outcomes of the present study indicated that L. plantarum, L. pentosus, and L. fermentum were the dominant lactobacilli in the honey stomach of honeybee A. dorsata collected during the dry season from Malaysia forest area - specifically "Melaleuca in Terengganu".

Matched MeSH terms: Lactobacillus/isolation & purification*
Plasmid profiling and curing of Lactobacillus strains isolated from the gastrointestinal tract of chicken

Chin SC, Abdullah N, Siang TW, Wan HY

J Microbiol, 2005 Jun;43(3):251-6.
PMID: 15995642

In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.

Matched MeSH terms: Lactobacillus/isolation & purification
Lactobacillus nasalidis sp. nov., isolated from the forestomach of a captive proboscis monkey (Nasalis larvatus)

Suzuki-Hashido N, Tsuchida S, Hayakawa T, Sakamoto M, Azumano A, Seino S, et al.

Int J Syst Evol Microbiol, 2021 Apr;71(4).
PMID: 33906706 DOI: 10.1099/ijsem.0.004787

Three strains (YZ01T, YZ02 and YZ03) of Gram-stain-positive, facultatively anaerobic rods were isolated from the forestomach contents collected from a captive male proboscis monkey (Nasalis larvatus) at Yokohama Zoo in Japan. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belonged to the genus Lactobacillus. Based on the sequence similarity of the 16S rRNA gene, Lactobacillus delbrueckii subsp. indicus JCM 15610T was the closest phylogenetic neighbour to YZ01T. Sequence analyses of two partial concatenated housekeeping genes, the RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS) also indicated that the novel strains belonged to the genus Lactobacillus. The average nucleotide identity and digital DNA-DNA hybridization (dDDH) between L. delbrueckii subsp. indicus and YZ01T were 85.9 and 31.4 %, respectively. The phylogenetic tree based on the whole genomic data of strains YZ01T, YZ02 and YZ03 suggested that these three strains formed a single monophyletic cluster in the genus Lactobacillus, indicating that it belonged to a new species. The DNA G+C content of strain YZ01T was 51.6 mol%. The major fatty acids were C16 : 0 and C18 : 1 ω9c. Therefore, based on phylogenetic, phenotypic and physiological evidence, strains YZ01T, YZ02 and YZ03 represent a novel species of the genus Lactobacillus, for which the name Lactobacillus nasalidis sp. nov. is proposed with the type strain YZ01T (=JCM 33769T=DSM 110539T).

Matched MeSH terms: Lactobacillus/isolation & purification
Metagenomics analysis reveals significant modulation of cecal microbiota of broilers fed palm kernel expeller diets

Chen WL, Tang SGH, Jahromi MF, Candyrine SCL, Idrus Z, Abdullah N, et al.

Poult Sci, 2019 Jan 01;98(1):56-68.
PMID: 30137571 DOI: 10.3382/ps/pey366

The potential use of palm kernel expeller (PKE) as an alternative energy source in broiler diets is limited by the high fiber content. Although enzymatic treatment could alleviate the fiber component and increase the nutritive value of PKE, this apparent improvement is not reflected in the growth response of birds fed with the treated-PKE. As chicken's ceca are the most heavily populated with microflora within their gastrointestinal tract, it was hypothesized that any modulation of the intestinal environment by dietary treatments should be reflected by the composition and activities of the cecal microflora. There is a correlation between cecal microbiota composition and the efficiency of the host to extract energy from the diet and to deposit that energy into improved feed conversion ratio. At present, little is known about the changes on cecal microflora of broilers fed with PKE diets. Hence, this study was designed to assess the effects of feeding different forms of PKE; namely untreated PKE (UPKE), enzyme-treated PKE (EPKE), and oligosaccharides extracted from PKE (OligoPKE), on the cecal microbiota of broiler chickens at 14 d old (day 14) and 28 d old (day 28) using 16S rRNA gene high-throughput next-generation sequencing method. The results showed that temporal changes in cecal microbiota of broiler chickens were evident on day 14 and day 28. The relative abundance of phylum Firmicutes, known to be involved in nutrient uptake and absorption in both age groups was higher in the UPKE as compared to EPKE group. In addition, supplementation of OligoPKE increased (P < 0.05) the relative abundance of Lactobacillus on both D14 and D28, signifying its effect as prebiotics in enhancing growth of indigenous Lactobacillus. Our results showed that cecal microbiota was significantly modulated by dietary treatments and that the lower relative abundance of phylum Firmicutes in chickens fed with EPKE could be a reason why broiler chickens fed with EPKE of higher metabolizable energy (ME) content did not show improvement in their growth performance.

Matched MeSH terms: Lactobacillus/isolation & purification
Physio-chemical, microbiological properties of tempoyak and molecular characterisation of lactic acid bacteria isolated from tempoyak

Chuah LO, Shamila-Syuhada AK, Liong MT, Rosma A, Thong KL, Rusul G

Food Microbiol, 2016 Sep;58:95-104.
PMID: 27217364 DOI: 10.1016/j.fm.2016.04.002

This study aims to determine physio-chemical properties of tempoyak, characterise the various indigenous species of lactic acid bacteria (LAB) present at different stages of fermentation and also to determine the survival of selected foodborne pathogens in tempoyak. The predominant microorganisms present in tempoyak were LAB (8.88-10.42 log CFU/g). Fructobacillus durionis and Lactobacillus plantarum were the dominant members of LAB. Other LAB species detected for the first time in tempoyak were a fructophilic strain of Lactobacillus fructivorans, Leuconostoc dextranicum, Lactobacillus collinoides and Lactobacillus paracasei. Heterofermentative Leuconostoc mesenteroides and F. durionis were predominant in the initial stage of fermentation, and as fermentation proceeded, F. durionis remained predominant, but towards the end of fermentation, homofermentative Lb. plantarum became the predominant species. Lactic, acetic and propionic acids were present in concentrations ranging from 0.30 to 9.65, 0.51 to 7.14 and 3.90 to 7.31 mg/g, respectively. Genotyping showed a high degree of diversity among F. durionis and Lb. plantarum isolates, suggesting different sources of LAB. All tested Lb. plantarum and F. durionis (except for one isolate) isolates were multidrug resistant. Salmonella spp., Listeria monocytogenes and Staphylococcus aureus were not detected. However, survival study showed that these pathogens could survive up to 8-12 days. The results aiming at improving the quality and safety of tempoyak.

Matched MeSH terms: Lactobacillus/isolation & purification*
Identification of lactic acid bacteria from chili bo, a Malaysian food ingredient

Leisner JJ, Pot B, Christensen H, Rusul G, Olsen JE, Wee BW, et al.

Appl Environ Microbiol, 1999 Feb;65(2):599-605.
PMID: 9925588

Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28 degreesC with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg-1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.

Matched MeSH terms: Lactobacillus/isolation & purification
Inhibition of Staphylococcus aureus by crude and fractionated extract from lactic acid bacteria

Wong CB, Khoo BY, Sasidharan S, Piyawattanametha W, Kim SH, Khemthongcharoen N, et al.

Benef Microbes, 2015 Mar;6(1):129-39.
PMID: 25213027 DOI: 10.3920/BM2014.0021

Increasing levels of antibiotic resistance by Staphylococcus aureus have posed a need to search for non-antibiotic alternatives. This study aimed to assess the inhibitory effects of crude and fractionated cell-free supernatants (CFS) of locally isolated lactic acid bacteria (LAB) against a clinical strain of S. aureus. A total of 42 LAB strains were isolated and identified from fresh vegetables, fresh fruits and fermented products prior to evaluation of inhibitory activities. CFS of LAB strains exhibiting a stronger inhibitive effect against S. aureus were fractionated into crude protein, polysaccharide and lipid fractions. Crude protein fractions showed greater inhibition against S. aureus compared to polysaccharide and lipid fractions, with a more prevalent effect from Lactobacillus plantarum 8513 and L. plantarum BT8513. Crude protein, polysaccharide and lipid fractions were also characterised with glycine, mannose and oleic acid being detected as the major component of each fraction, respectively. Scanning electron microscopy revealed roughed and wrinkled membrane morphology of S. aureus upon treatment with crude protein fractions of LAB, suggesting an inhibitory effect via the destruction of cellular membrane. This research illustrated the potential application of fractionated extracts from LAB to inhibit S. aureus for use in the food and health industry.

Matched MeSH terms: Lactobacillus/isolation & purification
Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced-based PCR fingerprinting

Lee CM, Sieo CC, Cheah YK, Abdullah N, Ho YW

J Sci Food Agric, 2012 Feb;92(3):660-6.
PMID: 21919004 DOI: 10.1002/jsfa.4627

Four repetitive element sequence-based polymerase chain reaction (rep-PCR) methods, namely repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), polytrinucleotide (GTG)₅ -PCR and BOX-PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep-PCR methods and their combination (composite rep-PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D).

Matched MeSH terms: Lactobacillus/isolation & purification
Fulltext Probiotic Properties of Exopolysaccharide-Producing Lactobacillus Strains Isolated from Tempoyak

Khalil ES, Abd Manap MY, Mustafa S, Alhelli AM, Shokryazdan P

Molecules, 2018 Feb 13;23(2).
PMID: 29438288 DOI: 10.3390/molecules23020398

Tempoyak is a functional Malaysian food (an acid-fermented condiment) which is produced from the pulp of the durian (Durio zibethinus) fruit. The current study aimed to isolate and identify potential exopolysaccharide (EPS)-producing Lactobacillus strains from tempoyak for potential use as probiotics. Seven isolates (DUR2, DUR4, DUR5, DUR8, DUR12, DUR18, and DUR20) out of 44 were able to produce EPS, and exhibited resistance to acid and bile salt compared to the reference strains Lactobacillus rhmnosus (ATCC53103) and L. plantarum (ATCC8014). The seven isolated strains belonged to five different species-L. plantarum, L. fermentum, L. crispatus, L. reuteri, and L. pentosus-which were identified using API 50 CHL and 16S rRNA gene sequences (Polymerase chain reaction, PCR - based). The seven strains displayed different ability to produce EPS (100-850 mg/L). Isolates exhibited a high survivability to acid (pH 3.0), bile salts (0.3%), and gastrointestinal tract model (<70%). Results showed that the auto-aggregation and cell surface hydrophobicity ranged from 39.98% to 60.09% and 50.80% to 80.53%, respectively, whereas, the highest co-aggregation value (66.44%) was observed by L. fermentum (DUR8) with Pseudomonas aeruginosa. The isolates showed good inhibitory activity against tested pathogens, high antioxidant activity (32.29% to 73.36%), and good ability to reduce cholesterol (22.55% to 75.15%). Thus, the seven tested strains have value as probiotics.

Matched MeSH terms: Lactobacillus/isolation & purification

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