Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances.

Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.

Prevalence of bla(PenA) and bla(OXA) in Burkholderia pseudomallei Isolated from Patients at Sappasitthiprasong Hospital and Their Susceptibility to Ceftazidime and Carbapenems.

BACKGROUND: Burkholderia pseudomallei is a causative agent of melioidosis. Ceftazidime is the preferred drug of choice for treatment. However, the motility rate is high in endemic areas. OBJECTIVE: This study aimed to determine the susceptibility tofour different antimicrobial agents and to detect the ß-lactamase genes in B. pseudomallei isolates from patients admitted to Sappasitthiprasong Hospital. MATERIAL AND METHOD: 85 B. pseudomallei isolates from patients admitted to Sappasitthiprasong Hospital between November 2010 and May 2011 were determined for antimicrobial susceptibility by standard disk diffusion and minimum inhibitory concentration (MIC). Real-time polymerase chain reaction (PCR) was used for the detection of bla(penA) and bla(OXA) in ß-lactamase genes. RESULTS: Almost all of the clinical isolates ofB. pseudomallei were susceptible to ceftazidime and imipenem. Cefatzidime MIC was ≤ 1-16 µg/ml and imipenem MIC was ≤ 1-4 µg/ml. The real-time PCR revealed that more than 90% of B. pseudomallei isolates carried bla(PenA) and bla(OXA). CONCLUSION: Although the clinical isolates of B. pseudomallei were susceptible to ceftazidime and imipenem, this study showed B. pseudomallei had a gene that produced beta-lactamase enzyme and may be poorly effective in the use of beta-lactam drugs.

Isolation, Identification, and Evaluation of Novel Probiotic Strains Isolated from Feces of Breast-Fed Infants.

OBJECTIVE: To isolate, identify, and evaluate the probiotic properties of lactic acid bacteria (LAB) isolated from the feces of breast-fed infants. MATERIAL AND METHOD: The probiotic tests included investigation of hemolysis activity, survival in simulated gastrointestinal tract conditions (acid and bile salt tolerance), susceptibility to antibiotics, and ability to inhibit selected bacterial pathogens (Escherichia coli O157:H7, Vibrio cholerae and Salmonella enterica subsp enterica serovar Typhimurium). The bacterial species identification was performed by both carbohydrate utilization and partial 16S ribosomal RNA sequencing. RESULTS: Five of fifty LAB isolates (UBU-03, UBU-06, UBU-09, UBU-34, and UBU-37) showed good probiotic properties. These five isolates showed non-hemolysis type (gamma-hemolysis), susceptibility to all antibiotics tested except for vancomycin, ability to survive in the simulated gastrointestinal conditions of both acid and bile salt solution, and ability to inhibit growth of E. coli O157: H7 and V. cholerae. Bacterial species identification revealed that all five isolates were firmly identified as Lactobacillus rhamnosus species. CONCLUSION: The L. rhamnosus strains that were isolated and characterized in this study could be considered as probiotic strains, and then used for further probiotic characterization in human cell cultures or animal models.

Antibacterial Activity of Excretions-Secretions from Chrysomya megacephala Against Escherichia coli.

BACKGROUND: The blowfly, Chrysomya megacephala, is distributed worldwide. Previous studies found maggot excretions-secretions from other blowfly species inhibited pro-inflammatory response and antimicrobial activity. OBJECTIVE: This study aimed to test the bactericidal activity of excretions-secretions from C. megacephala larvae. MATERIAL AND METHOD: A total of 1,500 3-day-old larvae were used to collect excretions-secretions (ES) modified by the Barnes method. The bactericidal activity ofthe excretions-secretions was test by Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli using suitable liquid culture assay. Scanning electron microscope (SEM) was used to investigate the morphological change ofthe bacteria. RESULTS: E. coli were significantly inhibited by excretions-secretions from C. megacephala larvae. P. aeruginosa and S. aureus were not found to inhibit growth. CONCLUSION: The excretions-secretions from C. megacephala larvae may have a medical property for the inhibition of bacterial growth.

Antibiotic resistance patterns of Enterococcus spp. isolated from Musca domestica and Chrysomya megacephala in ubon Ratchathani.

BACKGROUND: The housefly Musca domestica and the blowfly Chrysomya megacephala are found worldwide and are medically significant as mechanical vectors of various pathogens from unsanitary locations to food, resulting in diseases in humans. OBJECTIVES: This study aimed to test the antimicrobial activity against Enterococcus spp. isolated from M. domestica and C. megacephala by standard disk diffusion and minimal inhibitory concentration (MIC), and to study the potential of M. domestica and C. megacephala to transfer multi-drug resistant enterococcus to humans. MATERIAL AND METHOD: Seven hundred adult flies were collected from fresh-food markets, garbage piles, restaurants, school cafeterias, and rice paddy fields in Muang Ubon Ratchathani and Warinchamrap in Ubon Ratchathani Province. Antimicrobial susceptibility for Enterococcus spp. isolated from adult flies was performed by disk diffusion test and minimum inhibitory concentration (MIC) determination. RESULTS: One hundred and twenty isolates of Enterococcus spp. were taken from 67 M. domestica and 53 C. megacephala. Standard disk diffusion showed the Enterococcus spp. isolates exhibited susceptibility to ampilcillin (99.2%), chloramphenicol (74.20%), tetracycline (75.0%), vancomycin (50.8%), and erythromycin (42.5%). The MICs of antimicrobial agents for all isolates were < or = 0.25-8 microg/mL for vancomycin, 1- > 16 microg/mL for tetracycline, 4- > 16 microg/mL for chloramphenicol, and 0.5-8 microg/mL for ciprofloxacin. CONCLUSION: The study demonstrated the potential of M. domestica and C. megacephala to carry Enterococcus spp. Nine antimicrobial susceptibility patterns were obtained among the 120 enterococci isolates.

Evaluation of Prebiotic Potential of Crude Polysaccharides Extracted from Wild Lentinus polychrous and Lentinus squarrosulus and Their Application for a Formulation of a Novel Lyophilized Synbiotic.

Edible mushrooms, including wild mushrooms, are currently being investigated as natural sources to evaluate their prebiotic potential. This study aimed to evaluate the prebiotic potential of crude polysaccharides (CPSs) extracted from wild Lentinus squarrosulus UBU_LS1 and Lentinus polychrous UBU_LP2 and their application as cryoprotectants in the freeze-drying process to formulate a novel synbiotic product. Based on fruiting body morphology and molecular identification, two wild edible mushrooms named UBU_LS1 and UBU_LP2 were identified as Lentinus squarrosulus and Lentinus polychrous, respectively. L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 contained high amounts of CPS after hot water extraction. Monosaccharide component analysis showed that CPS_UBU_LS1 and CPS_UBU_LP2 were typical heteropolysaccharides. CPS_UBU_LS1 and CPS_UBU_LP2 showed hydrolysis tolerance to the simulated human gastric acidic pH solution, indicating that these CPSs are capable of reaching the lower gastrointestinal tract. Antioxidant activity determined using the 1,1-diphenyl-2-picrylhydrazyl assay revealed that the CPS_UBU_LS1 and CPS_UBU_LP2 displayed greater antioxidant activity comparable with that of ascorbic acid. It was found that CPS_UBU_LS1 and CPS_UBU_LP2 have a high potential for stimulating growth in all probiotic strains. Moreover, both CPS compounds could possibly be used as cryoprotectants in freeze drying, since the viability of the selected probiotic L. fermentum 47-7 exhibited cell survival of greater than 70% after 90 days of storage at 4 °C. These results highlight that wild edible mushrooms L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 are potential natural sources of prebiotics and can be applied as cryoprotectants in the freeze-drying process. The crude polysaccharide derived from this study could also be considered as a potent antioxidative compound. Therefore, our study provides evidence to support the application of CPSs from wild edible mushrooms in synbiotic product development and in various functional foods. Finally, further evaluation of these prebiotics, including the determination of the potential rehabilitation of beneficial gut microbes in diseased individuals, is currently being conducted by our research group.

Sequencing and analysis of three plasmids from Lactobacillus casei TISTR1341 and development of plasmid-derived Escherichia coli-L. casei shuttle vectors.

Pyrosequencing followed by conventional PCR and sequencing was used to determine the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2, and pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were found to be almost identical, respectively, to those of pLA106, pLA105, and pLA103 from Lactobacillus acidophilus strain TK8912, suggesting that these strains may be related. Sequence analysis and comparison indicated that pRCEID2.9 replicates by a rolling circle (RC) mechanism, while pRCEID3.2 and pRCEID13.9 probably follow a theta-type mode of replication. Replicons of pRCEID2.9 and pRCEID13.9 were used to develop Escherichia coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds. Real-time quantitative PCR analysis showed copy numbers of around 4 and 15, respectively, for the pRCEID13.9- and pRCEID2.9-derived shuttle vectors per chromosome equivalent. The functionality of vector pRCEID-LC13.9 was proved by cloning and expressing in L. casei of a green fluorescent protein gene variant from Aequorea victoria under the control of the promoter from a homologous lactate dehydrogenase gene. The new vectors might complement those currently in use for the exploitation of L. casei as a cellular factory and in other biotechnological applications.

Evaluation of Nano-Wall Material for Production of Novel Lyophilized-Probiotic Product.

Lyophilization is one of the most used methods for bacterial preservation. In this process, the cryoprotectant not only largely decreases cellular damage but also plays an important part in the conservation of viability during freeze-drying. This study investigated using cryoprotectant and a mixture of the cryoprotectant to maintain probiotic activity. Seven probiotic strains were considered: (Limosilactobacillus reuteri KUKPS6103; Lacticaseibacillus rhamnosus KUKPS6007; Lacticaseibacillus paracasei KUKPS6201; Lactobacillus acidophilus KUKPS6107; Ligilactobacillus salivarius KUKPS6202; Bacillus coagulans KPSTF02; Saccharomyces cerevisiae subsp. boulardii KUKPS6005) for the production of a multi-strain probiotic and the complex medium for the lyophilized synbiotic production. Cholesterol removal, antioxidant activity, biofilm formation and gamma aminobutyric acid (GABA) production of the probiotic strains were analyzed. The most biofilm formation occurred in L. reuteri KUKPS6103 and the least in B. coagulans KPSTF02. The multi-strain probiotic had the highest cholesterol removal. All the probiotic strains had GABA production that matched the standard of γ-aminobutyric acid. The lyophilized synbiotic product containing complex medium as a cryoprotectant and wall material retained a high viability of 7.53 × 108 CFU/g (8.89 log CFU/g) after 8 weeks of storage. We found that the survival rate of the multi-strain probiotic after freeze-drying was 15.37% in the presence of complex medium that was used as high performing wall material. Our findings provided a new type of wall material that is safer and more effective and, can be extensively applied in relevant food applications.

Antibiotic Resistance-Susceptibility Profiles of Enterococcus faecalis and Streptococcus spp. From the Human Vagina, and Genome Analysis of the Genetic Basis of Intrinsic and Acquired Resistances.

The spread of antibiotic resistance is a major public health concern worldwide. Commensal bacteria from the human genitourinary tract can act as reservoirs of resistance genes playing a role in their transfer to pathogens. In this study, the minimum inhibitory concentration of 16 antibiotics to 15 isolates from the human vagina, identified as Enterococcus faecalis, Streptococcus anginosus, and Streptococcus salivarius, was determined. Eight isolates were considered resistant to tetracycline, five to clindamycin and quinupristin-dalfopristin, and four to rifampicin. To investigate the presence of antimicrobial resistance genes, PCR analysis was performed in all isolates, and five were subjected to whole-genome sequencing analysis. PCR reactions identified tet(M) in all tetracycline-resistant E. faecalis isolates, while both tet(M) and tet(L) were found in tetracycline-resistant S. anginosus isolates. The tet(M) gene in E. faecalis VA02-2 was carried within an entire copy of the transposon Tn916. In S. anginosus VA01-10AN and VA01-14AN, the tet(M) and tet(L) genes were found contiguous with one another and flanked by genes encoding DNA mobilization and plasmid replication proteins. Amplification and sequencing suggested the lsaA gene to be complete in all E. faecalis isolates resistant to clindamycin and quinupristin-dalfopristin, while the gene contain mutations rendering to a non-functional LsaA in susceptible isolates. These results were subsequently confirmed by genome analysis of clindamycin and quinupristin-dalfopristin resistant and susceptible E. faecalis strains. Although a clinical breakpoint to kanamycin for S. salivarius has yet to be established, S. salivarius VA08-2AN showed an MIC to this antibiotic of 128 µg mL-1. However, genes involved in kanamycin resistance were not identified. Under the assayed conditions, neither tet(L) nor tet(M) from either E. faecalis or S. anginosus was transferred by conjugation to recipient strains of E. faecalis, Lactococcus lactis, or Lactobacillus plantarum. Nonetheless, the tet(L) gene from S. anginosus VA01-10AN was amplified by PCR, and cloned and expressed in Escherichia coli, to which it provided a resistance of 48-64 µg mL-1 to tetracycline. Our results expand the knowledge of the antibiotic resistance-susceptibility profiles of vaginal bacteria and provide the genetic basis of their intrinsic and acquired resistance.

Genome Sequence of Lactobacillus fermentum 47-7, a Good In Vitro Probiotic Strain Isolated from a Healthy Thai Infant.

We report the genome sequence of Lactobacillus fermentum 47-7, a good in vitro probiotic strain isolated from an infant. Its genome size is 1.83 Mb, it is assembled from 180 contigs, and it consists of 1,636 protein-coding genes, 15 rRNAs, 57 tRNAs, and 4 noncoding RNAs. This genome sequence will be useful for a variety of applications.

Secretion of M2e:HBc fusion protein by Lactobacillus casei using Cwh signal peptide.

The ability to serve as a delivery vehicle for various interesting biomolecules makes lactic acid bacteria (LAB) very useful in several applications. In the medical field, recombinant LAB expressing pathogenic antigens at different cellular locations have been used to elicit both mucosal and systemic immune responses. Expression-secretion vectors (ESVs) with a signal peptide (SP) are pivotal for protein expression and secretion. In this study, the genome sequence of Lactobacillus casei ATCC334 was explored for new SPs using bioinformatics tools. Three new SPs of the proteins Cwh, SurA and SP6565 were identified and used to construct an ESV based on our Escherichia coli-L. casei shuttle vector, pRCEID-LC13.9. Functional testing of these constructs with the green fluorescence protein (GFP) gene showed that they could secrete the GFP. The construct with CwhSP showed the highest GFP secretion. Consequently, CwhSP was selected to develop an ESV construct carrying a synthetic gene encoding the extracellular domain of the matrix 2 protein fused with the hepatitis B core antigen (M2e:HBc). This ESV was shown to efficiently express and secrete the M2e:HBc fusion protein. The identified SPs and the developed ESVs can be exploited for expression and secretion of homologous and heterologous proteins in L. casei.

Development of an Escherichia coli-Lactobacillus casei shuttle vector for heterologous protein expression in Lactobacillus casei.

There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles.

Cloning and expression of a codon-optimized gene encoding the influenza A virus nucleocapsid protein in Lactobacillus casei.

Lactic acid bacteria (LAB) species are envisioned as promising vehicles for the mucosal delivery of therapeutic and prophylactic molecules, including the development of oral vaccines. In this study, we report on the expression of a synthetic nucleocapsid (NP) gene of influenza A virus in Lactobacillus casei. The NP gene was re-designed based on the tRNA pool and the codon usage preference of L. casei BL23. The codon-optimized NP gene was then cloned and expressed in L. casei RCEID02 under the control of a constitutive promoter, that of the lactate dehydrogenase (ldh) gene. The synthetic NP gene was further expressed in L. casei EM116 under the control of an inducible promoter, that of the structural gene of nisin (nisA) from Lactococcus lactis. Based on Western blot analysis, the specific protein band of NP, with a molecular mass of 56.0 kDa, was clearly detected in both expression systems. Thus, our study demonstrates the success of expressing a codon-optimized influenza A viral gene in L. casei. The suitability of the recombinant LAB strains for immunization purposes is currently under evaluation.

Cloning and expression of a codon-optimized gene encoding the infl uenza A virus nucleocapsid protein in Lactobacillus casei

Lactic acid bacteria (LAB) species are envisioned as promising vehicles for the mucosal delivery of therapeutic and prophylactic molecules, including the development of oral vaccines. In this study, we report on the expression of a synthetic nucleocapsid (NP) gene of influenza A virus in Lactobacillus casei. The NP gene was re-designed based on the tRNA pool and the codon usage preference of L. casei BL23. The codon-optimized NP gene was then cloned and expressed in L. casei RCEID02 under the control of a constitutive promoter, that of the lactate dehydrogenase (ldh) gene. The synthetic NP gene was further expressed in L. casei EM116 under the control of an inducible promoter, that of the structural gene of nisin (nisA) from Lactococcus lactis. Based on Western blot analysis, the specific protein band of NP, with a molecular mass of 56.0 kDa, was clearly detected in both expression systems. Thus, our study demonstrates the success of expressing a codon-optimized influenza A viral gene in L. casei. The suitability of the recombinant LAB strains for immunization purposes is currently under evaluation (AU)

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