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ABSTRACT: The genetic context of the blaNDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was investigated. Sequencing of the cosmid selected for blaNDM-1 gene revealed the presence of two blaNDM-1 copies in the genome of P. aeruginosa MMA83 in a unique genetic environment. Additionally, mating assays, DNA-DNA hybridization and an S1 nuclease assay strongly suggest that the blaNDM-1 gene in P. aeruginosa MMA83 is chromosome-borne.
Antimicrobial Agents and Chemotherapy 04/2013; · 4.84 Impact Factor
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ABSTRACT: Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The cmbT gene (1377 bp) was cloned and overexpressed in L. lactis NZ9000. Results from cell growth studies revealed that the CmbT protein has an effect on host cell resistance to lincomycin, cholate, sulbactam, ethidium bromide, Hoechst 33342, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametoxazole. Moreover, in vivo transport assays showed that overexpressed CmbT-mediated extrusion of ethidium bromide and Hoechst 33342 was higher than in the control L. lactis NZ9000 strain. CmbT-mediated extrusion of Hoechst 33342 was inhibited by the ionophores nigericin and valinomycin known to dissipate proton motive force. This indicates that CmbT-mediated extrusion is based on a drug-proton antiport mechanism. Taking together results obtained in this study, it can be concluded that CmbT is a novel major facilitator multidrug resistance transporter candidate in L. lactis, with a possible signaling role in sulfur metabolism.
Research in Microbiology 09/2012; · 2.76 Impact Factor
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ABSTRACT: Adhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation (aggL) and possible interaction with mucin (mbpL), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL. In vivo and in vitro experiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated the in vitro and in vivo aggregation of the BGKP1-20 carrying plasmid with aggL to binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the other hand, the presence of AggL in the strain reduced its ability to adhere to the ileum. Moreover, MbpL protein showed an affinity to bind gastric type mucin proteins such as MUC5AC. This protein did not contribute to the binding of the strain to the ileal or colonic part of the intestine. Different potential functions of lactococcal AggL and MbpL proteins in the process of adhesion to the gastrointestinal tract are proposed.
Applied and environmental microbiology 09/2012; 78(22):7993-8000. · 3.69 Impact Factor
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ABSTRACT: New Delhi metallo-beta-lactamase 1 (NDM-1) is a newly-described metallo-β-lactamase (MBL), first identified in 2008 in single isolates of Klebsiella pneumoniae and Escherichia coli, both recovered from a patient repatriated to Sweden after treatment in a hospital in New Delhi, India (8).…
Antimicrobial Agents and Chemotherapy 08/2012; 56(11):6062-3. · 4.84 Impact Factor
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ABSTRACT: Ten lactobacilli and one leuconostoc showing auto-aggregation ability were isolated from artisanal cheeses. Furthermore, non-aggregation
strains were isolated from the same cheese sample, if existed. The analysis of factor(s) possibly involved in auto-aggregation
was performed. The pretreatment of cells with proteinase K resulted in the disappearance of auto-aggregation ability. Moreover,
cells also lost aggregation ability after three-times, successive washing in distilled water. Testing the ability of strain
Lactobacillus paracasei subsp. paracasei BGSJ2-8 and its aggregation-deficient derivative BGSJ2-81 to co-aggregate with Listeria innocua ATCC33090, Escherichia coli ATCC25922 or Salmonella typhimurium TR251 showed that strain BGSJ2-8 co-aggregated with these strains, but derivative BGSJ2-81 was not. However, the treatment
of L. paracasei subsp. paracasei BGSJ2-8 with proteinase K prior to co-aggregation tests resulted in losing co-aggregation ability. Surface properties of
selected strains were analyzed by MATS (microbial adhesion to solvents) method. It was noticed that the strains with auto-aggregation
ability were highly hydrophobic in comparison with aggregation-deficient ones. Comparative analyses of the surface features
of strain L. paracasei subsp. paracasei BGSJ2-8 and its derivative BGSJ2-81 revealed notable difference.
Keywords
Lactobacillus
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Leuconostoc
-Auto-aggregation-Co-aggregation-Surface properties
European Food Research and Technology 04/2012; 231(6):925-931. · 1.57 Impact Factor
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ABSTRACT: Aggregation may play a main role in the adhesion of bacteria to the gastrointestinal epithelium and their colonization ability, as well as in probiotic effects through co-aggregation with intestinal pathogens and their subsequent removal. The aggregation phenomenon in lactococci is directly associated with the sex factor and lactose plasmid co-integration event or duplication of the cell wall spanning (CWS) domain of PrtP proteinase.
Lactococcus lactis subsp. lactis BGKP1 was isolated from artisanal semi-hard homemade cheese and selected due to its strong auto-aggregation phenotype. Subsequently, non-aggregating derivative (Agg-) of BGKP1, designated as BGKP1-20, was isolated, too. Comparative analysis of cell surface proteins of BGKP1 and derivative BGKP1-20 revealed a protein of approximately 200 kDa only in the parental strain BGKP1. The gene involved in aggregation (aggL) was mapped on plasmid pKP1 (16.2 kb), cloned and expressed in homologous and heterologous lactococci and enterococci. This novel lactococcal aggregation protein was shown to be sufficient for cell aggregation in all tested hosts. In addition to the aggL gene, six more ORFs involved in replication (repB and repX), restriction and modification (hsdS), transposition (tnp) and possible interaction with mucin (mbpL) were also located on plasmid pKP1.
AggL is a new protein belonging to the collagen-binding superfamily of proteins and is sufficient for cell aggregation in lactococci.
BMC Microbiology 12/2011; 11:265. · 3.04 Impact Factor
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ABSTRACT: This work reports, for the first time, the presence of New Delhi metallo-β-lactamase 1 (NDM-1) in Pseudomonas aeruginosa. Moreover, this is the first report of the NDM-1 presence in the Balkan region. Cosmid gene libraries of carbapenem-nonsusceptible Pseudomonas aeruginosa clinical isolates MMA83 and MMA533 were screened for the presence of metallo-β-lactamases. Accordingly, both MMA83 and MMA533 carried the bla(NDM-1) gene. Pulsed-field gel electrophoresis (PFGE) analysis indicated that strains MMA83 and MMA533 belonged to different clonal groups. Five additional isolates from different patients clonally related to either MMA83 or MMA533 were found to be NDM-1 positive.
Antimicrobial Agents and Chemotherapy 06/2011; 55(8):3929-31. · 4.84 Impact Factor
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ABSTRACT: Pseudomonas sp. strain ATCC19151 is a natural isolate from sewage with the ability to degrade detergents. Genes encoding potential choline sulfatase (betC), substrate-binding ABC transporter protein (betD), sulfate transporter (betE), and divergent putative transcriptional regulator (betR) were cloned and characterized from strain ATCC19151. In silico analysis revealed that (1) the BetC protein belongs to alkPPc superfamily and shares CXPXR sequence with the cysteine sulfatases of group I, (2) BetR belongs to the LysR family of transcriptional regulators, (3) BetD is part of the PBPb superfamily of periplasmic and membrane-associated proteins, and (4) BetE is a permease and contains STAS domain. Insertional mutagenesis and genetic complementation show that betC gene encodes a functional choline sulfatase. Analysis of the betC (P(betC)) and betR (P(betR)) promoters revealed that they are inducible. BetR activates betC and betR transcription in the presence of choline sulfate, whilst in the absence of choline sulfate, BetR represses its own transcription. It was further established that BetR directly binds to betC-betR intergenic region in vitro, with higher affinity in the presence of choline sulfate as cofactor. Transcription of betC and betR was not induced in the presence of high concentration of NaCl.
Archives of Microbiology 03/2011; 193(6):399-405. · 1.43 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa isolates (n=526), collected from a paediatric hospital in Belgrade during 2007-2008, were screened for susceptibility to imipenem. Against 163 imipenem non-susceptible isolates, polymyxins were the most active compounds (100%), followed by levofloxacin (61.3%), ciprofloxacin (49.7%), aztreonam (42.3%) and amikacin (36.2%). Among them, 29.4% of isolates were pan-resistant. Imipenem-non-susceptible isolates were screened for presence of metallo-β-lactamase genes by PCR with primers for bla(IMP), bla(VIM), bla(SPM), bla(GIM) and bla(SIM). MBL genes were detected in five isolates (3%) and all of them were VIM-2-like and integrated into chromosome. VIM-2-producing isolates belonged to three clonal groups as determined by PFGE.
Journal of Medical Microbiology 02/2011; 60(Pt 6):868-9. · 2.50 Impact Factor
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ABSTRACT: A new shuttle-cloning vector, pA13, was constructed and successfully introduced into Escherichia coli, Lactobacillus and Lactococcus strains. It showed high segregational and structural stability in all three hosts. The natural plasmid pSJ2-8 from L. paracasei subsp. paracasei BGSJ2-8 was cloned into pA13 using BamHI to obtain the construct, pB5. Sequencing and in silico analysis of pB5 revealed fifteen open reading frames (ORF). Plasmid pSJ2-8 harbours genes encoding the production of two bacteriocins, BacSJ and acidocin 8912. Combined N-terminal amino acid sequencing of BacSJ in combination with DNA sequencing of the bacSJ2-8 gene enabled determination of the primary structure of bacteriocin BacSJ. The bacSJ2-8 gene encodes 68-amino-acid peptide with a double-glycine leader peptide consisting of 18 amino acids, followed by the orf2 (bacSJ2-8i) which encodes the immunity protein of BacSJ. The production and functional expression of BacSJ in homologous and heterologous hosts suggest that bacSJ2-8 and bacSJ2-8i together with the genes encoding the ABC transporter and accessory protein are the minimal requirements for production of BacSJ. Biochemical and genetic analyses showed that BacSJ belongs to class II bacteriocins.
International journal of food microbiology 06/2010; 140(2-3):117-24. · 3.01 Impact Factor
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ABSTRACT: Kajmak is an artisanal Serbian dairy product made by fermentation of milk fat. Overall, 374 bacterial isolates were collected from six kajmak samples of different ages produced in the households located in distinct regions of Serbia. In order to identify lactic acid bacteria present in chosen samples of kajmak, total 349 Gram-positive and catalase-negative isolates were analyzed. The recognition of isolates was performed by phenotypic characterization followed by molecular identification using (GTG)(5)-PCR and sequence analysis of 16S rRNA gene. Leuconostoc mesenteroides and Enterococcus faecium were the most frequently isolated species from kajmak samples. In contrast, leuconostocs and enterococci were found in BGMK3 and BGMK1 kajmak respectively, only after using enrichment technique for isolation suggesting they are present in low numbers in these kajmaks. Lactococcus lactis, Lactococcus raffinolactis and Lactococcus garvieae were also found in those samples but in lower proportion. Results showed that Lactobacillus plantarum, Lb. paracasei and Lb. kefiri were the most frequently isolated Lactobacillus species in analyzed kajmaks.
International Journal of Food Microbiology 09/2008; 127(3):305-11. · 3.33 Impact Factor
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ABSTRACT: The sigmas subunit of RNA polymerase is a central regulator which governs the expression of a host of stationary phase-induced and osmotically regulated genes in Gram-negative bacteria. The Pseudomonas putida rpoS gene is transcribed as a monocistronic rpoS mRNA with a 368 nucleotide-long 5' untranslated region (5' UTR). In this study, we investigate the posttranscriptional control of RpoS synthesis using rpoS-lacZ transcriptional and translational fusions consisting of the native promoter and deletions of 5' UTR or insertion into UTR. The differing activity of constructed translational fusions strongly indicated that the 5' UTR is involved in the translational regulation of RpoS expression in the stationary phase. The results obtained herein demonstrated that the structure of UTR performs an important function in the translational regulation of the rpoS gene.
The Journal of Microbiology 03/2008; 46(1):56-61. · 1.10 Impact Factor
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ABSTRACT: A large chromosomal inversion that confers resistance to high concentrations of the antibiotic spectinomycin in Lactococcus lactis subsp. lactis bv. diacetylactis S50 was identified by pulsed field gel electrophoresis. The same type of inversion was identified in 4 independent experiments and in 4 different derivatives of strain S50, indicating the same position and the same mechanism of recombination as a response to antibiotic selective pressure in all derivatives. An analysis of ribosomal operons in strain S50 and mutants revealed that ribosomal operons are not endpoints of the recombination. Spectinomycin-resistant mutants appeared in a population of S50 derivatives at a high frequency of 2 x 10(-7). These spectinomycin-resistant mutants were not able to compete successfully with the wild-type strain during 25 generations (48 h) of co-culture in vitro, indicating that inversion had a significant fitness cost. Results demonstrate that as a mechanism of genome plasticity, inversion can be directly involved in one-step development of the adaptation to a high concentration of spectinomycin.
Canadian Journal of Microbiology 03/2008; 54(2):143-9. · 1.36 Impact Factor
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ABSTRACT: The Bukuljac cheese is traditionally homemade cheese, produced from heat-treated goat's milk without the addition of any bacterial starter culture. The presence of lactic acid bacteria (LAB) in Bukuljac cheese has been analyzed by using a polyphasic approach including microbiological and molecular methods such as rep-PCR with (GTG)5 primer. Lactobacillus paracasei subsp. paracasei represents a dominant strain in the microflora of analyzed cheese. Out of 55 Gram-positive and catalase-negative isolates, 48 belonged to L. paracasei subsp. paracasei species. Besides lactobacilli, five Lactococcus lactis subsp. lactis and two Enterococcus faecalis were found. Results of PCR-denaturing gradient gel electrophoresis (DGGE) of DNA extracted directly from the fresh cheese revealed the presence of Leuconostoc mesenteroides. Only lactobacilli showed a high proteolytic activity and hydrolyzed alpha(s1)- and beta-caseins. They are also producers of diacetyl. In addition, 34 out of 55 isolates, all determined as lactobacilli, showed the ability of auto-aggregation. Among 55 isolates, 50 also exhibited antimicrobial activity.
International Journal of Food Microbiology 03/2008; 122(1-2):162-70. · 3.33 Impact Factor
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ABSTRACT: Screening the collection of natural isolates from semi-hard homemade cheese resulted in isolation and characterization of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8. The strain BGSJ2-8 harbors several important phenotypes, such as bacteriocin production, aggregation phenomenon, and production of proteinase. Bacteriocin SJ was purified by three-step chromatography. Mass spectrometry established molecular mass of the active peptide at 5372 Da. The auto-aggregation phenotype of wild-type (WT) strain was mediated by secreted aggregation-promoting factor (protein of molecular mass > 200 kDa), probably acting in cooperation with other cell surface protein(s). Comparative study of WT and its spontaneous nonaggregating derivative revealed that aggregation factor was responsible for the observed differences in the bacteriocin and proteinase activities. Bacteriocin SJ activity and resistance to different stresses were higher in the presence of aggregating factor. In contrast, proteinase activity was stronger in the nonaggregating derivative.
Current Microbiology 09/2007; 55(3):266-71. · 1.82 Impact Factor
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ABSTRACT: In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.
Canadian Journal of Microbiology 12/2006; 52(11):1110-20. · 1.36 Impact Factor
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ABSTRACT: The PsrA transcriptional regulator is involved in stationary phase induced transcriptional regulation of rpoS and in negative auto-regulation in Pseudomonas aeruginosa. This study was designed to determine whether other loci were regulated by PsrA in P. aeruginosa. Computer search was performed of the PsrA binding motif (G/CAAAC N(2-4) GTTTG/C) against the P. aeruginosa genome sequence. Four of 14 analysed promoters responded to and bound PsrA; (i) divergent promoters controlling PA2952/PA2951 and PA2953, (ii) promoter of PA0506 and (iii) upstream region of PA3571. Promoters PA0506 and PA2952-PA2951 were regulated negatively whereas promoters of PA2953 and PA3571 were regulated positively by PsrA. Two dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D SDS-PAGE) analysis on total proteins from P. aeruginosa PAO1 and psrA knock-out derivative was also performed resulting in the identification of 11 protein spots which were differentially regulated. These studies have indicated PsrA as a global regulator.
FEMS Microbiology Letters 06/2005; 246(2):175-81. · 2.04 Impact Factor
