Jelena Begovic

Emergence of NDM-1 Metallo-β-Lactamase in Pseudomonas aeruginosa Clinical Isolates from Serbia

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 blaNDM-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.

Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's milk cheese

The Bukuljac cheese is traditionally homemade cheese, produced from heat-treated goats 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.

A survey of the lactic acid bacteria isolated from Serbian artisanal dairy product kajmak

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.

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

Aims:  Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics.

Different Roles for Lactococcal Aggregation Factor and Mucin Binding Protein in Adhesion to Gastrointestinal Mucosa

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.

Cloning and expression of a novel lactococcal aggregation factor from Lactococcus lactis subsp. lactis BGKP1

BackgroundAggregation 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.ResultsLactococcus 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.ConclusionAggL is a new protein belonging to the collagen-binding superfamily of proteins and is sufficient for cell aggregation in lactococci.

Interaction of Lactobacillus fermentum BGHI14 with Rat Colonic Mucosa: Implications for Colitis Induction

ABSTRACT The present study was carried out to test the colonic mucosal response of rats to oral supplementation with Lactobacillus fermentum BGHI14 and to correlate the tissue reaction to trinitrobenzenesulfonate (TNBS)-induced colitis with mucosal barrier alterations caused by bacterial ingestion. An immune cell-mediated reaction of healthy colonic tissue was noticed after bacterial feeding. After prolonged bacterial treatment, the observed reaction had retreated to normality, but the mRNA levels of proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) remained elevated. These data point to the chronic low-grade inflammation that could be caused by long-term probiotic consumption. Although no detrimental effects of bacterial pretreatment were noticed in colitic rats, at least in the acute state of disease, the results obtained in our study point to the necessity of reassessment of existing data on the safety of probiotic preparations. Additionally, probiotic effects in experimental colitis models might depend on time coordination of disease induction with treatment duration.

Technological and probiotic potential of BGRA43 a natural isolate of Lactobacillus helveticus

Lactobacillus helveticus BGRA43 is a human intestinal isolate showing antimicrobial activity, amongst others, against Yersinia enterocolitica, Shigella sonnei, Shigella flexneri, and Streptococcus pneumoniae. BGRA43 produces PrtH proteinase with proteolytic activity on both casein and β-lactoglobulin (BLG). BGRA43 is able to reduce the allergenicity of BLG. Bioactive peptides released in BGRA43 fermented milk are potent modulators of innate immunity by modulating the production of proinflammatory cytokines IL-6 and TNF-α. BGRA43 is able to survive in simulated gastric and intestinal conditions. The growth of BGRA43 in milk results in a fast acidification lowering the milk pH to 4.53 generating mild, homogeneous, and viscous yogurt-like product. The strain BGRA43 grows suitably in pure cow or goat’s milk as well as in milk containing inulin or nutrim even when they are used as the sole carbon source. It is suggested that strain BGRA43 could be used as a single-strain culture for the preparation of yogurt-like products from bovine or caprine milk. Overall, L. helveticus BGRA43 could be considered as a potential probiotic candidate with appropriate technological properties attractive for the dairy industry.

The Clinical Isolate Pseudomonas aeruginosa MMA83 Carries Two Copies of the blaNDM-1 Gene in a Novel Genetic Context

ABSTRACT The genetic context of the blaNDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was investigated. Sequencing of the cosmid selected for the 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.

Genotypic diversity and virulent factors of Staphylococcus epidermidis isolated from human breast milk.

Staphylococcus epidermidis strains were isolated from the expressed human breast milk (EHM) of 14 healthy donor mothers. Genetic diversity was evaluated using RAPD-PCR REP-PCR and pulse-field gel electrophoresis (PFGE). PFGE allowed the best discrimination of the isolates, since it provided for the greatest diversity of the analyzed genomes. Among the S. epidermidis strains, resistance to gentamicin, tetracycline, erythromycin, clindamycin or vancomycin was detected, whilst four isolates were multiresistant. The results from our study demonstrate that staphylococci from EHM could be reservoirs of resistance genes, since we showed that tetK could be transferred from EHM staphylococci to Gram-negative Escherichia coli. Most of the staphylococcal strains displayed excellent proteolytic and lipolytic activities. Additionally, the presence of ica genes, which was related to their ability to form a biofilm on tissue culture plates, and the presence of virulence factors including autolysin/adhesin AtLE, point to their pathogenic potential.