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New Delhi metallo-β-lactamase (NDM) is a serious challenge to the treatment of infections and public health. Serbia has been designated as an endemic region for isolates carrying the bla NDM-1 gene, as well as one of several commonly proposed countries of origin. This is the first report of NDM-1-positive Escherichia coli from Serbia. A carbapenem-resistant clinical isolate of E. coli strain IMD989, isolated from the blood culture of a pediatric patient with leukemia, was subjected to antimicrobial susceptibility tests, molecular typing, and conjugation experiments. The strain exhibited resistance to meropenem and was classified as a novel sequence type, ST5123, belonging to E. coli phylogenetic group A. ST5123 showed similarity to veterinary isolates ST93 and ST3977. The bla NDM-1 gene was detected by polymerase chain reaction (PCR) and sequencing. Cloning and sequencing of genomic clones confirmed that strain IMD989 produces an NDM-1 variant. Conjugation experiments, pulsed-field gel electrophoresis, and Southern blot hybridization revealed that bla NDM-1 was located in IMD989 on a transmissible 80 kb plasmid, designated as pIMD989. PCR analysis confirmed that pIMD989 belongs to the IncF plasmid family. Propagation of IMD989 and selected transconjugants carrying pIMD989 over 14 days in solid media with and without antibiotic selection showed that pIMD989 is a stable plasmid.

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.

Proteinases and bacteriocins are of great importance to the dairy industry, but their interactions have not been studied so far. Lactococcus lactis subsp. lactis BGMN1-5 is a natural isolate from homemade semi-hard cheese which produces two bacteriocins (Lactococcin B and LsbB), as well as proteinase PrtP. A medium-dependent increase in the bacteriocin LcnB activity of L. lactis BGMN1-501, a derivate of L. lactis subsp. lactis BGMN1-5, was shown to be accompanied by a decrease in its promoter activity. A similar effect of media components on gene expression was reported for proteinase PrtP, whose gene is co-localized on the same plasmid as the lcnB gene. Thus, the PrtP-LcnB interplay was investigated. Single gene knockout mutants were constructed with disrupted prtP or lcnB genes. PrtP- mutants showed higher bacteriocin activity that had lost its growth medium dependence, which was in contrast to the original strain. When LcnB from this mutant was combined with proteinase from the LcnB- mutant in vitro, its activity was rendered to the original level, suggesting that proteinase reduces bacteriocin activity. We propose a new model of medium dependent expression of these genes with regard to the effects of their interaction in vivo. [ABSTRACT FROM AUTHOR]

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New Delhi metallo-β-lactamase (NDM) is a serious challenge to the treatment of infections and public health. Serbia has been designated as an endemic region for isolates carrying the blaNDM-1 gene, as well as one of several commonly proposed countries of origin. This is the first report of NDM-1-positive Escherichia coli from Serbia. A carbapenem-resistant clinical isolate of E. coli strain IMD989, isolated from the blood culture of a pediatric patient with leukemia, was subjected to antimicrobial susceptibility tests, molecular typing, and conjugation experiments. The strain exhibited resistance to meropenem and was classified as a novel sequence type, ST5123, belonging to E. coli phylogenetic group A. ST5123 showed similarity to veterinary isolates ST93 and ST3977. The blaNDM-1 gene was detected by polymerase chain reaction (PCR) and sequencing. Cloning and sequencing of genomic clones confirmed that strain IMD989 produces an NDM-1 variant. Conjugation experiments, pulsed-field gel electrophoresis, and Southern blot hybridization revealed that blaNDM-1 was located in IMD989 on a transmissible 80 kb plasmid, designated as pIMD989. PCR analysis confirmed that pIMD989 belongs to the IncF plasmid family. Propagation of IMD989 and selected transconjugants carrying pIMD989 over 14 days in solid media with and without antibiotic selection showed that pIMD989 is a stable plasmid. [ABSTRACT FROM AUTHOR]

Plasmids are autonomous episomally replicating genetic elements, which carry backbone genes important for the replication and maintenance within their host, and accessory genes that might confer an advantage to their host under specific selective pressure in its ecological niche. The genome of dairy isolate L. lactis subsp. lactis bv. diacetylactis S50 was sequenced using the PacBio SMRT Cell Seq-RSII platform and revealed to possess one of the largest plasmidomes among L. lactis strains studied so far, harboring six plasmids: pS6 (5553 bp), pS7a (7308 bp), pS7b (7266 bp), pS19 (19,027 bp), pS74 (74,256 bp) and pS127 (127,002 bp) in total representing 8.9% of genome size (240,412 bp). Based on predicted plasmid replication proteins and origins it appears that all six plasmids replicate via the theta-type mechanism. The two the largest plasmids (pS74 and pS127), carry a number of genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as bacteriocin production, protein degradation, magnesium and cobalt/nickel transporters, selenium binding, exopolysaccharides (EPS) production, bacteriophage and stress resistance. Beside genes for replication, the small plasmids (pS6, pS7a, pS7a, and pS19) also carry genes important for mobilization and host survival such as type I restriction-modification (R-M) system, metal transporters, enzymes and transcriptional regulators. All plasmids in S50 strain are mobilizable, containing an oriT sequences, while pS127 is self-conjugative and allows for mobilization of the other plasmids. Small plasmids are prone to structural and segregational instability, while pS127 appeared to be segregationally stable thanks to the possession of two partition systems. The main characteristic of plasmid pS74 is EPS production, while plasmid pS127 is characterized by proteinase and multiple bacteriocins, tra locus, phage abortive systems and metal transporters. In addition to LcnA and LcnB, plasmid pS127 encodes several bacteriocin-pheromone molecules and a new bacteriocin named LcnS50, with narrow spectrum of action limited to lactococci, that has been successfully cloned and heterologously expressed. • Plasmidome of S50 strain is the largest among lactococci, representing 13% of genome. • Plasmids of S50 strain indicate niche adaptation of lactococci to milk environment. • Technologically relevant traits in strain S50 are encoded by plasmids. • New bacteriocin LcnS50 is encoded by genes located on the largest plasmid pS156 in strain S50. [ABSTRACT FROM AUTHOR]