The plasmid complement of the cheese isolate Lactococcus garvieae IPLA 31405 revealed adaptation to the dairy environment

Lactococcus garvieae is a lactic acid bacterium found in raw-milk dairy products as well as a range of aquatic and terrestrial environments. The plasmids in L. garvieae have received little attention compared to those of dairy Lactococcus lactis, in which the genes carried by these extrachromosomal elements are considered of adaptive value. The present work reports the sequencing and analysis of the plasmid complement of L. garvieae IPLA 31405, a strain isolated from a traditional, Spanish, starter-free cheese made from raw-milk. It consists of pLG9 and pLG42, of 9,124 and 42,240 nucleotides, respectively. Based on sequence and structural homology in the putative origin of replication ( ori) region, pLG9 and pLG42 are predicted to replicate via a theta mechanism. Real-time, quantitative PCR showed the number of copies per chromosome equivalent of pLG9 and pLG42 to be around two and five, respectively. Sequence analysis identified eight complete open reading frames (orfs) in pLG9 and 36 in pLG42; these were organized into functional modules or cassettes containing different numbers of genes. These modules were flanked by complete or interrupted insertion sequence (IS)-like elements. Among the modules of pLG42 was a gene cluster encoding specific components of a phosphoenolpyruvate-phosphotransferase (PEP-PTS) system, including a phospho-β-galacosidase. The cluster showed a complete nucleotide identity respect to that in plasmids of L. lactis. Loss of pLG42 showed this to be involved in lactose assimilation. In the same plasmid, an operon encoding a type I restriction/ modification (R/M) system was also identified. The specificity of this R/M system might be broadened by different R/M specificity subunits detected in pLG9 and in the bacterial chromosome. However, challenges of L. garvieae IPLA 31405 against L. lactis phages proved that the R/M system was not involved in phage resistance. Together, these results support the hypothesis that, as in L. lactis, pLG42 contribute towards the adaptation of L. garvieae to the dairy environment. © 2015 Flórez, Mayo.This research was partially funded by projects from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. AGL2011-24300) and Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) (Ref. RM2011-00005-00- 00). AB Flórez was supported by research contracts under Juan de la Cierva Program from the Consejo Superior de Investigaciones Científicas (CSIC) (Ref. JCI-2010-07457).Peer Reviewe

Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis

This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml− 1) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird–Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5–1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods.The study was partially supported by projects from the Spanish Ministry of Economy and Competitiveness (Ref. AGL2014-57820-R) and Plan for Science, Technology and Innovation 2013–2017 of the Asturias Principality, co-funded by FEDER (Ref. GRUPIN14-137). A.B. Flórez was supported by a research contract from CSIC under the JAE-Doc Program.Peer reviewe

Draft genome sequence of three antibiotic-resistant Leuconostoc mesenteroides strains of dairy origin

Leuconostoc mesenteroides is a lactic acid bacterium (LAB) commonly associated with fermented foods. Here, we report the genome sequence of three selected dairy strains, showing atypical antibiotic resistances (AR). Genome analysis provided a better understanding of the genetic bases of AR in Leuconostoc and its potential transferability among foodborne bacteria.Peer Reviewe

Diversity and biofilm-forming capability of bacteria recovered from stainless steel pipes of a milk-processing dairy plant

Bacteria may adhere to and develop biofilm structures onto dairy surfaces trying to protect themselves from adverse conditions such as pasteurization and CIP processes. Thus, biofilms are considered common sources of food contamination with undesirable bacteria. The purpose of this study was to evaluate the diversity of the microbiota attached to stainless steel surfaces in pre- and post-pasteurization pipe lines of a milk-processing plant. Seventy Gram-positive isolates were identified as Enterococcus faecalis (33), Bacillus cereus (26), Staphylococcus hominis (8), Staphylococcus saprophyticus (2), and Staphylococcus epidermidis-Staphylococcus aureus (1) species. Fifty-five Gram-negative isolates were identified to the species Escherichia coli (18), Klebsiella pneumoniae (13), Acinetobacter calcoaceticus (6), Serratia marcescens (6), Enterobacter spp. (5), Pseudomonas aeruginosa (4), Escherichia vulneris (2), and Proteus mirabilis (1). Fifty-five different strains were detected by the RAPD technique. These were subjected to an in vitro assay to evaluate their biofilm-forming capability. E. faecalis (7), A. calcoaceticus (4), K. pneumoniae (3), S. hominis (3), and P. aeruginosa (2) were the species in which more biofilm producer strains were encountered. The adhered microbiota was also assessed by the PCR-DGGE culture-independent technique. This analysis revealed a greater bacterial diversity than that revealed by culturing methods. In this way, in addition to the bacteria detected by culturing, DNA bands belonging to the genera Chrysobacterium and Streptomyces were also identified. This study emphasizes that knowledge of attached microorganisms to dairy surfaces may help develop strategies to improve optimal operational parameters for pasteurization and CIP processes in dairy plants.Financial support for this work was provided by projects from CICYT (Ref. AGL2011-24300-ALI) and INIA (Ref. RM2011-00005-00-00). A.B Flórez was supported by a research contract under Juan de la Cierva Program (Ref. JCI-2010-07457).Peer reviewe

Molecular Identification and Quantification of Tetracycline and Erythromycin Resistance Genes in Spanish and Italian Retail Cheeses

Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. This study reports the identification and quantification of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). The numbers of resistant bacteria varied widely among the antibiotics and the different cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-specific PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). The most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). The copy numbers of these genes, as quantified by qPCR, ranged widely between cheeses (from 4.94 to 10.18 log10/g). DGGE analysis revealed distinct banding profiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants.The study was partially supported by a Spain-Italy bilateral collaboration program (Ref. IT2009-0080 and IT105MD12L). Financial support was further provided by projects from CICYT (Ref. AGL2011-24300-ALI) and INIA (Ref. RM2011-00005-00-00). A. B. Flórez and S. Delgado were supported by research contracts under Juan de la Cierva Program (Ref. JCI-2010-07457 and JCI-2008-02391, resp.). A. Alegría was awarded a scholarship of the Severo Ochoa program from FICYT (Ref. BP08-053).Peer Reviewe

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strainsThis work was financed by the European Union STREP project ACE-ART (FP6-506214).Peer reviewe

Genetic and functional analysis of biogenic amine production capacity among starter and non-starter lactic acid bacteria isolated from artisanal cheeses

© 2015, Springer-Verlag Berlin Heidelberg. This work reports the capacity of 137 strains of starter and non-starter LAB belonging to nine species of the genera Lactobacillus, Lactococcus, Streptococcus and Leuconostoc (all isolated from artisanal cheeses) to produce histamine, tyramine, putrescine and β-phenylethylamine, the biogenic amines (BA) most commonly found in dairy products. Production assays were performed in liquid media supplemented with the appropriate precursor amino acid; culture supernatants were then tested for BA by (U)HPLC. In addition, the presence of key genes involved in the biosynthetic pathways of the target BA, including the production of putrescine via the agmatine deiminase pathway, was assessed by PCR. Twenty strains were shown to have genes involved in the synthesis of BA; these belonged to the species Lactobacillus brevis (4), Lactobacillus curvatus (3), Lactococcus lactis (11) and Streptococcus thermophilus (2). With the exception of the two S. thermophilus strains, all those possessing genes involved in BA production synthesized the corresponding compound. Remarkably, all the putrescine-producing strains used the agmatine deiminase pathway. Four L. brevis and two L. curvatus strains were found able to produce both tyramine and putrescine. There is increasing interest in the use of autochthonous LAB strains in starter and adjunct cultures for producing dairy products with ‘particular geographic indication’ status. Such strains should not produce BA; the present results show that BA production capacity should be checked by (U)HPLC and PCR.This work was funded by the Ministry of Economy and Competitiveness, Spain (AGL2013-45431-R), the Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología (FICYT), cofunded by FEDER (GRUPIN14-137) and the INIA (RM2011-00005-00-00).Peer Reviewe

Whole genome analysis as a tool for the safety assessment of antibiotic resistance in food-processing bacteria

Trabajo presentado en la 2nd EFSA Scientific Conference, celebrado en Milán, Italia, del 14 al 16 de octubre de 2015Acquisition of antibiotic resistances (AR) by pathogens leads ultimately to a failure of antibiotic therapy. The food chain is considered a key player in the transmission of AR determinants to pathogens from reservoirs in commensal and beneficial bacteria. Therefore, the absence of transmissible AR genes in bacteria used as starter and adjunct cultures for food and feed processing is considered to be critical (EFSA, 2012; EFSA Journal, 10:2740). Genome sequencing allows the inspection of the whole genetic makeup of bacteria in the search for the basis of desirable and undesirable traits, including that of AR. Thus, in silico sequence analysis and comparison against databases can be used as a tool for the safety assessment of microorganisms intended to be used in food systems. This communication reports on the genome analysis of three Leuconostoc mesenteroides strains of dairy origin showing atypical resistances to tetracycline (LbT16), erythromycin and clindamycin (LbE15), and kanamycin, streptomycin, tetracycline and virginiamycin (LbE16). Genes encoding for erythromycin [erm(B)] and tetracycline [tet(S)] resistance had already been detected by PCR. Genome analysis confirmed the presence of these genes and identified others which encode uncommon AR in lactic acid bacteria. Analysis of the genes and their flanking regions revealed a potential of some to be horizontally transferred to other bacteria. This study demonstrates the effectiveness of combining genome sequencing and bioinformatics analysis as an affordable tool for the safety assessment of food bacteria. This innovative approach could become a novel paradigm in the selection programs of starters for the food industry.Peer Reviewe

Two membrane proteins from Bifidobacterium breve UCC2003 constitute an ABC-type multidrug transporter

Intrinsic resistance to drugs is one of the main determining factors in bacterial survival in the intestinal ecosystem. This is mediated by, among others, multidrug resistance (MDR) transporters, membrane proteins which extrude noxious compounds with very different chemical structures and cellular targets. Two genes from Bifidobacterium breve encoding hypothetical membrane proteins with a high homology with members of the ATP-binding cassette (ABC) family of multidrug efflux transporters, were expressed separately and jointly in Lactococcus lactis. Cells co-expressing both proteins exhibited enhanced resistance levels to the antimicrobials nisin and polymyxin B. Furthermore, the drug extrusion activity in membrane vesicles was increased when both proteins were co-expressed, compared to membranes in which the proteins were produced independently. Both proteins were co-purified from the membrane as a stable complex in a 1 :1 ratio. This is believed to be the first study of a functional ABC-type multidrug transporter in Bifidobacterium and contributes to our understanding of the molecular mechanisms underlying the capacity of intestinal bacteria to tolerate cytotoxic compoundsThis work was financed by the European Union STREP project ACEART(FP6-506214), European Union FEDER funds, and the Spanish Plan Nacional de I+D (project AGL2004-06727-C02). J. A. Moreno was the recipient of a post-doctoral contract from CSIC (I3P programme), Spain. The work was also financially suported by the Department of Agriculture and Food FIRM programme (01/R&D/C/ 159), by the Higher Education Authority Programme for Research in Third Level Institutions, and by the SFI-funded Alimentary Pharmabiotic Centre.Peer reviewe

Two membrane proteins from Bifidobacterium breve UCC2003 constitute an ABC-type multidrug transporter

Intrinsic resistance to drugs is one of the main determining factors in bacterial survival in the intestinal ecosystem. This is mediated by, among others, multidrug resistance (MDR) transporters, membrane proteins which extrude noxious compounds with very different chemical structures and cellular targets. Two genes from Bifidobacterium breve encoding hypothetical membrane proteins with a high homology with members of the ATP-binding cassette (ABC) family of multidrug efflux transporters, were expressed separately and jointly in Lactococcus lactis. Cells co-expressing both proteins exhibited enhanced resistance levels to the antimicrobials nisin and polymyxin B. Furthermore, the drug extrusion activity in membrane vesicles was increased when both proteins were co-expressed, compared to membranes in which the proteins were produced independently. Both proteins were co-purified from the membrane as a stable complex in a 1 :1 ratio. This is believed to be the first study of a functional ABC-type multidrug transporter in Bifidobacterium and contributes to our understanding of the molecular mechanisms underlying the capacity of intestinal bacteria to tolerate cytotoxic compoundsThis work was financed by the European Union STREP project ACEART(FP6-506214), European Union FEDER funds, and the Spanish Plan Nacional de I+D (project AGL2004-06727-C02). J. A. Moreno was the recipient of a post-doctoral contract from CSIC (I3P programme), Spain. The work was also financially suported by the Department of Agriculture and Food FIRM programme (01/R&D/C/ 159), by the Higher Education Authority Programme for Research in Third Level Institutions, and by the SFI-funded Alimentary Pharmabiotic Centre.Peer reviewe