Dominant cultivable Lactobacillus species from the feces of healthy adults in northern Spain

The aim of this study was to identify numerically dominant cultivable lactobacilli species in the feces of healthy adults. Ten individuals from Asturias, northern Spain, were chosen. Bacterial colonies grown under anoxic conditions on MRS with cysteine were microscopically examined for lactobacilli. Isolates were subsequently grouped based on the analysis of their carbohydrate fermentation profiles and then identified by partial amplification, sequencing, and comparison of their 16S rRNA gene sequences. Lactobacilli varied from undetectable levels in three subjects (< 105 CFU/g feces) to around 109 CFU/g feces. Among the 71 isolates obtained from seven individuals, 12 Lactobacillus species were identified. High interindividual variation was observed in terms of total numbers, number of species, and dominant species. Lactobacillus paracasei was found in four of the seven individuals; L. gasseri, L. delbrueckii, and L. plantarum in three. Phenotyping showed that only one strain per species was in the majority in each individual. [Int Microbiol 2007; 10(2):141-145

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

Resistencia a antibióticos en bacterias ácido-lácticas y productos lácteos

Trabajo presentado en el XXVII Congreso Nacional de Microbiología, celebrado en Málaga (España), del 2 al 5 de julio de 2019Antibióticos y resistencia En la lucha contra las enfermedades infecciosas, el descubrimiento y la utilización de los antibióticos es sin duda uno de los logros más importantes de la medicina del siglo XX. Sin embargo, el éxito inicial se ha ido empañando con el paso del tiempo debido a la aparición y diseminación de cepas resistentes que complican y encarecen los tratamientos. De hecho, algunas bacterias patógenas de las especies Acinetobacter baumanii, Enterococcus faecalis y Klebsiella pneumoniae carecen hoy en día de tratamientos efectivos. Muchos autores vaticinan, en un futuro próximo, el incremento del número de patógenos resistentes y, por tanto, un aumento de la morbilidad y mortalidad de infecciones fácilmente curables en la actualidad. La resistencia a antibióticos es un fenómeno natural ligado a procesos evolutivos de adaptación de los microbios al medio ambiente causado por la presión selectiva que supone la presencia de antibióticos en los ecosistemas, incluyendo su utilización en clínica y veterinaria, pero también en agricultura, ganadería y acuicultura. En muchos casos, la aparición de resistencias ha precedido la utilización práctica de los antibióticos, de manera que existen otros fenómenos desconocidos que contribuyen también a la aparición y diseminación de resistencias en el medio ambiente. El impacto adverso para la salud humana de las resistencias puede abordarse también desde una perspectiva ecológica. Esta aproximación ha recibido poca atención hasta el momento y se centra en el papel que llevan a cabo los ecosistemas como consecuencia de la presencia en el medio ambiente de (i) antibióticos, (ii) bacterias resistentes a antibióticos, (iii) genes de resistencia a antibióticos y (iv) elementos genéticos móviles (plásmidos, integrones, transposones, etc.) implicados en la transferencia horizontal de genes de resistencia

Microbiota cultivable y microbiota total del estómago humano

Ponencia presentada en el III Workshop Probióticos, Prebióticos y Salud. Evidencia Científica, celebrado en Barcelona los días 15 y 16 de diciembre de 2011.Se han identificado 86 aislados de 16 especies distintas. Propio nibacterium acnes resultó ser el microorganismo mayoritario con 41 aislados. Destaca también la presencia de 19 aislados de lactobacilos de cinco especies y 11 aislados de tres especies de estafilococos. En la pirosecuenciación se obtuvieron 56.738 lecturas válidas que pertenecían a 59 familias y 69 géneros, indicando una diversidad muy superior a la obtenida por cultivo. Se apreció una gran variabilidad de secuencias entre las distintas muestras, y, tras diversos análisis bioinformáticos, éstas se distinguen de las descritas en otras posiciones del tracto gastrointestinal (boca, faringe o intestino), mostrando que el estómago contiene una composición bacteriana propia. En la actualidad estamos estudiando las propiedades probióticas de los lactobacilos, de las que destacamos su capacidad de inhibición de Helicobacter pylori.Peer Reviewe

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.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