Aurora Fernández-Astorga

Relationships between Escherichia coli cells and the surrounding medium during survival processes

In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur. On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC). An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates. The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule. The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane. Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake. Indeed, cell free supernatants supported the growth of several generations of a population of 104 cells ml–1. The survival of E. coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability. It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state.

gfp-Tagged cells as a useful tool to study the survival of Escherichia coli in the presence of the river microbial community

We have used an Escherichia coli strain DH5a containing pGreenTIR to study the survival of this bacterium in river water. As green fluorescence was maintained throughout survival both in dark and illuminated conditions, gfp-tagged E. coli cells were clearly distinguished from the microbial community of the river Butrón. gfp-tagged E. coli cells were monitored to estimate total density as well as the density of the culturable and viable (active electron transport system, CTC+) cells. Our results indicate that autochthonous bacteria and introduced E. coli are predated by flagellates. The autochthonous bacterial community behaves as predation-escaping prey, showing a tendency to cellular miniaturization and so maintaining the density of the population. In contrast, introduced E. coli behaves as predation-non-escaping prey, so E. coli was eliminated from the system. When comparing the elimination by predation of heat-treated and non-heated gfp-tagged E. coli cells we deduce that the flagellates do not discriminate between live and heat-treated cells. Finally, in the presence of the river microbial community, the E. coli cells appeared to be ingested before cellular deterioration could occur. Thus predation reduces the quantitative importance of the viable but nonculturable (VBNC) population of E. coli in the aquatic systems.

PCR-Restriction Fragment Length Polymorphism Assay for Detection of gyrA Mutations Associated with Fluoroquinolone Resistance in Campylobacter coli

ABSTRACT A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.

Prevalence and genetic diversity of arcobacter in food products in the north of Spain.

The bacterial contamination of food products can cause serious public health problems. Interest in Arcobacter contamination has increased due to the relationship between these bacteria and human enteritis. We studied the prevalence and genetic diversity of Arcobacter species at the retail level in the province of Alava in Basque Country, Spain. The results showed a high genetic diversity and indicated the regular presence of the main Arcobacter spp. associated with human enteric illness in food products. Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii were detected with an overall prevalence close to 40% and were isolated from 15 (42.8%) fresh cows milk samples, 12 (73.3%) shellfish samples, 11 (55%) chicken samples, 2 (10%) pork samples, and 1 (5%) beef sample. The results indicate the need to investigate the impact of Arcobacter spp. on public health.

Comparison of Campylobacter fla-SVR genotypes isolated from humans and poultry in three European regions

Aims:  The genetic diversity of Campylobacter isolated from human infection and from poultry was assessed in strains originating in three different European regions in order to compare these two hosts and to investigate European regional differences.

Molecular Typing of Campylobacter jejuni Isolates Involved in a Neonatal Outbreak Indicates Nosocomial Transmission

ABSTRACT Genotypic typing by restriction fragment length polymorphism and pulsed-field gel electrophoresis showed that two neonates in a neonatal ward were infected with the same Campylobacter jejuni strain. Isolates from the mother and brother of the index patient were identical to each other but distinct from the neonatal type. Genotyping results therefore suggested that the neonatal C. jejuni infection was nosocomial in origin.

Detection and enumeration of viable but non‐culturable transconjugants of Escherichia coli during the survival of recipient cells in river water

Viable but non‐culturable transconjugant cells were detected by a modification of the direct viable count (DVC) method. This modification involved the addition of parental antimicrobial markers (kanamycin and streptomycin) to the elongation medium in order to promote selective elongation of the transconjugant cells. Presence of viable, other than culturable, transconjugants was demonstrated in matings with parental cells from TSB culture as well as with recipient cells from survival in river water (under illuminated and non‐illuminated systems). In matings with a recipient strain from illuminated systems, culturable transconjugants were not detected after the third day of recipient cell survival. In spite of this, viable transconjugants were detected in numbers that exceeded 105cells ml−1. These results clearly show that a fraction of non‐culturable recipient cells is able to receive and express plasmids by conjugation processes and form viable but non‐culturable transconjugant cells.

Disinfectant tolerance and antibiotic resistance in psychrotrophic Gram-negative bacteria isolated from vegetables

A total of 330 strains of psychrotrophic non‐fermenting Gram‐negative bacteria isolated from vegetables were studied. In spite of the wide range of antibiotic resistance occurring, less than 10% showed resistance patterns which included mezclocillin‐ticarcillin‐gentamicin or ceftizoxime‐norfloxacin. Reductions of > 5 log10 in the numbers of cfu were found when these strains were exposed for 30 min to a quaternary ammonium compound (1% w/v).

Multilocus sequence typing reveals genetic diversity of foodborne Arcobacter butzleri isolates in the North of Spain

The emerging pathogen Arcobacter butzleri is being increasingly isolated from different animal food products but the routes of its transmission to human are not well established yet. Typing methods would be useful in gaining such knowledge. Here we report the great genetic diversity observed among A. butzleri isolates from different food products. Forty-five isolates were analyzed by Multilocus Sequence Typing (MLST). A total of 157 alleles were identified across all seven loci, ranging from 16 alleles at glnA to 31 at glyA. MLST differentiated the isolates into 34 sequence types (STs), with the majority of isolates containing a unique sequence type. Seventy-four new alleles were identified, which resulted in the assignment of 33 new STs. No association of alleles or STs with food source was observed. For the first time, lateral gene transfer from Arcobacter skirrowii to A. butzleri at the glyA locus is also reported.

Spread of an Enterococcus faecalis sequence type 6 (CC2) clone in patients undergoing selective decontamination of the digestive tract.

Enterococcus faecalis (E. faecalis) is a common cause of nosocomial infection in immunocompromised patients. The presence and dissemination of high‐risk clonal complexes, such as CC2, is an ongoing problem in hospitals. The aim of this work was to characterize 24 E. faecalis isolates from ICU patients undergoing selective decontamination of the digestive tract (SDD) by phenotypical (antimicrobial susceptibility) and genotypical (presence of virulence genes, RAPD‐PCR and MLST) methods. Our results showed high prevalence of the ST6 E. faecalis clone (91.6%), especially adapted to the hospital environment, with a multidrug resistance pattern and a multitude of putative virulence genes. In addition, ST179 (4.2%) and ST191 (4.2%) were detected. By RAPD–PCR analysis, the 22 isolates identified as ST6 showed six different DNA patterns, while the two remaining isolates, ST179 and ST191, showed two additional profiles. CC2 is a known clonal complex with high adaptability to hospital environment and worldwide distribution. The high prevalence of the ST6 clone in the studied population could be related to the presence of gentamicin in the SDD mixture since most strains were gentamicin resistant. Consequently, strict surveillance should be applied for rapid detection and control of this clone to prevent future spread outside the ICU.