Montserrat Llagostera

Isolation and Characterization of Potentially Pathogenic Antimicrobial-Resistant Escherichia coli Strains from Chicken and Pig Farms in Spain

ABSTRACT To ascertain whether on animal farms there reside extended-spectrum β-lactamase (ESBL) and plasmidic class C β-lactamase-producing Escherichia coli isolates potentially pathogenic for humans, phylogenetic analyses, pulsed-field gel electrophoresis (PFGE) typing, serotyping, and virulence genotyping were performed for 86 isolates from poultry (57 isolates) and pig (29 isolates) farms. E. coli isolates from poultry farms carried genes encoding enzymes of the CTX-M-9 group as well as CMY-2, whereas those from pig farms mainly carried genes encoding CTX-M-1 enzymes. Poultry and pig isolates differed significantly in their phylogenetic group assignments, with phylogroup A predominating in pig isolates and phylogroup D predominating in avian isolates. Among the 86 farm isolates, 23 (26.7%) carried two or more virulence genes typical of extraintestinal pathogenic E. coli (ExPEC). Of these, 20 were isolated from poultry farms and only 3 from pig farms. Ten of the 23 isolates belonged to the classic human ExPEC serotypes O2:H6, O2:HNM, O2:H7, O15:H1, and O25:H4. Despite the high diversity of serotypes and pulsotypes detected among the 86 farm isolates, 13 PFGE clusters were identified. Four of these clusters contained isolates with two or more virulence genes, and two clusters exhibited the classic human ExPEC serotypes O2:HNM (ST10) and O2:H6 (ST115). Although O2:HNM and O2:H6 isolates of human and animal origins differed with respect to their virulence genes and PFGE pulsotypes, the O2:HNM isolates from pigs showed the same sequence type (ST10) as those from humans. The single avian O15:H1 isolate was compared with human clinical isolates of this serotype. Although all were found to belong to phylogroup D and shared the same virulence gene profile, they differed in their sequence types (ST362-avian and ST393-human) and PFGE pulsotypes. Noteworthy was the detection, for the first time, in poultry farms of the clonal groups O25b:H4-ST131-B2, producing CTX-M-9, and O25a-ST648-D, producing CTX-M-32. The virulence genes and PFGE profiles of these two groups were very similar to those of clinical human isolates. While further studies are required to determine the true zoonotic potential of these clonal groups, our results emphasize the zoonotic risk posed especially by poultry farms, but also by pig farms, as reservoirs of ESBL- and CMY-2-encoding E. coli.

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Use of a bacteriophage cocktail to control Salmonella in food and the food industry.

The use of lytic bacteriophages for the biocontrol of food-borne pathogens in food and in the food industry is gaining increasing acceptance. In this study, the effectiveness of a bacteriophage cocktail composed of three different lytic bacteriophages (UAB_Phi 20, UAB_Phi78, and UAB_Phi87) was determined in four different food matrices (pig skin, chicken breasts, fresh eggs, and packaged lettuce) experimentally contaminated with Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis. A significant bacterial reduction (>4 and 2 log/cm(2) for S. Typhimurium and S. Enteritidis, respectively; p≤0.005) was obtained in pig skin sprayed with the bacteriophage cocktail and then incubated at 33 °C for 6h. Significant decreases in the concentration of S. Typhimurium and S. Enteritidis were also measured in chicken breasts dipped for 5 min in a solution containing the bacteriophage cocktail and then refrigerated at 4 °C for 7 days (2.2 and 0.9 log10 cfu/g, respectively; p≤0.0001) as well as in lettuce similarly treated for 60 min at room temperature (3.9 and 2.2 log10 cfu/g, respectively; p≤0.005). However, only a minor reduction of the bacterial concentration (0.9 log10 cfu/cm(2) of S. Enteritidis and S. Typhimurium; p≤0.005) was achieved in fresh eggs sprayed with the bacteriophage cocktail and then incubated at 25 °C for 2 h. These results show the potential effectiveness of this bacteriophage cocktail as a biocontrol agent of Salmonella in several food matrices under conditions similar to those used in their production.

Multiresistance in Pasteurella multocida Is Mediated by Coexistence of Small Plasmids

ABSTRACT In most gram-negative bacteria, acquired multiresistance is conferred by large plasmids compiling numerous antimicrobial resistance genes. Here, we show an evolutionary alternative strategy used by Pasteurella multocida to become resistant to multiple clinically relevant antibiotics. Thirteen β-lactam-resistant clinical isolates, concomitantly resistant to tetracyclines and/or streptomycin as well as to sulfonamides, were studied. Pulsed-field gel electrophoresis analysis revealed different profiles among the isolates, showing that clonal dissemination was not the sole event responsible for the spread of multiresistance. Each P. multocida strain carried two or three small plasmids between 4 and 6 kb in size. A direct association between resistance profile and plasmid content was found. Complete nucleotide sequencing of all plasmids revealed seven different replicons, six of them belonging to the ColE1 superfamily. All plasmids carried one, or a maximum of two, antimicrobial resistance determinants. Plasmids pB1000 and pB1002 bore blaROB-1, pB1001 carried tet(B), pB1003 and pB1005 carried sul2 and strA, pB1006 harbored tet(O), and p9956 bore the tet(H) gene. All plasmids except pB1002 and pB1006 were successfully transformed into Escherichia coli. pB1000, also involved in β-lactam resistance in Haemophilus parasuis (A. San Millan et al., Antimicrob. Agents Chemother. 51:2260-2264, 2007), was mobilized in E. coli using the conjugation machinery of an IncP plasmid. Stability experiments proved that pB1000 was stable in P. multocida but highly unstable in E. coli. In conclusion, blaROB-1 is responsible for β-lactam resistance in P. multocida in Spain. Coexistence and the spread of small plasmids are used by P. multocida to become multiresistant.

Single DNA molecule detection in an optical trap using surface-enhanced Raman scattering

Raman spectra from single DNA molecules in their natural aqueous environment are presented. A DNA molecule that is anchored between two optically trapped dielectric beads is suspended in a solution with nanosized silvercolloid particles. The nonspecific binding of the metal to the DNA enhances the Raman scattering that is excited by a near-infrared beam. A Raman spectrum is first recorded followed by a force-extension curve that verifies the presence of a single DNA molecule.

The high‐affinity zinc‐uptake system znuACB is under control of the iron‐uptake regulator (fur) gene in the animal pathogen Pasteurella multocida

The Pasteurella multocida znuACB genes encoding a high-affinity zinc-uptake system have been identified and cloned. In contrast to what happens in Escherichia coli, znuA is not physically linked to znuCB. Through lacZ transcriptional fusions it has been demonstrated that zinc negatively regulates both znuA and znuCB operons. Nevertheless, and contrary to that determined so far for all other znuACB bacterial systems known, P. multocida znuACB genes are not under control of the zur gene, which is absent in this bacterial species, but rather are under its iron-uptake regulator (fur) gene. Furthermore, construction of defective mutants has demonstrated that P. multocida znuA and znuCB transcriptional units are required for virulence of this organism in a mouse model.

Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry

ABSTRACT Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time.

A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa.

Escherichia coli LexA protein is the repressor of a gene network whose members are directly involved in the repair of damaged DNA and in the survival of bacterial cells until DNA lesions have been eliminated. The lexA gene is widely present in bacteria, although the sequences of only three LexA-binding sites are known: Gram-positive, alpha Proteobacteria and some members of gamma Proteobacteria represented by E. coli. Taking advantage of the fact that the genome sequence of the plant-pathogenic bacterium Xylella fastidiosa has been determined, its lexA gene has been cloned and overexpressed in E. coli to purify its product. After demonstration that X. fastidiosa lexA and recA genes are co-transcribed, gel mobility shift assays and directed mutagenesis experiments using the promoter of the lexA-recA transcriptional unit demonstrated that the X. fastidiosa LexA protein specifically binds the imperfect palindrome TTAGN(6)TACTA. This is the first LexA binding sequence identified in the gamma Proteobacteria differing from the E. coli-like LexA box. Although a computational search has revealed the presence of TTAGN(6)TACTA-like motifs upstream of X. fastidiosa genes other than lexA, X. fastidiosa LexA only binds the promoter of one of them, XF2313, encoding a putative DNA-modification methylase. Moreover, X. fastidiosa LexA protein does not bind any of the other genes whose homologues are regulated by the LexA repressor in E. coli (uvrA, uvrB, ssb, ruvAB, ftsK, dinG, recN and ybfE). RT-PCR quantitative analysis has also demonstrated that lexA-recA and XF2313 genes, as well as the X. fastidiosa genes which are homologues to those of E. coli belonging to the LexA regulon, with the exception of ssb, are DNA damage-inducible in X. fastidiosa.

Dissemination of extended-spectrum β-lactamase-producing bacteria: the food-borne outbreak lesson

OBJECTIVES Commensal and opportunistic bacteria producing extended-spectrum beta-lactamases (ESBL-PB) have undergone a broad and rapid spread within the general population; however, the routes of dissemination have not been totally elucidated. The aim of this study was to determine whether individuals involved in an outbreak of acute gastroenteritis, in addition to the enteropathogenic microorganism, share an ESBL-PB as indirect demonstration of its transmission from a common food source. METHODS From 2003 to 2004 in Barcelona, Spain, stool samples from 905 people involved in 132 acute gastroenteritis outbreaks and 226 food handlers related to the outbreaks were investigated. RESULTS In 31 outbreaks, 58 diners carrying one or more ESBL-PB were detected. In 10 outbreaks, two or more diners shared the same ESBL-PB, and in four of them, the strain was shared with the food handlers. CONCLUSIONS This study provides circumstantial evidence that foods can be a transmission vector for ESBL-PB, probably from two reservoirs, food animals and food handlers.

Analysis of the Protective Capacity of Three Streptococcus suis Proteins Induced under Divalent-Cation-Limited Conditions

ABSTRACT Streptococcus suis is a gram-positive pathogen that causes serious diseases in pigs and, in some cases, humans. Three genes of the virulent S. suis 89/1591 strain, encoding putative divalent-cation-binding lipoproteins, were isolated based on information obtained from the draft annotation files of this organisms genome. The products of these genes, which are inducible by divalent-cation deprivation, were subsequently purified, and their immunogenic and protective abilities were analyzed. All three proteins (SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237) were found to be immunogenic, but only one of them (SsuiDRAFT 0103) induced a significant protective response (87.5%, P = 0.01) against the same S. suis strain. Furthermore, the S. suis ssuiDRAFT 1240 gene (adcR), which encodes a predicted regulator of Zn2+ and/or Mn2+ uptake in streptococci, was cloned, and its protein product was purified. Electrophoretic mobility shift assays with purified S. suis AdcR protein showed experimentally, for the first time, that the AdcR DNA-binding sequence corresponds to the TTAACNRGTTAA motif. In addition, a requirement for either Zn2+ or Mn2+, but not Fe2+, to establish in vitro binding of AdcR to its target sequence and the ability of AdcR to bind the ssuiDRAFT 0103 and ssuiDRAFT 1237 gene promoters but not the promoter of the ssuiDRAFT 0174 gene were demonstrated. Taken together, these data suggest that SsuiDRAFT 0103 is a good candidate for vaccines against S. suis and support preliminary results indicating that bacterial envelope proteins involved in the uptake of divalent cations other than iron may be useful for protective purposes.