Fluorescence microscopy to monitor wine malolactic fermentation.

Malolactic fermentation (MLF) is a natural and biological deacidification of wines and a required step for making premium red wines. MLF is carried out by lactic acid bacteria (LAB) that are present in the fermenting wines. Currently, real-time control of MLF is an issue of great interest as the classical plate count technique for assessing bacterial populations requires long incubation times that are not compatible with a tight control of MLF. The aim of this study was to apply fluorescence microscopy and the bacteria staining kit Live/Dead BacLight™ to quantify viable LAB populations in red wines undergoing MLF. This method proved to be a fast and reliable culture-independent method to monitor wine MLF. Moreover, comparison of bacterial population data obtained by fluorescence microscopy and classical plate counts of LAB populations allowed discriminating a population of fully active and culturable cells, from total viable cells that include cells in an intermediate unculturable state.

Production and Antimicrobial Activity of Nisin Under Enological Conditions.

Lactic acid bacteria (LAB) are responsible for the malolactic fermentation of wines, and, therefore, controlling the growth of these bacteria is a key factor for elaborating premium wines. Sulfur dioxide has been traditionally used as an efficient antimicrobial and antioxidant agent, however, nowadays consumers' demand tends toward a reduction of sulfur dioxide levels in wine and other fermented foods. A previous study of our research group had demonstrated the effectiveness of the bacteriocin nisin to inhibit the growth of enological LAB, and its activity had been tested in culture broths. The aim of this study was to investigate the possibility of controlling the growth of bacteria in wine by the use of nisin in combination with sulfur dioxide, and to study nisin production by the natural producer Lactococcus lactis LM29 under enological conditions. Our results showed that L. lactis LM29 produced nisin in the presence of 2 and 4% ethanol (v/v), while higher concentrations of ethanol fully inhibited the production of nisin. We obtained a nisin enriched active extract (NAE) from the cell-free supernatant of a culture of L. lactis LM29 in MRS broth containing 60% (v/v) sterile grape juice, and the extract was fully active in inhibiting the growth of the enological LAB tested by the microtiter method. Moreover, the nisin concentration of the obtained NAE could actually prevent the formation of an undesirable biofilm of LAB strains. Finally, our results of wine ageing under winery conditions showed that the use of 50 mg/L nisin decreased fourfold the concentration of sulfur dioxide required to prevent LAB growth in the wines.

Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress.

This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a bacterial adaptive response to the presence of 2% ethanol in the culture broth and differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway (20-40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis strains were studied and compared to those of regulator-deletion mutants MG∆argR and MG∆ahrC. The results showed that in the presence of 2% ethanol those strains with an active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with an active ADI pathway consumed arginine and produced ornithine in the presence of 2% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in an ethanol containing medium.

Potential spoilage yeasts in winery environments: Characterization and proteomic analysis of Trigonopsis cantarellii.

Wine microbiota is complex and includes a wide diversity of yeast species. Few of them are able to survive under the restrictive conditions of dry red wines. In our study we detected and identified seven yeast species of the order Saccharomycetales that can be considered potential spoilers of wines due to physiological traits such as acidogenic metabolism and off-odor generation: Arthroascus schoenii, Candida ishiwadae, Meyerozyma guilliermondii, Pichia holstii, Pichia manshurica, Trigonopsis cantarellii, and Trigonopsis variabilis. Based on the prevalence of T. cantarellii isolates in the wine samples of our study, we further characterized this species, determined molecular and phenotypic features, and performed a proteomic analysis to identify differentially expressed proteins at mid-exponential growth phase in the presence of ethanol in the culture broth. This yeast species is shown to be able to grow in the presence of ethanol by expressing heat shock proteins (Hsp70, Hsp71) and a DNA damage-related protein (Rad24), and to be able to confer spoilage characteristics on wine.

Lineages and virulence gene content among extended-spectrum ß-lactamase-producing Escherichia coli strains of food origin in Tunisia.

Nineteen extended-spectrum ß-lactamase (ESBL)-positive Escherichia coli strains recovered from food samples in Tunisia were characterized by multilocus sequence typing and phylogenetic typing, and the virulence gene and plasmid content were also determined. These strains presented unrelated pulsed-field gel electrophoresis patterns and contained genes coding for the following ESBLs (the number of strains is in parentheses): CTX-M-1 (15), CTX-M-14 (2), CTX-M-8 (1), and SHV-5 (1). Twelve different sequence types (STs) were identified among the 19 ESBL-positive strains, which included two new STs (ST2022 in 2 bla(CTX-M-14)-containing strains and ST1970 in 2 bla(CTX-M-1)-containing strains). ST155 and ST602 were detected in four and three bla(CTX-M-1)-containing strains, respectively, and ST405 was detected in one bla(CTX-M-8)-producing strain. All ESBL-positive strains were ascribed to the phylogenetic groups A and B1. Most of the bla(CTX-M-1)-containing strains harbored an IncI1 plasmid, except for the four bla(CTX-M-1)-positive strains of beef origin and ST155, which harbored an IncN plasmid. The two bla(CTX-M-14)-containing strains contained an IncI1 plasmid. The virulence gene fimA was detected in all strains. Most strains also carried the aer gene, and six strains carried the eae gene. All strains were negative for the virulence genes sxt, papG-III, papC, hly, cnf1, and bfp. We conclude that ESBL-producing E. coli strains of food origin in Tunisia show high diversity and that plasmids harboring ESBL genes could be implicated in the dissemination of this resistance phenotype.

Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus and other staphylococci of animal and human origin.

OBJECTIVES: To detect the presence of lnu genes in staphylococcal strains with the unusual phenotype lincosamide resistance/macrolide susceptibility (L(R)/M(S)), and to determine their locations and genetic environments. METHODS: Six staphylococcal strains of human and animal origin with the phenotype L(R)/M(S) were studied. The presence of 15 resistance genes was tested by PCR. SCCmec typing was performed for all methicillin-resistant strains. agr typing, spa typing and multilocus sequence typing were carried out for Staphylococcus aureus strains. Transformation experiments were carried out by electrotransformation. Plasmid or chromosomal gene location was determined by Southern blot analysis and the genetic environments of the lnu genes were studied in all strains. RESULTS: Three methicillin-resistant staphylococcal strains contained the lnu(A) gene. The presence of the pLNU1 plasmid carrying lnu(A) was confirmed in one methicillin-resistant S. aureus (MRSA) ST398-t108 and one methicillin-resistant Staphylococcus sciuri. A novel lnu(A)-carrying plasmid (pUR5425) was identified in one MRSA ST125-t067 strain. Transformants of the three lnu(A)-positive strains presented increased lincomycin MIC values. The remaining three studied staphylococcal strains harboured the lnu(B) gene: two methicillin-susceptible S. aureus (MSSA) ST9-t337 and one MRSA ST398-t011. The lnu(B) gene was embedded in the chromosome in the two MSSA strains and in a large-sized plasmid in the MRSA strain. The same lnu(B) genetic environment was detected in these three strains. CONCLUSIONS: The resistance phenotype L(R)/M(S) seems to be related to S. aureus animal-associated clonal lineages (ST398 and ST9). A novel lnu(A)-carrying plasmid was identified and this is the first detection of the lnu(B) gene in MRSA ST398.

Diversity of clones and genotypes among vancomycin-resistant clinical Enterococcus isolates recovered in a Spanish hospital.

Forty-three vancomycin-resistant enterococci (VRE) from different patients were recovered in a Spanish Hospital (2003-2010), representing 0.4% of the total of enterococci recovered. Mechanisms detected were vanA (five Enterococcus faecium, two E. faecalis), vanB2 (seven E. faecium, five E. faecalis), vanB1 (one E. faecalis), and vanC1/2 (22 E. gallinarum, 1 E. casseliflavus). Four different Tn1546 structures were found among the seven vanA strains, three of them with insertions (ISEf1 or IS1542) or deletions. Most of the VRE presented a multiresistance phenotype and harbored different resistance genes [erm(B), tet(M), tet(L), ant(6)-Ia, aac(6')-aph(2''), aph(3')-IIIa, and catA]. Sixteen unrelated pulsotypes were detected among the 20 vanA/vanB E. faecalis and E. faecium isolates by pulsed-field-gel-electrophoresis and 11 unrelated pulsotypes among the 22 E. gallinarum isolates. Six different sequence types (ST) were demonstrated among the 12 vancomycin-resistant E. faecium strains (one of them new), and 5 were included into the clonal-complex (CC) CC17. Five different ST were detected among the eight E. faecalis strains. The esp gene was detected in 58% and 25% of E. faecium and E. faecalis strains, respectively, and the hyl gene in 78% and 89%, respectively. A high diversity of clones and genotypes of VRE were detected in this hospital.

Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria.

Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC(50) = 19 ng/ml) than the other LAB species (IC(50) = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking.

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method.

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) - PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth.

Vancomycin-resistant enterococci from Portuguese wastewater treatment plants.

The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in sludge and sewage of urban and poultry-slaughterhouse wastewater treatment plants. A total of 17 vancomycin resistant enterococci (eight vanA -containing Enterococcus faecium and nine vanC1/vanC2 -containing Enterococcus gallinarum/casseliflavus) were found among 499 isolates of sewage and sludge samples of 14 urban and nine poultry-slaughterhouse wastewater treatment plants. These seventeen VRE isolates showed resistance to kanamycin (n = 8), tetracycline (n = 7), erythromycin (n = 7), ciprofloxacin (n = 7), ampicillin (n = 7), streptomycin (n = 6), and gentamicin (n = 2). The tetM gene, related with tetracycline resistance, was found in six of eight van A-containing isolates, in all seven vanC-1 isolates and in one of two vanC-2 isolates. The ermB gene in seven erythromycin-resistant isolates; and the aac6 '-aph2 ″ gene in the two high-level-gentamicin-resistant isolates. Moreover, two vanA -containing E. faecium isolates harbored the hyl virulence gene, and three isolates the entA bacteriocin gene. The purK-1 allele was detected in our urban vanA -containing E. faecium isolate, and we found as well the purK-6 allele in one poultry-slaughterhouse vanA -containing E. faecium isolate. This study suggests that the wastewater treatment plants might be an important source of dissemination of antibiotic-resistant enterococci in Portugal.

Detection, molecular characterization, and clonal diversity of methicillin-resistant Staphylococcus aureus CC398 and CC97 in Spanish slaughter pigs of different age groups.

The objective of this study was to determine the frequency of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in slaughter pigs, to characterize the recovered isolates, and to investigate their genomic relatedness. Nasal swabs were collected from 53 finishing-pigs (F-pigs) and 53 suckling-piglets (S-piglets) at two different abattoirs in La Rioja (Northern Spain) coming from six production holdings. MRSA isolates were characterized by spa−, agr−, SCCmec−, and multilocus sequence typing, pulsed-field gel electrophoresis (PFGE)-ApaI, toxin gene profiling, antimicrobial susceptibility, and determination of antimicrobial resistance genes. MRSA isolates were recovered from 11 F-pigs (14 isolates) and 26 S-piglets (30 isolates). Forty of the 44MRSA presented the spa-types t011, t108, t1197, and t2346, which corresponded to the sequence type ST398 and to the clonal complex CC398. Interestingly, the remaining four isolates from F-pigs presented the spa-type t3992, and they were ascribed to a new sequence type named ST1379 (a single-locus variant of ST97), which was included in clonal complex CC97. Five PFGE-ApaI clusters with up to nine individual patterns detected among our MRSA and low genomic relatedness was observed between F-pig and S-piglet isolates. All MRSA were positive for hla, hld, and hlg hemolysin genes. ST1379 isolates harbored eta, lukE/D, and hlg-2 toxin genes, whereas ST398 isolates were positive for hlb. A great variety of distinct resistance gene patterns were observed, most of them coming from F-pig isolates. MRSA virulence properties seem to be dependent of the isolate clonal lineage. This study showed that slaughter pigs are frequently colonized by MRSA CC398; moreover, the detection of strains belonging to CC97 underlines that other lineages are also able to spread in livestock. Further studies should assess the risk of CC398 and non-CC398 MRSA to enter the food chain as well as the human health implications.

Tn1546 structures and multilocus sequence typing of vanA-containing enterococci of animal, human and food origin.

OBJECTIVES: To characterize the Tn1546 structure and to perform the genetic typing of 51 PFGE-unrelated vanA-containing enterococci of different origin (clinical, food and faecal samples of healthy humans and healthy poultry). METHODS: Tn1546 structure was characterized by a PCR primer walking strategy and sequencing. Multilocus sequence typing (MLST) was performed for Enterococcus faecium and Enterococcus faecalis strains, and esp and hyl genes were detected by PCR. RESULTS: Nine different Tn1546 structures were identified in the studied strains. Type I was the most prevalent structure (75%) (identical to GenBank M97297). Two new Tn1546 structures were identified (in three clinical and animal strains), containing two new insertion sequences (ISs; ISEfa11 disrupting vanS and ISEfa10 disrupting orf1). An additional new Tn1546 structure was found in one animal strain, containing ISEf1 interrupting vanY and IS1542 in the orf2-vanR region. A high diversity of sequence types (STs) was detected among clinical (6 ST/18 strains) and non-clinical E. faecium strains (18 ST/24 strains). STs associated with clonal complexes CC17 and CC9 were mainly detected among clinical and non-clinical E. faecium strains, respectively. Seven new STs were identified in non-clinical strains. The esp and hyl genes were only found among clinical E. faecium strains. CONCLUSIONS: A moderate variability in Tn1546 structure has been detected among unrelated vanA-containing enterococci of different origins, showing three new structures including two new ISs. A high diversity of STs was detected among E. faecium strains, especially among non-clinical strains, and new STs have been identified.

Effect of yeast mannoproteins and grape polysaccharides on the growth of wine lactic acid and acetic acid bacteria.

Polysaccharides constitute one of the main groups of wine macromolecules, and the difficulty in separating and purifying them has resulted in them being less studied than other wine macromolecules. In this study, the biological activity of a number of polysaccharide fractions obtained from yeast lees, must, and wine has been analyzed against a large collection of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) of enological origin. Results showed that a high proportion of AAB strains (60-88%) was inhibited by concentrations lower than 50 mg/L polysaccharide fractions containing intermediate- (6-22 kD) and small-molecular-weight (<6 kD) mannoproteins and oligosaccharide fragments derived from cellulose and hemicelluloses. Results also showed that, in contrast, yeast mannoproteins in concentrations up to 200 mg/L activated the growth of 23-48% of the studied LAB strains when ethanol was present in the culture broth. Specially, yeast commercial mannoproteins of intermediate molecular weight (6-22 kD) were active in increasing Oenococcus oeni growth (81.5% of the studied O. oeni strains) in the presence of ethanol in the culture broth. These effects of wine polysaccharides on bacterial growth provide novel and useful information for microbiological control of wines and winemaking biotechnology.

Genetic diversity of the pln locus among oenological Lactobacillus plantarum strains.

A total of 33 Lactobacillus plantarum strains obtained from grape musts and wines during alcoholic and malolactic fermentations were submitted to PCR analysis with specific primers directed to 27 genes of the plantaricin (pln) locus previously described for L. plantarum strains. The number of genes that were detected varied depending on the strain, and cluster analysis of results rendered seven groups, named plantaritypes (similarity within each group >89%) that included all the 33 oenological strains, four L. plantarum type strains (C11, NC8, J23 and J51) that had been previously described, and strain WCFS1 whose genome had been fully sequenced. The common features for most strains (74%) were the presence of the plnABCD regulatory system (which includes genes of the inducing peptide, its coupled membrane-located histidine protein kinase and two response regulators), the two-peptide bacteriocin plnEF genes and the genes of a membrane-bound ABC transport system. The pln locus is shown to be widespread among oenological strains (94% of appearance), as well as to possess a remarkable plasticity and variable regions related to its regulation and bacteriocin production.

Detection of vanA and vanB2-containing enterococci from food samples in Spain, including Enterococcus faecium strains of CC17 and the new singleton ST425.

Two-hundred-twenty-nine food samples of animal origin were tested to know the prevalence of vancomycin-resistant enterococci (VRE) after a decade of avoparcin ban as animal growth promoter in Spain. VRE with acquired mechanism of resistance were detected in 9 of these 229 samples (3.9%, obtained from chicken, veal and rabbit), and one VRE per food sample was further characterized. The vanA gene was identified in seven isolates (2 E. faecium, 3 E. durans, and 2 E. hirae), and the vanB2 gene in the remaining 2 isolates (identified as E. faecium). The two vanB2 isolates showed a phenotype of multiresistance that included, in addition to vancomycin, also ampicillin, erythromycin, tetracycline, streptomycin, kanamycin, ciprofloxacin, chloramphenicol and trimethoprim-sulfamethoxazole and contained, among others, erm(B), tet(M), ant(6), and aph(3')-III genes. Most of vanA enterococci showed erythromycin and tetracycline resistance and contained the erm(B) and tet(M) genes. One vanA- and both vanB2-positive E. faecium isolates were classified by MLST analysis into the CC17 clonal complex (ST17 and ST78), and one additional vanA isolate was included in a new sequence type named ST425 (singleton). Co-transference by conjugation of erm(B) and vanA genes was demonstrated in one vanA-positive E. faecium isolate. The inclusion of vanB2 cluster into Tn5382 structure was demonstrated in the two vanB2 isolates, as well as the linkage pbp5-Tn5382, and beta-haemolysis and gelatinase production was identified in one of them. Food sample of animal origin could be a vehicle of transference of VRE of vanA and vanB2 type that could be transferred to humans.

Prevalence and mechanisms of erythromycin resistance in Streptococcus agalactiae from healthy pregnant women.

We sought to determine the resistance phenotypes for erythromycin and clindamycin and the mechanisms implicated in 93 Streptococcus agalactiae isolates recovered from healthy pregnant women. Susceptibility testing for erythromycin, clindamycin, penicillin, cefotaxime, vancomycin, quinupristin-dalfopristin, choramphenicol, ofloxacin, and meropenen was carried out by disc-diffusion test, and the E-test was also applied for erythromycin and clindamycin. The constitutive MLS(B) resistance (cMLS(B)) and inducible MLS(B) resistance (iMLS(B)) phenotypes, respectively, as well as the M resistance phenotype were determined by the erythromycin-clindamycin double-disc test. The presence of ermA, ermB, ermC, msrA, and mef(A/E) macrolide resistance genes was studied by PCR. Resistance to erythromycin and clindamycin was found in 15% and 9.6% of the isolates, respectively. The resistance phenotypes detected among the 14 erythromycin-resistant isolates were as follows (number of isolates): cMLS(B) (9), iMLS(B) (3), and M (2). The MICs for erythromycin and clindamycin were as follows: cMLS(B) isolates (128-256 and >or=32 mg/L, respectively), iMLS(B) isolates (16-256 and 1 mg/L), and M isolates (2-8 and 1 mg/L). The following combination of genes were detected among isolates with cMLS(B) or iMLS(B) phenotypes: erm(B) (6 isolates), ermA + ermTR (3), ermA + ermB + ermTR (1), and none of these genes (2). The two isolates with M phenotype harbored the mef(A/E), and msrA gene was also found in one of them.

Comparative study of the pln locus of the quorum-sensing regulated bacteriocin-producing L. plantarum J51 strain.

Lactobacillus plantarum J51 strain was isolated from a Rioja red wine and it showed bacteriocin activity against a wide range of lactic acid bacteria of oenological importance. These characteristics conferred L. plantarum J51 a high interest both in wine microbiology and in the study of bacteriocin production. In this work the bacteriocin production regulated under the "quorum-sensing" mechanism is observed and the pln locus of the bacteriocin-producing L. plantarum J51 is fully characterized. A 20,667 bp fragment was completely sequenced (GenBank accession number DQ340868), and showed five operons (plNC8betaalphac, plnLR-like, plnABCD, plnEFI, plnGHSTUVW) and a new region containing a putative operon with three new orfs that could encode a putative two-peptide bacteriocin.

Prevalence and diversity of integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain.

OBJECTIVES: To analyse the prevalence and diversity of integrons in faecal Escherichia coli isolates from healthy humans in Spain. METHODS: One hundred E. coli isolates were obtained in Levine agar plates from faecal samples of 100 healthy humans during March to October 2007. Susceptibility to 16 antimicrobial agents was determined by the disc diffusion method. The presence and characterization of class 1, 2 and 3 integrons, as well as the presence of other antimicrobial resistance genes, were performed by PCR and DNA sequencing. RESULTS: Integrases associated with class 1 and/or class 2 integrons were identified in 29 E. coli isolates (intI1 gene in 26 isolates, intI2 in 1 isolate and intI1 + intI2 in 2 isolates), the remaining 71 isolates being free of these integrons. Seven different gene cassette arrangements were demonstrated in 27 of the 28 intI1-positive isolates and were as follows (number of isolates): dfrA1 + aadA1 (12), aadA (8), dfrA17 + aadA5 (3), dfrA7 (1), dfrA5 (1), dfrA1 (1) and dfrA12 + orfF + aadA2 (1). Four isolates presented defective class 1 integrons lacking the 3'-conserved region. The three isolates containing class 2 integrons harboured the dfrA1 + sat + aadA1 gene cassette array in their variable region. Integron-positive isolates showed higher percentages of resistance to streptomycin, ampicillin, tetracycline, trimethoprim, sulfamethoxazole, chloramphenicol and nalidixic acid than integron-negative isolates. Sixty-five percent of the integron-positive isolates belonged to phylogenetic groups A or D. CONCLUSIONS: A high prevalence of integrons was detected in faecal E. coli of healthy humans. Individuals in the community could be a reservoir of integron-containing E. coli isolates.