Expression of the multiple antibiotic resistance operon (mar) during growth of Escherichia coli as a biofilm

The multiple antibiotic resistance (mar) operon is a global regulator controlling the expression of various genes in Escherichia coli which constitutes the mar regulon. Upregulation of mar leads to a multi-drug resistant phenotype, which includes resistance towards structurally unrelated antibiotics, organic solvents and the disinfectant pine oil. Biofilms also display similar decreases in susceptibility to antimicrobial agents. A marOII-lacZ fusion strain (SPC105) of E. coli was used to monitor mar expression under various growth conditions including batch, continuous and biofilm culture. In chemically-defined media (CDM), mar expression was maximal in mid-log and declined in the stationary phase. Conversely, in rich media (Luria-Bertani broth), minimal expression in mid-log was followed by an increase in the stationary phase. In continuous culture, expression was inversely related to specific growth rate (mu = 0.05-0.4 h-1). LacZ expression by the marOII-lacZ fusion was generally low within the total biofilm population and equivalent to that of stationary phase cultures grown in batch culture. When the expression of mar in CDM batch culture was compared with that in biofilm populations, beta-galactosidase activity was generally higher throughout batch culture than in the attached population. Overall, these results suggest that while mar expression will be greatest within the depths of a biofilm where growth rates are suppressed, its probable induction within biofilms cannot explain the elevated levels of antibiotic resistance observed.     

Detection of gentoxicity of benzidine and its derivatives with the Escherichia coli DJ 702 lacZ reversion mutagenicity assay

AIMS: The feasibility of Escherichia coli DJ 702 lacZ mutagenicity assay to detect genotoxicity of benzidine and its derivatives was evaluated. METHODS AND RESULTS: DJ 702 strain was grown overnight at 30 degrees C in Luria-Bertani (LB) medium containing some components, such as chloramphenicol, ampicillin, delta-aminolevulinic acid, isopropyl-beta-d-thiogalactoside, and trace element mix. The mixtures of a bacterial culture and tested chemical at indicated doses were incubated at 30 degrees C for 30 min. Subsequently, 2 ml of molten top agar was added and the resulting mixtures were immediately poured onto a minimal lactose (ML) plate. Plates were incubated at 30 degrees C for 48 h. The number of colonies was determined by visual scoring. In this study, results showed that all the tested chemicals were mutagenic to DJ 702 strain. CONCLUSIONS: E. coli lac mutagenicity assay using DJ 702 strain can detect the mutagenicity of benzidine and its derivatives. SIGNIFICANCE AND IMPACT OF THE STUDY: We detected the mutagenicity of benzidine and its derivatives in E. coli lac mutagenicity assay using DJ 702, indicating that this assay may be used to detect benzidine and its derivatives in a powerful, sensitive, and convenient mutagenesis assay.     

Identification of new genes regulated by the marRAB operon in Escherichia coli.

Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon. Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature. Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was represented by the mar operon. The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome. One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min. Cloning and sequencing of a portion of the fused genes showed all of them to be different. The phoA fusions at 77 min were located in a recently identified gene, slp, a lipoprotein of unknown function (D.M. Alexander and A. C. St. John, Mol. Microb. 11:1059-1071, 1994). The others showed no homology with any known genes of E. coli. The insertions caused small but reproducible changes in the antibiotic susceptibility profile. This approach has enabled the identification of new genes in E. coli which are regulated by the marRAB operon and involved in the Mar phenotype.     

A 210-min solid phase cytometry test for the enumeration of Escherichia coli in drinking water

A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies. The test involves membrane filtration through a 25-mm black polyester filter, induction of beta-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-beta-Dglucuronide as an enzyme substrate and laser scanning of the membrane filter. Scan results can be confirmed on-line by epifluorescence microscopy. Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated > or =90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar. In 5.4% of all samples examined, SPC detected between 1 and 11 E. coli per 100 ml, while the two plate methods yielded negative results. Cases of a negative SPC result but a positive E. coli count on both reference media were not observed. This test would primarily be useful for 'emergency' monitoring of drinking water when rapid results are crucial.     

Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis

AIMS: The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus. METHODS AND RESULTS: The 750 bp xylanase gene was amplified and subcloned into the unique NheI restriction enzyme site of pMG36e and subsequently transformed into competent Escherichia coli XLI-blue MRF cells and Lactococcus lactis cells. Bacterial culture containing pMG36e-Xy has an enzyme activity of 390 microg xylose ml(-1) culture 30 min(-1), respectively, when compared with 40 microg xylose ml(-1) culture 30 min(-1) for the negative control (plasmidless strain). CONCLUSIONS: The thermostable xylanase gene was successfully expressed in both E. coli and L. lactis. The activity of xylanase can be easily detected by the formation of visible clearing zones around the transformed colonies on Remazol Brilliant Blue-Xylan (RBB-Xylan) agar media. However, there were some significant differences in the optimum growth temperature and plasmid stability in the new clones. SIGNIFICANCE AND IMPACT OF THE STUDY: The constructed reporter vector has the potential to be used as a reporter system for Lactococcus as well as E. coli, and it is an addition to the pool of lactococcal vector systems.     

The correlation method for rapid monitoring of Escherichia coli in foods

AIMS: A new rapid method was developed to rapidly monitor Escherichia coli counts in foods. MATERIALS AND RESULTS: One ml of modified selective broth with 4-methylumbelliferyl beta-D-glucuronide and 1 ml of food sample were mixed in a sterile test tube and incubated at 37 degrees C. The positive reaction (fluorescence under u.v. light) was monitored at regular 30 min intervals. The positive reaction times in test tubes were compared with actual E. coli numbers from tested samples. The growth of E. coli in test tubes (broth) was much faster than growth on agar. The first experiment was performed to evaluate the rapid correlation method using pure E. coli cultures. The correlation between E. coli counts by the conventional plating method and positive reaction (fluorescence production) times in test tubes was highly agreeable (r(2) = 0 x 95). In the case of low E. coli numbers, such as 2 x 0 log10 cfu ml(-1), the rapid correlation method detected their presence after 10 h incubation. When highly contaminated samples were assayed (8 log10 cfu ml(-1)), the rapid correlation method detected the presence of E. coli after 4 h incubation. In the ground beef experiment, the correlation between fluorescence production time and actual E. coli numbers was also strongly agreeable (r(2) = 0 x 92). CONCLUSIONS: From these results, it is obvious that the new rapid method can rapidly monitor E. coli counts in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the new method saved about 10-14 h incubation time compared to conventional plating methods. The rapid correlation method required much shorter incubation times compared to conventional plating methods for monitoring E. coli.     

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

Growth of Escherichia coli O157:H7 during sprouting of alfalfa seeds

AIMS: Escherichia coli O157:H7 was monitored daily during sprouting of alfalfa seeds inoculated at high (3.92 log10 cfu g(-1)) and low (1.86 log10 cfu g(-1)) levels to assess the extent of pathogen growth during production. METHODS AND RESULTS: Sprouts and rinse water were tested by direct and membrane filter plating on modified sorbitol MacConkey agar and BCM O157:H7(+) agar; the antibody-direct epifluorescent filter technique; and rapid immunoassays. The pathogen reached maximum populations after one and two days of sprouting seeds inoculated at high and low levels, respectively; in either case, populations of 5-6 log10 cfu g(-1) were reached. Detection limits of two rapid immunoassays, Reveal and VIP, without enrichment were determined to be 5-7 log10 cfu ml(-1). CONCLUSION: These results show the ability of E. coli O157:H7 to grow to high levels during sprouting; however, because these levels may be below detection limits, it is necessary to include enrichment when monitoring sprout production for E. coli O157:H7 by the rapid test kits. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that sprouts may harbor high levels of pathogens. The appropriate use of rapid test methods for pathogen monitoring during sprouting is indicated.     

Simple conjugation and outgrowth procedures for tagging vibrios with GFP, and factors affecting the stable expression of the gfp tag

AIM: Our goal was to develop a simple system for tagging wild-type marine bacteria with gfp. METHODS AND RESULTS: Escherichia coli strain CC118lambdapir carrying the conjugative helper plasmid pEVS104 and the gfp-containing plasmid pKV111 was used to transfer gfp to Vibrio recipients. Four different media were tested for their ability to support the growth of recipients, but not the E. coli donor, to allow powerful enrichment of gfp-tagged wild-type vibrios from mating mixes. Forty-three vibrio strains, representing 39 different species, were successfully tagged with gfp using the conjugative transfer from E. coli followed by selective outgrowth at 15 degrees C on ZoBell 2216E agar containing 0.5% sodium alginate. Using this outgrowth medium, colonies of GFP-expressing vibrio clones were detectable within 4 days. The percentage of visibly fluorescent cells in three representative GFP-tagged vibrios was higher at 15 degrees C than at 20 or 25 degrees C (c. 50% vs. 45% or 40%, respectively), and was also higher during the aerobic rather than the anaerobic culturing (c. 50% vs. 35%, respectively). CONCLUSIONS: We found a simple selective outgrowth technique that enabled us to isolate a wide variety of GFP-tagged marine vibrios following the conjugative transfer of gfp from E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Tagging cells with GFP and related fluorescent proteins is a powerful approach for investigating the bacteria in situ, particularly during the colonization of hosts. The simple and cost-effective outgrowth condition described in this study could be applied to construct a wide variety gfp-tagged marine bacteria.     

Low-energy (30 keV) carbon ion induced mutation spectrum in the LacZ alpha gene of M13 mp 18 double-stranded DNA

Double-stranded M13 mp 18 DNA was irradiated with 30 ke V carbon ions in dry state under vacuum to investigate the low-energy heavy ion induced mutation spectra. When the irradiated DNA was used to transfect Escherichia coli JM 105, 3.6-5.7-fold increases in mutation frequency were observed, in contrast to the spontaneous group. Sequences of the 92 induced mutants showed that the carbon ions in this study could induce an interesting mutation spectrum in the lacZ alpha gene. One-base mutations (96.8%) and base pair substitutions (56.4%) were predominant, most of which involved G:C base pairs (90.6%), especially G:C --> T:A transversions (49.6%) and G:C --> A:T transitions (39.6%). This is similar to the spectra induced by gamma-rays in the same ds M13, wild type E. coli system. We also found a considerable amount of carbon ion induced one-base deletion (38.5%) and the mutation sites distribution on the target lacZ alpha gene was obviously non-random. We compared this study with previous data employing gamma-rays to discuss the possible causes of the mutation spectrum.     

Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals.

Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress.     

Development of a capture/enrichment sandwich ELISA for the rapid detection of enteropathogenic and enterohaemorrhagic Escherichia coli O26 strains

AIMS: To improve the sensitivity of a monoclonal antibody (MAb 2F3) based enteropathogenic Escherichia coli (EPEC)/enterohaemorrhagic E. coli (EHEC) serogroup O26-specific sandwich ELISA (sELISA), using a capture/enrichment format of the assay. METHODS AND RESULTS: The sELISA utilized an EPEC/EHEC O26-specific MAb 2F3 as the capture reagent and an E. coli serogroup O26 lipopolysaccharide-specific polyclonal antibody in the development stage. Wells containing faeces test samples from bovine enteritis cases and agar colony sweep cultures from human diarrhoea cases, after a 2-h capture stage, were washed and enrichment of the captured cells was encouraged by addition of tryptone soya broth. After overnight incubation, the contents of each well were transferred to sterile wells and the sELISA completed. Any sELISA positive samples were then subcultured onto blood agar to recover and further characterize the positive cultures. The assay had a sensitivity of 10(3) CFU ml(-1). ELISA positive samples consisted of 21 (4.8%) of the 442 bovine and 19 (3.7%) of the 519 human samples tested, and ELISA positive EPEC/EHEC O26 strains were isolated from 11 and three of these samples respectively. CONCLUSION: The capture/enrichment method improved the sensitivity of a MAb-based sELISA for the detection of EPEC/EHEC O26 strains, and also contributed to an improved isolation rate of the organism from field samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a specific MAb in a capture/enrichment format of the sELISA, provides a prospectively suitable screening method for the detection of pathogenic bacteria from mixed culture samples.     

Comparison of a new system (Compactdry SCD) with conventional methods for quantitative urine cultures

AIMS: Compactdry SCD, a new quantitative, ready-to-use and self-diffusible dry medium sheet urine culture system, was compared with conventional methods to evaluate the results of quantitative urine cultures. METHODS & RESULTS: Compactdry SCD was tested on 25 urine specimens, and results compared with those of traditional culture methods. The results from Compactdry SCD analysis correlated well with those from the standard plate count (SPC) method. In fact, the correlation was stronger than that dipslide systems and SPC. Even low-count bacteriuria (< 103 cfu ml(-1) and mixed bacteriuria were detected by Compactdry SCD. CONCLUSIONS: The Compactdry SCD system provides results comparable to those obtained by SPC: simple interpretation, ease of use, long-term storage and good sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report suggesting that the Compactdry SCD system has many advantages over traditional quantitative urine culture methods and that it is both appropriate and practical for clinical use.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Isolation of shiga toxin-producing Escherichia coli (STEC) from foods using EHEC agar

Enterohaemorrhagic Escherichia coli (EHEC) agar was evaluated for its ability to recover one isolate of each of three serotypes (O157:H7, O26 and O113:H21) of shiga toxin-producing E. coli (STEC) from raw mince, pasteurized milk and salami after enrichment. The method detected around one colony-forming unit (cfu) in 25 ml in milk, but was less sensitive with salami, requiring 10-1000 cfu 25 g-1 (depending on serotype) for detection. In raw minced beef any enterohaemolysin-producing colonies were outnumbered by other colonies and only one of 12 enrichments yielded the inoculum serotype. Additional tests were conducted on 15 retail meat products. One 25-g sample of each product was processed as purchased, while another was inoculated with 157-185 cfu of a cocktail of E. coli O157, O113 and O26 cultures. Recovery was easily achieved with cooked meat products and salami. Recovery from raw minced meat was again difficult, but sometimes possible. Testing more suspect colonies than were tested in this study would presumably increase the sensitivity of the method.     

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.     

The recovery of heat-stressed Escherichia coli in lake water microcosms

C.-H. LIM AND K.P. FLINT. 1995. Escherichia coli was heat stressed at 55, 60 or 65°C in sterile flasks of lake water. After 6 h at these temperatures the viable count on nutrient agar had dropped below the limits of detection (1 colony in 100 ml). The flasks were transferred to a 15°C incubator and left for 7 d. Recovery of the stressed E. coli was shown to occur within 48 h at this temperature. Recovery also occurred in microcosms amended with 5o (v/v) synthetic sewage. The stressed E. coli multiplied in the amended but not in the unamended microcosms.