Understanding the cause of false negative β‐d‐glucuronidase reactions in culture media containing fermentable carbohydrate

Aims:  To explain the basis for false negative β‐glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Methods and Results:  Escherichia coli strains were assessed for their reactions in culture media containing a β‐d ‐glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of β‐d ‐glucuronidase at pH 5·0 and pH 7·2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme β‐d ‐glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose‐containing media expressed virtually no β‐d ‐glucuronidase activity at pH 5·0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. Conclusions:  E. coli that failed to produce green colonies on MLGA produced lower levels of β‐d ‐glucuronidase than did strains that formed green colonies, the difference being greater at pH 5·0 than pH 7·2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Significance and Impact of the Study:  Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme β‐d ‐glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7·2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).     

Evaluation of Colilert-18 for the detection of coliforms and Escherichia coli in tropical fresh water

AIMS: To evaluate the suitability of Colilert-18 in detecting Escherichia coli and total coliforms in tropical freshwater samples. METHODS AND RESULTS: Target organisms were isolated from yellow-fluorescent and yellow wells of Colilert-18/Quanti-Tray using m-TEC agar and m-ENDO LES agar respectively. All the selected isolates were first identified based on their fatty acid methyl ester profile. Isolates showing contradictory results to that of the Colilert-18 procedure were re-identified using API 20E strips. A total of 357 isolates, 177 from yellow-fluorescent wells and 180 from yellow wells, were identified. CONCLUSIONS: The false-positive and -negative rates for E. coli detection using Colilert-18 were 36.4% and 11%, respectively, while for coliform detection the false-positive rate was 10.3%. SIGNIFICANCE AND IMPACT OF THE STUDY: The high false-positive rate of Colilert-18, tempers its value for E. coli detection when used for tropical freshwater samples.     

Evaluation of a new medium for the enumeration of total coliforms and Escherichia coli in Japanese surface waters

Aim: A new medium, EC‐Blue‐10, containing chromogenic and fluorogenic substrates, KNO₃ and sodium pyruvate has been developed for the rapid simultaneous detection and enumeration of total coliforms and Escherichia coli in water. Methods and Results:  Two evaluations of EC‐Blue‐10 were carried out. Firstly, EC‐Blue‐10 was compared with Colilert‐MPN for 96 water samples using MPN for total coliforms and E. coli. Secondly, the detection of coliforms and E. coli were compared using 2400 tubes of EC‐Blue‐10 and Colilert‐MPN. The regression coefficients between EC‐Blue‐10 and Colilert‐MPN for total coliforms and E. coli were 0·91 and 0·89, respectively. For the detection results, the Cohen’s kappa values between the two media were 0·79 for coliforms and 0·72 for E. coli. Conclusions: EC‐Blue‐10 is almost same as Colilert‐MPN for the detection of coliforms and E. coli in surface waters. Further evaluation for EC‐Blue‐10 is needed to verify in different geographical areas. Significance and Impact of the Study: EC‐Blue‐10 is useful method for the rapid and simultaneous detection of total coliforms and E. coli in water sample.     

A medium detecting β-glucuronidase for the simultaneous membrane filtration enumeration of Escherichia coli and coliforms from drinking water

A medium for the single membrane enumeration of Escherichia coli and coliforms from potable water was developed by the modification of the standard UK membrane filtration medium. The medium, membrane-Lactose Glucuronide Agar (m-LGA), employs a chromogenic substrate for the detection of β-glucuronidase activity and sodium pyruvate to enhance recovery of chlorine-stressed coliforms. Escherichia coli identification was significantly improved on m-LGA with 98.6% of presumptive isolates confirming. Recovery of coliforms from drinking water samples was also significantly improved.     

Comparative evaluation of chromogenic agar CM1046 and mFC agar for detection of E. coli and thermotolerant coliform bacteria from water samples

Aims: The equivalence of Oxoid (CM 1046) Brilliance^(TM)E. coli/coliform selective agar to mFC agar, as used in the Australian/New Zealand Standard Method to detect thermotolerant coliforms and Escherichia coli in water samples, was assessed. Methods and Results: A total of 244 water samples were analysed in parallel over a 5‐month period. Sewage effluent samples (n = 131, sites = 43), freshwater (n = 62, sites = 18) and marine/brackish water samples (n = 51, sites = 23) were analysed. The Wilcoxon matched‐pairs signed‐ranks test showed a varying degree of statistical difference between the two methods. All matrices had a higher recovery in the trial method. Enterococci faecalis, Aeromonas spp. and Vibrio spp. did not grow on the CM1046 agar, and Pseudomonas aeruginosa and Enterobacter aerogenes were inhibited. Conclusions: The use of CM 1046 for the detection and enumeration of E. coli and thermotolerant coliforms in water samples is a suitable alternative to the AS/NZS Standard Method. Significance and Impact of the study: The use of CM1046 agar was less labour intensive and time consuming, as no secondary confirmation steps were required. Confirmed results could be reported within 24 h of sample analysis, as compared to 48 h with the reference method. Public health concerns can be addressed in a more efficient manner.     

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Characterization of cyanobacterial β‐carotene ketolase and hydroxylase genes in Escherichia coli,and their application for astaxanthin biosynthesis

Carotenoid biosynthesis is highly conserved and well characterized up to the synthesis of β‐carotene. Conversely, the synthesis of astaxanthin from β‐carotene is less well characterized. Regardless, astaxanthin is a highly sought natural product, due to its various industrial applications and elevated antioxidant capacity. In this article, 12 β‐carotene ketolase and 4 β‐carotene hydroxylase genes, isolated from 5 cyanobacterial species, are investigated for their function, and potential for microbial astaxanthin synthesis. Further, this in vivo comparison identifies and applies the most promising genetic elements within a dual expression vector, which is maintained in Escherichia coli. Here, combined overexpression of individual β‐carotene ketolase and β‐carotene hydroxylase genes, within a β‐carotene accumulating host, enables a 23.5‐fold improvement in total carotenoid yield (1.99 mg g^?1), over the parental strain, with >90% astaxanthin. Biotechnol. Bioeng. 2009;103: 944–955. © 2009 Wiley Periodicals, Inc.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Efficient experimental design and micro‐scale medium enhancement of 6‐deoxyerythronolide B production through Escherichia coli

The recent use of heterologous hosts to produce natural products has shown significant potential, although limitations still exist regarding optimal production titers. In this study, we utilize micro‐scale cultures and well‐defined screening methods to identify key medium components that influence the heterologous production of the complex polyketide 6‐deoxyerythronolide B (6dEB) through E. coli. It was determined that tryptone had a significant effect on 6dEB production and could supplement substrate requirements and improve recombinant protein levels of the essential deoxyerythronolide B synthase (DEBS) which catalyze 6dEB conversion. As a result, the study (1) demonstrates the feasibility of micro‐scale cultures to study E. coli 6dEB production and effectively model larger‐scale cultures; (2) identifies an enhanced medium which generates over 160 mg L^?1 6dEB (a 22‐fold improvement over current culture media); and (3) provides new insight and understanding related to the heterologous production of 6dEB from E. coli. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

New tools for recombinant protein production in Escherichia coli: A 5‐year update

The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost‐effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.     

Loop‐mediated isothermal amplification assay targeting the blaCTX‐M9 gene for detection of extended spectrum β‐lactamase‐producing Escherichia coli and Klebsiella pneumoniae

Extended‐spectrum β‐lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β‐lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL‐producing bacteria is necessary for identification, prevention and treatment. Loop‐mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL‐producing bacteria by a LAMP technique. ESBLs‐producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double‐disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla_CTX‐M9 gene was designed based on bla_CTX‐M9 from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder‐like patterns of band sizes from 226 bp of the bla_CTX‐M9 DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla_CTX‐M9 was greater than that of the PCR method by at least 10,000‐fold. These results showed that the LAMP primers specifically amplified only the bla_CTX‐M9 gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.     

α‐Haemolysin production,as a single factor,causes fulminant sepsis in a model of Escherichia coli‐induced bacteraemia

α‐Haemolysin (HlyA) from uropathogenic Escherichia coli has been demonstrated to be a significant virulence factor for ascending urinary tract infections. Once the E. coli reach the well‐vascularised kidneys, there is a high risk of bacteraemia and a subsequent septic host response. Despite this, HlyA has the potential to accelerate the host response both directly and via its ability to facilitate adenosine triphosphate release from cells. It has not been settled whether HlyA aggravates bacteraemia into a septic state. To address this, we used an E. coli strain in a model of acute urosepsis that was either transfected with a plasmid containing the full HlyA operon or one with deletion in the HlyA gene. Here, we show that HlyA accelerates the host response to E. coli in the circulation. Mice exposed to HlyA‐producing E. coli showed massively increased proinflammatory cytokines, a substantial fall in circulating thrombocytes, extensive haematuria, and intravascular haemolysis. This was not seen in mice exposed to either E. coli that do not secrete HlyA or vehicle controls. Consistent with the massive host response to the bacteria, the mice exposed to HlyA‐producing E. coli died exceedingly early, whereas mice exposed to E. coli without HlyA production and vehicle controls survived the entire observation period. These data allow us to conclude that HlyA is a virulence factor that accelerates a state of bacteraemia into fulminant sepsis in a mouse model.     

Molecular characterization of the β‐lactamases in Escherichia coli and Klebsiella pneumoniae from a tertiary care hospital in Riyadh,Saudi Arabia

The widespread use of antimicrobials has increased the occurrence of multidrug resistant microbes. The commonest mechanism of antimicrobial resistance in Enterobacteriaceae is production of β‐lactamases such as metallo‐β‐lactamases (MBL) and extended spectrum β‐lactamases (ESBL). Few studies have used a molecular approach to characterize the prevalence of β‐lactamases. Here, the prevalence of different β‐lactamases was characterized by performing three multiplex PCRs targeting genes similar to those described in earlier publications. Antimicrobial susceptibility tests for all isolates were performed using the agar dilution method. β‐lactamase was detected in 72% of the isolates, the detection rate being 64% in 2011 and 75% in 2012. The isolates were highly resistant to carbapenems such as meropenem and imipenem and susceptible to colistin and tigecycline. In this study, 22% of isolates contained both MBL and ESBL. ESBL was detected more frequently in Escherichia coli isolates, whereas carbapenemase was detected more frequently in Klebsiella pneumoniae isolates. These findings suggest the spread of multi‐resistant ESBL and MBL producers in the community. Our results have implications for patient treatment and also indicate the need for increased surveillance and molecular characterization of isolates.