Antimicrobial‐resistant faecal Escherichia coli in wild mammals in central Europe: multiresistant Escherichia coli producing extended‐spectrum beta‐lactamases in wild boars

Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli in populations of wild mammals in the Czech Republic and Slovakia. Methods and Results: Rectal swabs or faeces collected during 2006–2008 from wild mammals were spread on MacConkey agar and MacConkey agar containing 2 mg l^?1 of cefotaxime. From plates with positive growth, one isolate was recovered and identified as E. coli. Susceptibility to 12 antibiotics was tested using the disk diffusion method. Resistance genes, class 1 and 2 integrons and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). Extended‐spectrum beta‐lactamases (ESBL) were further characterized by DNA sequencing, macrorestriction profiling and determination of plasmid sizes. Plasmid DNA was subjected to EcoRV digestion, transferability by conjugation and incompatibility grouping by multiplex PCR. The prevalence of resistant isolates was 2% in small terrestrial mammals (rodents and insectivores, n_E. coli = 242), 12% in wild ruminants and foxes (n_E. coli = 42), while no resistant isolates were detected in brown bears (n_E. coli = 16). In wild boars (Sus scrofa) (n_E. coli = 290), the prevalence of resistant isolates was 6%. Class 1 and 2 integrons with various gene cassettes were recorded in resistant isolates. From wild boars, five (2%, n_{rectal smears} = 293) multiresistant isolates producing ESBL were recovered: one isolate with bla_CTX‐M‐1 + bla_TEM‐1, three with bla_CTX‐M‐1 and one with bla_TEM‐52b. The bla_CTX‐M‐1 genes were carried on approx. 90 kb IncI1 conjugative plasmids. Conclusions: Antibiotic‐resistant E. coli occured in populations of wild mammals in various prevalences. Significance and Impact of the Study: Wild mammals are reservoirs of antibiotic‐resistant E. coli including ESBL‐producing strains which were found in wild boars.     

Prevalence and diversity of class 1 integrons and resistance genes in antimicrobial-resistant Escherichia coli originating from beef cattle administered subtherapeutic antimicrobials

Aims: To characterize class 1 integrons and resistance genes in tetracycline‐resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control). Methods and Results: Tetracycline‐resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher (P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 (r = 0·44), aadA1 (r = 0·61), cat (r = 0·58) and intI1(r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays. Conclusions: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline‐resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline‐resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons. Significance and Impact of the Study: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline‐resistant E. coli isolated from feedlot cattle.     

Occurrence and transmission of class 1 and 2 integrons among phenotypic highly ampicillin-resistant avian <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from Pakistan

Thirty four avian Escherichia coli isolates were collected from different cities of Punjab province, Pakistan during 2008–2009. Twenty five phenotypic highly ampicillin-resistant (MICs ≥ 256 μg/ml) avian E. coli strains were selected for the investigation of occurrence and transmission of class 1, 2 and 3 integrons and β-lactamase genes. Amoxicillin, sulfonamide, trimethoprim, enrofloxacin, pefloxacin and tetracycline were the most common phenotypic resistant among ampicillin-resistant avian E. coli strains. Integrons and β-lactamase were found 60 and 72% respectively. Class 1 and 2 integrons were found 52 and 8%, while class 3 integrons were not found in all strains. All class 1 positive strains had variable fragments associated with gene cassettes dfrA7, dfrA1-aadA1, aadA1, aadA22 and dfrA12-orfF-aadA2 respectively, which confer resistance to trimethoprim and streptomycin. Class 2-positive strains had similar gene cassettes array dfrA1-sat1-aadA1 conferring resistance to trimethoprim, streptothricin and spectinomicin/streptomycin. Integrons are frequently found in β-lactamase positive isolates and widely disseminate multidrug resistance genes but they do not play role in the spreading of β-lactamase genes. Class 1 integrons gene cassette aadA22 is reported for the first time in avian E. coli. Findings of this study may provide important and useful information reflecting specific antibiotic selective pressure in Punjab province, Pakistan.     

Distribution of integron-associated trimethoprim–sulfamethoxazole resistance determinants among Escherichia coli from humans and food-producing animals

Aims: To compare the distribution of integrons and trimethoprim–sulfamethoxazole resistance genes among Escherichia coli isolates from humans and food‐producing animals. Methods and Results: A collection of 174 multidrug‐resistant E. coli isolates obtained from faecal samples of food‐producing animals (n = 64) and humans (n = 59), and patients with urinary tract infections (n = 51) in Hong Kong during 2002–2004 were studied. The strains were analysed for their phylogenetic groups, the presence of sul genes (sul1 and sul2), integrons (intl1 and intl2) and class 1 integron‐associated dfr cassette genes by PCR, restriction enzyme analysis and sequencing. Integrons were identified in 110 (63·2%) isolates. The prevalence of integrons was significantly different according to the specimen sources (animal faecal 84·4%, human faecal 67·8% and human urinary 31·4%) and phylogenetic groups (B1 80·8%, A 77·6%, D 54·1% and B2 11·5%). Faecal isolates (both human and animal) are more likely to belong to group A and B1. In contrast, most urinary isolates were either groups B2 and D. Among dfr containing isolates, dfrA1 and dfrA12 were almost exclusively found in strains of phylogenetic groups A and B1; and were present in animal and human faecal isolates. In contrast, dfrA17 was found in both faecal and urinary isolates and comprised strains from all phylogenetic groups. The sul1 and sul2 genes were equally prevalent among the isolates irrespective of the specimen source and phylogenetic group status. Pulsed‐field gel electrophoresis analysis of isolates with identical cassette genes showed that they were genetically diverse. Conclusions: More animal faecal isolates carry class 1 integrons than human faecal and human urinary isolates, and the distribution of phylogenetic groups is common across animal and human faecal isolates but different from human urinary isolates. Significance and Impact of the Study: Commensal isolates from food‐producing animals are an important reservoir for integrons carrying antibiotic resistance genes.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

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.     

Establishing microbial co‐cultures for 3‐hydroxybenzoic acid biosynthesis on glycerol

Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment‐friendly, and cost‐effective production of these value‐added products without the reliance on petroleum. In this study, rationally designed E. coli–E. coli co‐culture systems were established for converting glycerol to 3‐hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. The co‐culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co‐culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with the E. coli mono‐culture, the optimized co‐culture showed 5.3‐fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co‐culture engineering for addressing the challenges of aromatic compound biosynthesis.     

Occurrence and molecular characteristics of antimicrobial resistance of Escherichia coli from broilers,pigs and meat products in Thailand and Cambodia provinces

Nine hundred and forty‐one samples were collected in Sa Keao, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) from July 2014 to January 2015. A total of 667 Escherichia coli isolates (381 isolates from Sa Keao and 286 isolates from Banteay Meanchey) were obtained and examined for antimicrobial susceptibility, class 1 integrons, ESBL genes and horizontal transfer of resistance determinants. Prevalence of E. coli in pig and broiler carcass samples from slaughterhouses and fresh markets was 36–85% in Sa Keao and 11–69% in Banteay Meanchey. The majority of these isolates were multidrug resistant (75.3%). Class 1 integrons were common in both Thai (47%) and Cambodian (62%) isolates, of which four resistance gene cassette arrays including aadA1, dfrA1‐aadA1, dfrA12‐aadA2 and aadA2‐linF were identified. Class 1 integrons in two broiler isolates from Sa Keao (dfrA12‐aadA2) and one broiler isolate from Banteay Meanchey (dfrA1‐aadA1) were horizontally transferable. Sixteen isolates were confirmed to be ESBL‐producing strains with ESBL gene bla_CTX‐M‐15, broad spectrum β‐lactamase gene bla_TEM‐1 and the AmpC gene bla_CMY‐2 being detected. The bla_TEM‐1 gene was most prevalent and located on a conjugative plasmid.     

VIM‐1 carbapenemase‐producing Escherichia coli in gulls from southern France

Acquired carbapenemases currently pose one of the most worrying public health threats related to antimicrobial resistance. A NDM‐1‐producing Salmonella Corvallis was reported in 2013 in a wild raptor. Further research was needed to understand the role of wild birds in the transmission of bacteria resistant to carbapenems. Our aim was to investigate the presence of carbapenem‐resistant Escherichia coli in gulls from southern France. In 2012, we collected 158 cloacal swabs samples from two gull species: yellow‐legged gulls (Larus michahellis) that live in close contact with humans and slender‐billed gulls (Chroicocephalus genei) that feed at sea. We molecularly compared the carbapenem‐resistant bacteria we isolated through culture on selective media with the carbapenem‐susceptible strains sampled from both gull species and from stool samples of humans hospitalized in the study area. The genes coding for carbapenemases were tested by multiplex PCR. We isolated 22 carbapenem‐resistant E. coli strains from yellow‐legged gulls while none were isolated from slender‐billed gulls. All carbapenem‐resistant isolates were positive for bla_VIM‐1 gene. VIM‐1‐producing E. coli were closely related to carbapenem‐susceptible strains isolated from the two gull species but also to human strains. Our results are alarming enough to make it urgently necessary to determine the contamination source of the bacteria we identified. More generally, our work highlights the need to develop more bridges between studies focusing on wildlife and humans in order to improve our knowledge of resistant bacteria transmission routes.     

Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline‐amended and ciprofloxacin‐amended growth media

Aims: The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants. Methods and Results: Numerous bacterial strains were isolated from three municipal wastewater treatment facilities on tetracycline‐ (n = 164) and ciprofloxacin‐amended (n = 65) growth media. These bacteria were then characterized with respect to their resistance to as many as 10 different antimicrobials, the presence of 14 common genes that encode resistance to tetracycline, the presence of integrons and/or the ability to transfer resistance via conjugation. All of the characterized strains exhibited some degree of multiple antimicrobial resistance, with nearly 50% demonstrating resistance to every antimicrobial that was tested. Genes encoding resistance to tetracycline were commonly detected among these strains, although intriguingly the frequency of detection was slightly higher for the bacteria isolated on ciprofloxacin‐amended growth media (62%) compared to the bacteria isolated on tetracycline‐amended growth media (53%). Class 1 integrons were also detected in 100% of the queried tetracycline‐resistant bacteria and almost half of the ciprofloxacin‐resistant strains. Conjugation experiments demonstrated that at least one of the tetracycline‐resistant bacteria was capable of lateral gene transfer. Conclusions: Our results demonstrate that multiple antimicrobial resistance is a common trait among tetracycline‐resistant and ciprofloxacin‐resistant bacteria in municipal wastewater. Significance and Impact of the Study: These organisms are potentially important in the proliferation of antimicrobial resistance because they appear to have acquired multiple genetic determinants that confer resistance and because they have the potential to laterally transfer these genetic determinants to strains of clinical importance.     

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Herein, we report the development of a microbial bioprocess for high‐level production of 5‐aminolevulinic acid (5‐ALA), a valuable non‐proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5‐ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5‐ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl‐CoA for enhanced 5‐ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high‐level 5‐ALA biosynthesis from glycerol (~30 g L^?1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L^?1 (36.9% of the theoretical maximum yield) and 6.93 g L^?1 (50.9% of the theoretical maximum yield) 5‐ALA, respectively. This study represents one of the most effective bio‐based production of 5‐ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio‐based production.     

A comprehensive genome‐scale reconstruction of Escherichia coli metabolism—2011

The initial genome‐scale reconstruction of the metabolic network of Escherichia coli K‐12 MG1655 was assembled in 2000. It has been updated and periodically released since then based on new and curated genomic and biochemical knowledge. An update has now been built, named iJO1366, which accounts for 1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. iJO1366 was (1) updated in part using a new experimental screen of 1075 gene knockout strains, illuminating cases where alternative pathways and isozymes are yet to be discovered, (2) compared with its predecessor and to experimental data sets to confirm that it continues to make accurate phenotypic predictions of growth on different substrates and for gene knockout strains, and (3) mapped to the genomes of all available sequenced E. coli strains, including pathogens, leading to the identification of hundreds of unannotated genes in these organisms. Like its predecessors, the iJO1366 reconstruction is expected to be widely deployed for studying the systems biology of E. coli and for metabolic engineering applications.     

Association between the Presence of Class 1 Integrons, Virulence Genes, and Phylogenetic Groups of Escherichia coli Isolates from River Water

Ninety-six class 1 integron-positive and 96 integron-negative Escherichia coli isolates cultured from the water of the Warta River, Poland, were characterized for their phylogenetic group affiliation and for the presence of genes associated with virulence. Most strains belonged to phylogenetic group A, but phylogenetic group affiliation was not related with the presence of integrons. The occurrence of heat-stable toxin gene of enterotoxigenic E. coli, S fimbriae subunit gene sfaS, and siderophore receptor genes, fyuA and iutA, was associated with the presence of class 1 integrons. Moreover, virulence factor score (the total number of virulence-associated genes) was associated with the presence of integrons in groups. The results bring new insight into relations between the presence of integrons in E. coli, virulence traits, as well as phylogenetic group affiliation.     

Whole‐cell‐based CYP153A6‐catalyzed (S)‐limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL

Living microbial cells are considered to be the catalyst of choice for selective terpene functionalization. However, such processes often suffer from side product formation and poor substrate mass transfer into cells. For the hydroxylation of (S)‐limonene to (S)‐perillyl alcohol by Pseudomonas putida KT2440 (pGEc47ΔB)(pCom8‐PFR1500), containing the cytochrome P450 monooxygenase CYP153A6, the side products perillyl aldehyde and perillic acid constituted up to 26% of the total amount of oxidized terpenes. In this study, it is shown that the reaction rate is substrate‐limited in the two‐liquid phase system used and that host intrinsic dehydrogenases and not CYP153A6 are responsible for the formation of the undesired side products. In contrast to P. putida KT2440, E. coli W3110 was found to catalyze perillyl aldehyde reduction to the alcohol and no oxidation to the acid. Furthermore, E. coli W3110 harboring CYP153A6 showed high limonene hydroxylation activities (7.1 U g). The outer membrane protein AlkL was found to enhance hydroxylation activities of E. coli twofold in aqueous single‐phase and fivefold in two‐liquid phase biotransformations. In the latter system, E. coli harboring CYP153A6 and AlkL produced up to 39.2 mmol (S)‐perillyl alcohol L within 26 h, whereas no perillic acid and minor amounts of perillyl aldehyde (8% of the total products) were formed. In conclusion, undesired perillyl alcohol oxidation was reduced by choosing E. coli's enzymatic background as a reaction environment and co‐expression of the alkL gene in E. coli represents a promising strategy to enhance terpene bioconversion rates. Biotechnol. Bioeng. 2013; 110: 1282–1292. © 2012 Wiley Periodicals, Inc.     

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.     

Prevalence and antimicrobial resistance in Salmonella enterica isolated from broiler chickens,pigs and meat products in Thailand–Cambodia border provinces

This study aimed to examine the prevalence and antimicrobial resistance (AMR) of Salmonella isolates from broiler chickens, pigs and their associated meat products in the Thailand–Cambodia border provinces. A total of 941 samples were collected from pigs and broiler chickens at slaughter houses and from carcasses at local fresh markets in Sa Kaeo, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) in 2014 and 2015. From these samples, 345 Salmonella isolates were collected from Sa Keao (n = 145; 23%) and Banteay Meanchey (n = 200; 47%) and assayed for antimicrobial susceptibility, class 1 integrons and extended‐spectrum β‐lactamase (ESBL) genes. Serovars Typhimurium (29%) and Rissen (29%) were the most common serotypes found in Thai and Cambodian isolates, respectively. Multidrug resistance was detected in 34% and 52% of isolates from Sa Keao and Banteay Meanchey, respectively. The majority of the Thai isolates were resistant to ampicillin (72.4%), whereas most Cambodian isolates were resistant to sulfamethoxazole (71%). Eleven isolates from Sa Keao and 44 from Banteay Meanchey carried class 1 integrons comprising resistance gene cassettes. The most common gene cassette array was dfrA12‐aadA2 (61.1%). Six isolates were ESBL producers. The β‐lactamase genes found included bla_TEM‐1, bla_CTX‐M‐55 and bla_CMY‐2. Some of these class 1 integrons and ESBL genes were located on conjugative plasmid. In conclusion, multidrug‐resistant Salmonella are common in pigs, chickens and their products in the Thailand–Cambodia border provinces. Our findings indicate that class 1 integrons play a role in spread of AMR in the strains in this study.     

Effective antimicrobial activity of Cbf‐K16 and Cbf‐A7A13 against NDM‐1‐carrying Escherichia coli by DNA binding after penetrating the cytoplasmic membrane in vitro

New Delhi metallo‐beta‐lactamase‐1(NDM‐1)‐carrying isolates, which are resistant to most clinical used antibiotics except for tigecycline and colistin, have been found worldwide. Cathelicidin‐BF (BF‐30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Cbf‐K₁₆ and Cbf‐A₇A₁₃ were obtained by mutating Lys¹⁶, Ala⁷, and Ala¹³ of BF‐30, respectively. To investigate their antimicrobial activities against NDM‐1 carrying bacteria, recombinant Escherichia coli BL21 (DE3)‐NDM‐1 with high NDM‐1 activity was constructed by inserting the Klebsiella pneumoniae NDM‐1 gene (GenBank accession no. HQ328085) into a pET28a vector and transforming it into E. coli BL21 (DE3). The peptides showed effective antimicrobial activities against NDM‐1‐carrying E. coli, and the minimum inhibitory concentrations of Cbf‐K₁₆ and Cbf‐A₇A₁₃ were only 4 and 8 µg/ml, whereas those of minimum bactericidal concentrations were 8 and 16 µg/ml, respectively. A time course experiment showed that colony forming unit counts rapidly decreased, and bacteria were thoroughly eliminated within 3 and 6 h by the Cbf‐K₁₆ and Cbf‐A₇A₁₃ treatments, respectively. The peptides penetrated the bacterial cell membrane and enabled β‐galactosidase leakage, and caused the cytoplasmic membrane to become permeable, and finally bound to the DNA. The genomic DNA of E. coli was completely unable to migrate on an agarose gel after Cbf‐K₁₆ treatment (8 µg/ml). These data demonstrated that Cbf‐K₁₆ and Cbf‐A₇A₁₃ possess effective antimicrobial activity against drug‐resistant strains, including NDM‐1 carrying E. coli BL21 (DE3)‐NDM‐1, by binding to DNA after penetrating the cytoplasmic membrane in vitro, which may have potential therapeutic value for the treatment of NDM‐1‐carrying bacterial infections. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose‐Derived Sugars

Currently, microbial conversion of lignocellulose‐derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome‐scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole‐genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations’ involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next‐generation biofuels.     

Evaluation of the double‐disk synergy test for New Delhi metallo‐β‐lactamase‐1 and other metallo‐β‐lactamase producing gram‐negative bacteria by using metal‐ethylenediaminetetraacetic acid complexes

New Delhi metallo‐β‐lactamase‐1 (NDM‐1), one of the metallo‐β‐lactamases (MBLs), has been identified from clinical isolates worldwide. Rapid detection of NDM‐1 producers is necessary to prevent their dissemination. Seven types of EDTA complexes were evaluated as MBL inhibitors in double‐disk synergy tests (DDSTs), resulting in detection of the first isolate of NDM‐1‐producing Escherichia coli (NDM‐1 Dok01) in Japan. NDM‐1 Dok01 was detected when EDTA magnesium disodium salt tetrahydrate (Mg‐EDTA), EDTA calcium disodium salt dihydrate, EDTA cobalt disodium salt tetrahydrate and EDTA copper disodium salt tetrahydrate were used as MBL inhibitors. The sensitivity and specificity of DDSTs using Mg‐EDTA for 75 MBL producers and 25 non‐MBL producers were 96.0% and 100%, respectively. These findings indicate that the DDST method using Mg‐EDTA can detect MBL‐producing strains, including NDM‐1 producers.