Coli DH10B Research Articles

1437. Biochemical characterization of L1 and L2 β-lactamases from clinical isolates of Stenotrophomonas maltophilia

Background Stenotrophomonas maltophilia is a Gram-negative, non-fermenting opportunistic pathogen. Two β-lactamases provide intrinsic resistance to β-lactams: a class B Metallo- β-lactamase L1, and a class A serine β-lactamase (SβL) L2. Recently, we described novel variants of the L1 and L2 in a collection of clinical S. maltophilia isolates collected in the US, and showed through analyses of the amino acid sequences that L1 and L2 grouped into 4 (A-D, B, C, and E) and 2 (A and D) clades, respectively. We aimed to characterize the new L1 and L2 clinical variants biochemically.MethodsRepresentative blaL1 and blaL2 genes from each of the identified clades were cloned into pBC-SK and pET24 vectors and transformed into E. coli DH10B and BL21 (DE3) cells, respectively. Minimal inhibitory concentrations (MICs) were determined using CLSI approved methods. Cell-based assays and biochemical characterization performed on purified enzymes, including circular dichroism (CD), thermal stability, and steady-state kinetics assays, were performed.ResultsSusceptibility testing results using DH10-B E. coli strains expressing the L1 and L2 variants are shown in Table 1. Remarkably, while all L1 variants confer the same level of resistance to carbapenems, L2B conferred higher MICs to 3rd gen cephalosporins and aztreonam than L2D. Kinetics assays confirmed differences in the kcat of both enzymes to ceftazidime (32s-1 for L2B vs. 7s-1 for L2D) and avibactam inhibition constant Ki (1.7 μM for L2B vs. 4.5 μM for L2D). Structurally, L2B and L2D present distinctive CD spectra and thermal stabilities (ΔTm 5°C).Table 1 ConclusionAs opposed to the L2 variants, our results suggest that the L1 variants may not be functionally nor structurally different. Differences between L2B and L2D might have arisen due to the use of cephalosporins and SβL inhibitors. Further experiments are on the way to determine the structural basis of these observations and the implication of these for the design of novel β-lactamase inhibitors.Disclosures Krisztina M. Papp-Wallce, PhD, Entasis (Grant/Research Support)Merck (Grant/Research Support)Venatorx (Grant/Research Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Cloning and expression of pigC gene in Escherichia coli

Prodigiosin (Pg), which is particularly of interest because of anticancer and antimicrobial activities, can be produced through the PigC-catalyzed condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Therefore, the PigC protein plays an important role in prodigiosin biosynthetic pathway. However, studies related to PigC protein have not been carried out in Vietnam yet. In this work, the pigC gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Using PCR and universal primers, we amplified a fragment of 3 kb covering entire coding region of the pigC gene from Serratia sp. strain M5. The pigC gene was inserted into pJET1.2 vector, and then transformed into E. coli DH10B. The sequence of a recombinant vector pJET1.2/pigC was evaluated by using whole colony PCR amplification. Sequence alignment results revealed that the obtained pigC gene possesses 71.5% and 75.4% of nucleotide identity in comparison with two strains, Serratia 39006 and Serratia sp. AS9 published in GenBank with their respective accession numbers of AJ833001 and CP002773. The recombinant vector pJET1.2/pigC was used to reamplify pigC, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing recombinant vector pET22b/pigC was expressed in the auto-induced medium. The presence of PigC protein in the lysate was identified as a 100 kDa band through Western Blot analysis using anti his-tag antibody. Afterward, the PigC protein was purified by Ni-NTA column, and its expression level was quantified through SDS-PAGE analysis. The results of our study provide a potential material for producing prodigiosin from recombinant protein in Vietnam.

Characterization of Inducible ccdB Gene as a Counterselectable Marker in Escherichia coli Recombineering.

Recombineering is a homologous-based DNA cloning and modification technique. Recombineering-mediated chromosomal gene knock-in usually involves a selectable/counterselectable cassette. Though a variety of selectable/counterselectable cassettes were developed; however, a specifically designed gene deletion strain or minimal medium is often required. Herein, we describe a novel selectable/counterselectable cassette Plac-ccdB-aacC1 in which aacC1 (gentamicin resistance gene) is used as the selectable marker for the homologous arm-flanked cassette knock-in, while the IPTG inducible ccdB gene is used as the counterselectable marker for chromosomal gene knock-in. The counterselection is achieved via supplementing 1mM IPTG in the LB agar medium. An oligonucleotide designed to evade the mismatch repair system was utilized to engineer an Escherichia coli DH10B-derived gyrA462 strain that was used to as the host for the plasmid harboring the Plac-ccdB-aacC1 cassette. By using the Plac-ccdB-aacC1 cassette, a linear-linear homologous recombination (LLHR) system was generated by knocking a 6.2kb araC-PBAD-redγ-recET-recA DNA fragment into the E. coli DH10B chromosome. The functional of the LLHR recombineering system was characterized by cloning of the target DNA from PCR product as well as from the genomic DNA mixture. The Plac-ccdB-aacC1 cassette will be a useful tool in E. coli recombineering.

Plasmids containing the same origin of replication are useful tools to perform biotechnological studies in Pseudomonas putida U and in E. coli DH10B

Plasmids containing the same origin of replication are useful tools to perform biotechnological studies in Pseudomonas putida U and in E. coli DH10B

High Heterogeneity of Escherichia coli Sequence Types Harbouring ESBL/AmpC Genes on IncI1 Plasmids in the Colombian Poultry Chain.

BackgroundEscherichia coli producing ESBL/AmpC enzymes are unwanted in animal production chains as they may pose a risk to human and animal health. Molecular characterization of plasmids and strains carrying genes that encode these enzymes is essential to understand their local and global spread.ObjectivesTo investigate the diversity of genes, plasmids and strains in ESBL/AmpC-producing E. coli from the Colombian poultry chain isolated within the Colombian Integrated Program for Antimicrobial Resistance Surveillance (Coipars).MethodsA total of 541 non-clinical E. coli strains from epidemiologically independent samples and randomly isolated between 2008 and 2013 within the Coipars program were tested for antimicrobial susceptibility. Poultry isolates resistant to cefotaxime (MIC ≥ 4 mg/L) were screened for ESBL/AmpC genes including blaCTX-M, blaSHV, blaTEM, blaCMY and blaOXA. Plasmid and strain characterization was performed for a selection of the ESBL/AmpC-producing isolates. Plasmids were purified and transformed into E. coli DH10B cells or transferred by conjugation to E. coli W3110. When applicable, PCR Based Replicon Typing (PBRT), plasmid Multi Locus Sequence Typing (pMLST), plasmid Double Locus Sequence Typing (pDLST) and/or plasmid Replicon Sequence Typing (pRST) was performed on resulting transformants and conjugants. Multi Locus Sequence Typing (MLST) was used for strain characterization.ResultsIn total, 132 of 541 isolates were resistant to cefotaxime and 122 were found to carry ESBL/AmpC genes. Ninety-two harboured blaCMY-2 (75%), fourteen blaSHV-12 (11%), three blaSHV-5 (2%), five blaCTX-M-2 (4%), one blaCTX-M-15 (1%), one blaCTX-M-8 (1%), four a combination of blaCMY-2 and blaSHV-12 (4%) and two a combination of blaCMY-2 and blaSHV-5 (2%). A selection of 39 ESBL/AmpC-producing isolates was characterized at the plasmid and strain level. ESBL/AmpC genes from 36 isolates were transferable by transformation or conjugation of which 22 were located on IncI1 plasmids. These IncI1 plasmids harboured predominantly blaCMY-2 (16/22), and to a lesser extend blaSHV-12 (5/22) and blaCTX-M-8 (1/22). Other plasmid families associated with ESBL/AmpC-genes were IncK (4/33), IncHI2 (3/33), IncA/C (2/33), IncΒ/O (1/33) and a non-typeable replicon (1/33). Subtyping of IncI1 and IncHI2 demonstrated IncI1/ST12 was predominantly associated with blaCMY-2 (12/16) and IncHI2/ST7 with blaCTX-M-2 (2/3). Finally, 31 different STs were detected among the 39 selected isolates.ConclusionsResistance to extended spectrum cephalosporins in E. coli from Colombian poultry is mainly caused by blaCMY-2 and blaSHV-12. The high diversity of strain Sequence Types and the dissemination of homogeneous IncI1/ST12 plasmids suggest that spread of the resistance is mainly mediated by horizontal gene transfer.

Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect

This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

Detection of the plasmid-mediated KPC-2 carbapenem-hydrolysing enzyme in three unusual species of the Enterobacteriaceae family in Israel

Sir, Carbapenem-resistant Enterobacteriaceae (CRE) pose a major threat to public health worldwide. One of the most frequently encountered mechanisms is the Ambler class A Klebsiella pneumoniae carbapenemase (KPC)-type enzyme. Since the initial report of a KPC-producing K. pneumoniae in 2001, these organisms have spread worldwide, mainly due to the spread of a single successful clone, sequence type 258 (ST258). In addition, KPC enzymes have been detected in multiple genera and species of the Enterobacteriaceae family, including Salmonella spp., Klebsiella oxytoca, Enterobacter spp., Citrobacter freundii, Escherichia coli, Proteus mirabilis and Serratia marcescens. The blaKPC gene is typically located on the Tn4401 transposon, which may be carried on various plasmids. Transfer of mobile genetic elements may serve as a vector for inter-species transmission and thus poses a serious challenge for infection control measures. In this report, we describe three unusual Enterobacteriaceae species carrying the blaKPC-2 gene: Leclercia adecarboxylata, Kluyvera cryocrescens and Kluyvera ascorbata. The molecular and microbiological characteristics of these isolates are discussed. All strains in this research were isolated from rectal surveillance cultures of patients from three medical centres: Laniado Medical Center (LMC), Tel Aviv Sourasky Medical Center (TASMC) and Rambam Medical Center (RMC). Table 1 summarizes the molecular and microbiological characteristics of the isolates. Bacterial identification was performed with the VITEK-2 system using GN-ID and GN09 cards (bioMerieux, Marcy l’Etoile, France) and confirmed by 16S rDNA sequencing. We identified the isolates as K. cryocrescens (two isolates), K. ascorbata (one isolate) and L. adecarboxylata (one isolate). All isolates carried the blaKPC-2 gene. 5 Plasmid DNAs purified from the four isolates were transformed into E. coli DH10B. Transformants were selected on lysogeny broth (LB) agar supplemented with 100 mg/L ampicillin and confirmed by PCR. Susceptibility to ertapenem, imipenem and meropenem was tested by agar dilution and interpreted based on the 2012 CLSI criteria. All isolates were resistant to ertapenem, imipenem and meropenem. In the transformants, the MICs of ertapenem, imipenem and meropenem were 2–8, 4–8 and 2–4 mg/L, respectively. Determination of plasmid sizes was done as previously described, revealing various sizes of the blaKPC-2-harbouring plasmids. The plasmid incompatibility group (Inc) was identified as IncN. The blaKPC-2 gene was located on the Tn4401 transposon and was typed by sequencing as Tn4401c. The presence of the Tn4401c variant in these isolates is in contrast with isolates belonging to the Israeli epidemic clone, K. pneumoniae ST258, which harbours the Tn4401a variant on the pKpQIL plasmid, which belongs to the IncFII incompatibility group. It was shown previously that isolates harbouring Tn4401c had a 10-fold reduction in their blaKPC expression. This also correlated with lower MICs of carbapenems. The identical plasmid incompatibility group and Tn4401 type in all isolates suggests horizontal inter-species transmission of the Tn4401c element between different plasmids of the same type, as recently described by Mathers et al. Kluyvera spp. and L. adecarboxylata are present ubiquitously in the environment in water and soil and were also described as part of the normal gastrointestinal flora in humans. Human infections with these bacteria are rare. Several cases of L. adecarboxylata infections in humans have been reported, especially in immunocompromised patients. In such patients, L. adecarboxylata was reported to cause bacteraemia, sepsis, peritonitis, cellulitis, endocarditis and cholecystitis. Reported human infections caused by Kluyvera spp. include bacteraemia, soft tissue infections, intra-abdominal abscesses and urinary tract infections. Recently, Ribeiro et al. reported on the first isolate of a carbapenem-resistant Kluyvera georgiana possessing the blaKPC-2 gene. Although clinical infections were absent in all of these cases, the spread of blaKPC-carrying plasmids into unusual Enterobacteriaceae species is of concern from an infection control perspective. These bacteria, though commonly regarded as avirulent, could serve as a reservoir for the spread of resistance genes between different species, leading to dissemination of resistant bacteria between patients and in the environment.

Characterization of Escherichia coli urinary tract infection isolates in remote northern Saskatchewan communities: the Northern Antibiotic Resistance Partnership

Antimicrobial resistance is a growing concern especially in many remote northern communities of Canada where antimicrobials are liberally used. In this study, 1418 Escherichia coli urinary tract infection (UTI) isolates, obtained over a 2.5-year period (October 2005–March 2008), from 3 remote northern sites in Saskatchewan, Canada, were identified. Antimicrobial susceptibility testing of the first 544 clinically significant isolates revealed high prevalence of resistance to trimethoprim–sulfamethoxazole (TMP-SXT) (30.7%). Pulsed-field gel electrophoresis (PFGE) of 165 TMP-SXT–resistant isolates revealed a heterogeneous population. Multilocus sequence typing identified 7 STs from 9 identified PFGE clusters, which included separate PFGE clusters of fluoroquinolone-resistant and -susceptible ST131 isolates. The majority of TMP-SXT–resistant isolates (85.5%) were found to carry class 1 integrons, and plasmids from 62 (81%) of 77 representative isolates were successfully transformed into E. coli DH10B. Overall, ampicillin was the most common plasmid-encoded resistance phenotype transferred with TMP-SXT at 60% (37/62). Further characterization of 52 plasmids by restriction fragment length polymorphism and replicon typing revealed the presence of many plasmid lineages, suggesting that the elevated rates of TMP-SXT resistance in these communities are most likely attributed to the horizontal transfer of class 1 integrons. Results from this study emphasize the importance of continued surveillance of remote northern communities in order to optimize the efficacy of empiric UTI treatment.

Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.

Plasmid-encoded OXA-48 carbapenemase in Escherichia coli from Israel

Sir, Carbapenem resistance among Enterobacteriaceae in Israel emerged in 2004 and was observed mainly in Klebsiella pneumoniae, but also in Enterobacter species and Escherichia coli. Since its emergence, carbapenem resistance in these species, both in clinical and colonizing isolates, has been rendered by the production of plasmid-mediated K. pneumoniae carbapenemase (KPC). In late 2007, a woman in her early thirties previously diagnosed with acute lymphoblastic leukaemia, was admitted to the haematology ward in Tel Aviv Sourasky Medical Center. The patient had arrived in Israel from Jordan in order to undergo chemotherapy and, later, bone marrow transplantation. During the first month after transplantation the patient was intermittently febrile and was treated with various antimicrobials, including piperacillin/tazobactam, amikacin, ciprofloxacin, vancomycin, imipenem and voriconazole. Two months after admission, while being treated with imipenem and voriconazole, the patient suffered from fever, dyspnoea and renal failure, and was transferred to the intensive care unit. During that period a carbapenem-resistant E. coli strain (E. coli 1736) was isolated from a Hickman catheter, leading to removal of the catheter and further treatment with ceftazidime and colistin. Treatment with these antibacterial agents cleared the OXA-48-producing E. coli, yet unfortunately the patient died 3 months later from a systemic Pseudomonas infection. E. coli 1736 was multidrug resistant, showing resistance to penicillins, piperacillin/tazobactam, aminoglycosides, quinolones and carbapenems, but susceptibility to all cephalosporins, aztreonam, tigecycline and colistin (Table 1). Analytical isoelectric focusing (IEF) performed on crude enzyme preparations revealed the presence of two b-lactamases with pIs of 5.4 and 7.2 (data not shown). PCR screening for the presence of b-lactamases in E. coli 1736 indicated the presence of blaTEM-1 and blaOXA-48 genes corresponding to the pIs of the b-lactamases visualized by IEF. Plasmid analysis of E. coli 1736 revealed four plasmids, three of around ≤50 kb in size and a larger plasmid of around 100 kb. Plasmid DNA was purified and transformed into E. coli DH10B. Transformant colonies that were screened positive for blaOXA-48 by PCR harboured the 50 kb plasmid. Southern analysis of plasmid DNA derived from these transformants using a blaOXA-48-labelled probe demonstrated the presence of blaOXA-48 on the acquired 50 kb plasmid. Acquisition of this plasmid increased the MICs of imipenem, meropenem and ertapenem without conferring full resistance (Table 1). PCR mapping of the genetic environment surrounding blaOXA-48 was performed in collaboration with the laboratory of Professor P. Nordmann (Hospital de Bicetre, Paris, France). blaOXA-48 was found to be located inside Tn1999.2, similar to the structure described for other enteric strains, such as the E. coli strain from Turkey and the K. pneumoniae strain from Lebanon. E. coli 1736 was not resistant to all b-lactam antibiotics as reported for other OXA-48-producing strains, yet it presented a high level of resistance to the commonly used carbapenems (MICs ≥16 mg/L), higher than usually seen in KPC-producing E. coli strains isolated in our hospital. These high carbapenem MICs suggested the presence of additional resistance mechanisms together with OXA-48 carbapenemase. Outer membrane protein (OMP) produced by E. coli 1736 was determined by PCR and sequencing of ompA, ompC and ompF genes. Further OMP analysis was performed by protein extraction and separation on Tris–Tricine gels using SDS-PAGE followed by mass spectrometry (performed in the Biological Mass Spectrometry Facility at the Weizmann Institute of Science). Both methods indicated the absence of at least one major porin, OmpC. Until the isolation of E. coli 1736, carbapenem resistance in E. coli in our country was exclusively attributed to the Ambler class A carbapenemase KPC. This is the first identified Enterobacteriaceae isolate in our country possessing a carbapenem-hydrolysing oxacillinase. To seek the possible origin of this strain we performed multilocus sequence typing (MLST; http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi. html), which genotyped the strain as sequence type (ST) 2, an E. coli ST that has never been recorded previously in our Research letters

The genetic environment of armA on pHNE, an IncN plasmid, in one Escherichia coli isolate from a chicken

Sir, Methylation of 16S ribosomal RNA (rRNA) mediated by 16S rRNA methylase, which confers high-level aminoglycoside resistance (MICs .1024 mg/L) in Enterobacteriaceae, has recently emerged as a major medical problem worldwide. Until now, seven plasmid-encoded 16S rRNA methylases, ArmA, RmtA, RmtB, RmtC, RmtD, RmtE and NpmA, have been reported in various clinical Gram-negative isolates in multiple geographic regions. armA is one of the most prevalent 16S rRNA methylase genes found in several Gram-negative pathogens. Since it was initially identified in Klebsiella pneumoniae in 2003, two studies focusing on its genetic environment and dissemination in Enterobacteriaceae from both humans and animals have been conducted. Here, we identify a novel genetic environment of armA on pHNE, an IncN plasmid, in one Escherichia coli isolate from a chicken. Previously, we reported the emergence of 16S rRNA methylases encoded by armA and rmtB in E. coli isolates from chickens. In a routine survey of antimicrobial-resistant isolates from chickens in 2009, armA was detected in an E. coli isolate exhibiting high-level resistance to the aminoglycoside antibiotic amikacin (MICs .1024 mg/L) (n1⁄41/18) from a diseased flock in a typical traditional farm in Henan Province, China. Phylogenetic group analysis using multiplex PCR revealed that this armA E. coli isolate belonged to phylogenetic group D, which was associated with extra-intestinal virulence. In order to further elucidate the genetic environment of armA, plasmids extracted from this armA-positive isolate (using the Plasmid Midi Kit, Qiagen, Germany) were transferred to E. coli DH10B by electroporation to investigate whether the armA resistance determinant was localized on plasmids. The transformants were screened on a Luria–Bertani agar plate containing

Construction and functional characterization of an integrative form λ Red recombineering Escherichia coli strain

Lambda Red recombineering is a DNA cloning and engineering technique involving recombination between homologous regions. The homologous recombination is mediated by the lambda Red genes consisting of red alpha, red beta and gam. Three lambda Red recombineering systems are currently available; the first is the plasmid-based system, in which lambda Red genes were cloned into temperature-sensitive plasmids; the second is the prophage-based system, in which lambda Red genes containing prophage were integrated into the Escherichia coli genome; the third is the integrative form system, characterized by the integration of lambda Red genes (or their counterparts) into the E. coli genome. In this study, a novel integrative form recombineering host, E. coli LS-GR, was constructed through the integration of functional recombineering elements including lambda Red genes, recA, araC and aacC1 into the E. coli DH10B genome. LS-GR shows high recombination efficiency for medium copy number vector and single copy number BAC vector modifications. The results indicate that LS-GR could be used as a general recombineering host strain.

TEM-168, a Heretofore Laboratory-Derived TEM β-Lactamase Variant Found in an Escherichia coli Clinical Isolate

There have been over 160 variants of the TEM-1 and TEM-2 penicillinases described, most of which also exhibit activity against extended-spectrum cephalosporins (www.lahey.org/Studies/). It was noted in 1994 that a T265M mutation (Ambler numbering) was often associated with active-site mutations E104K, R164S, G238S, and E240K in some TEM variants (4). Comparison of MICs and kinetic studies of TEM-1 variants with the amino acid substitutions T265M, G238S, G238S:T265M, G238S:E240K, and G238S:G240K:T265M constructed by in vitro mutagenesis showed that the T265M mutation had no measurable effect on the TEM-1 wild type or on the G238S or G238S:240K variant. To date, no naturally occurring T265M variant of TEM-1 has been reported, though currently, 22 TEM variants contain an M265 residue along with other residue changes (www.lahey.org/Studies/). Escherichia coli N08-1503 obtained by the National Microbiology Laboratory for advanced testing was resistant to ampicillin, cefazolin, and ampicillin-sulbactam, as determined by the submitting laboratory. Antimicrobial testing was carried out by disk diffusion and broth microdilution according to CLSI guidelines and Etests (AB Biodisk) according to manufacturer's instructions (Table ​(Table1)1) (1). The CLSI methods indicated that E. coli N08-1503 did not harbor an extended-spectrum β-lactamase (ESBL), though a reduced susceptibility to ceftazidime was noted. Analysis using the ESBL Etest strips yielded similar results. Interestingly, the cefepime ESBL Etest strip yielded a positive result. This strip is recommended outside of the United States when testing a strain with an inducible ampC gene (e.g., Enterobacter) or when a nondeterminable result is obtained. Broth microdilution confirmed the reduced susceptibility to cefepime and synergy with clavulanic acid, though the cefepime MICs were one or two doubling dilutions higher than the Etest results were. Resistance to amoxicillin-clavulanic acid and piperacillin-tazobactam and intermediate susceptibility to cefoxitin were also noted for E. coli N08-1503 and DH10B transformed with a plasmid (pT168) harboring blaTEM-168 (see below). TABLE 1. Antimicrobial data for E. coli N08-1503 and the transformants harboring pT168a PCR analysis results for β-lactamase genes were positive for TEM and negative for SHV, CTX-M, OXA-1, and CMY-2 types. Sequence analysis of the TEM amplicon revealed a translation product with a single amino acid change from TEM-1, a T261M change (T265M by Ambler numbering) caused by a C782T transition. This variant has been assigned the name TEM-168 (www.lahey.org/Studies). In the blaTEM-168 promoter region, we detected the G162T change that defines the strong P4 promoter (3, 5). Plasmid analysis showed that an ∼13-kb plasmid, pT168, harboring blaTEM-168 could be transformed into E. coli DH10B. Isoelectric focusing of crude extracts from E. coli N08-1503 and the DH10B transformant identified a single band of β-lactamase activity with a pI of 5.4. The resistance to β-lactam inhibitor combinations was likely due to the hyperproduction of plasmidic TEM-168 expressed from the P4 promoter, similar to what has been shown for TEM-1 (5, 6). Since the TEM-168 did not test positive with standard ESBL tests, it is easy to understand why this variant has not been described previously in clinical isolates. It was the unusual result with the cefepime ESBL Etest strip that made us continue our investigation to further characterize the TEM harbored by E. coli N08-1503.

Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

Backgroundβ-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed.ResultsE. coli DH10B cells producing SHV-1 β-lactamase and a clinical isolate of K. pneumoniae producing SHV-5 β-lactamase were rendered membrane permeable, fixed and adhered to poly-L-lysine coated slides, and stained with purified polyclonal anti-SHV antibodies that were fluorescein labeled. E. coli DH10B cells without a blaSHV gene were used as a negative control. The procedure generated a fluorescence signal from those slides containing cells expressing SHV β-lactamase that was sufficient for direct imaging.ConclusionWe developed a rapid and accurate method of visualizing the SHV family of enzymes in clinical samples containing Gram-negative bacilli using a fluorescein-labeled polyclonal antibody.

Similar cefoxitin-resistance plasmids circulating in Escherichia coli from human and animal sources

The aim of this study was to determine the molecular epidemiology of cefoxitin-resistance Escherichia coli identified in cattle entering feedlots and determine if there were any similarities to E. coli causing human infections in Canadian hospitals. A total of 51 E. coli were isolated from a total of 2483 cattle entering four feedlots in southern Alberta, Canada. DNA fingerprinting using pulsed-field gel electrophoresis revealed thirty-two unique patterns with two major clusters observed comprised of Cluster A (11 strains) and Cluster B (7 strains). PCR and sequence analysis revealed 38 isolates (74.5%) harboured bla CMY-2, whereas the remainder were found to contain mutations in the promoter region of the chromosomal ampC gene, which has been previously associated with cefoxitin resistance. No resistance to nalidixic acid, ciprofloxacin, or amikacin was observed in the clinical isolates. bla CMY-2 harbouring plasmids were transferred to E. coli DH10B. All of the plasmids carrying bla CMY-2 contained the A/C replicon and also harboured other resistance genes. Plasmid fingerprinting using BglII revealed 17 unique patterns with all but one clustering within 70% similarity. Comparison of the plasmid fingerprints to those isolated from human clinically significant E. coli in Canada during a similar time period [Mulvey, M.R., Bryce, E., Boyd, D.A., Ofner-Agostini, M., Land, A.M., Simor, A.E, Paton, S., 2005. The Canadian Hospital Epidemiology Committee, and The Canadian Nosocomial Infection Surveillance Program, Health Canada. Molecular characterization of cefoxitin resistant Escherichia coli from Canadian hospitals. Antimicrob. Agents Chemother. 49, 358–365] revealed four strains that harboured bla CMY-2 A/C replicon type plasmid with fingerprint similarities of greater than 90% to the ones identified in E. coli from the cattle in this study. These findings highlight the potential linkage of multidrug resistant organisms in food producing animals and human infections in Canadian hospitals. The plasmids conferred resistance to multiple antibiotics which could limit options for the treatment of infections caused by these strains.

Improvement of Baculovirus Expression System and Purification of IL-6 Protein Expressed in Insect Cells

On the basis of site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid), which propagated in Escherichia coli, the Bac-to-Bac system provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. The efficiency of recombinant baculovirus infecting cells plays an important role in the protein expression. In this study, we have introduced an egfp expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp is then transformed into E. coli DH10B containing the transposition helper plasmid to construct a new transposition receipt strain E. coli DH10Bac-egfp. Because of the intact attTn7 sites in the lacZα cassette, the target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracking of the target protein expression and the recombinant Bacmid transfection and the recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac-egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coli DH10Bac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6×His tag was expressed and purified successfully from Sf9 cells infected with recombinant virus Bac-egfp-6×His-IL6 constructed by the improved Bac-to-Bac system.

Copper-dependent Interaction of Dynactin Subunit p62 with the N Terminus of ATP7B but Not ATP7A

The P-type ATPase affected in Wilson disease, ATP7B, is a key liver protein required to regulate and maintain copper homeostasis. When hepatocytes are exposed to elevated copper levels, ATP7B traffics from the trans-Golgi network toward the biliary canalicular membrane to excrete excess copper into bile. The N-terminal region of ATP7B contains six metal-binding sites (MBS), each with the copper-binding motif MXCXXC. These sites are required for the activity and copper-regulated intracellular redistribution of ATP7B. Two proteins are known to interact with the ATP7B N-terminal region: the copper chaperone ATOX1 that delivers copper to ATP7B, and COMMD1 (MURR1) that is potentially involved in vesicular copper sequestration. To identify additional proteins that interact with ATP7B and hence are involved in copper homeostasis, a yeast two-hybrid approach was employed to screen a human liver cDNA library. The dynactin subunit p62 (dynactin 4; DCTN4) was identified as an interacting partner, and this interaction was confirmed by co-immunoprecipitation from mammalian cells. The dynactin complex binds cargo, such as vesicles and organelles, to cytoplasmic dynein for retrograde microtubule-mediated trafficking and could feasibly be involved in the copper-regulated trafficking of ATP7B. The ATP7B/p62 interaction required copper, the metal-binding CXXC motifs, and the region between MBS 4 and MBS 6 of ATP7B. The p62 subunit did not interact with the related copper ATPase, ATP7A. We propose that the ATP7B interaction with p62 is a key component of the copper-induced trafficking pathway that delivers ATP7B to subapical vesicles of hepatocytes for the removal of excess copper into bile.

Expansion of Substrate Specificity of Cytochrome P450 2A6 by Random and Site-directed Mutagenesis*

The natural product indole is a substrate for cytochrome P450 2A6. Mutagenesis of P450 2A6 was done to expand its capability in the oxidization of bulky substituted indole compounds, which are not substrates for the wild-type enzyme or the double mutant L240C/N297Q, as determined in our previous work (Wu, Z.-L., Aryal, P., Lozach, O., Meijer, L., and Guengerich, F. P. (2005) Chem. Biodivers. 2, 51-65). Error-prone PCR and site-directed mutagenesis led to the identification of two critical amino acid residue changes (N297Q and I300V) that achieve the purpose. The new mutant (N297Q/I300V) was able to oxidize both 4- and 5-benzyloxy(OBzl)indoles to form colored products. Both changes were required for oxidation of these bulky substrates. The colored product derived from 5-OBzl-indole was mainly 5,5'-di-OBzl-indirubin, whereas the dominant blue dye isolated upon incubations with 4-OBzl-indole was neither an indigo nor an indirubin. Two-dimensional NMR experiments led to assignment of the structure as 4-OBzl-2-(4'-OBzl-1',7'-dihydro-7'-oxo-6'H-indol-6'-ylidene)indolin-3-one, in which a pyrrole ring and a benzene ring are connected with a double bond instead of the pyrrole-pyrrole connection of other indigoids. Monomeric oxidation products were also isolated and characterized; three phenols (4-OBzl-1H-indol-5-ol, 4-OBzl-1H-indol-6-ol, and 4-OBzl-1H-indol-7-ol) and one quinone (4-OBzl-1H-indole-6,7-dione, the postulated immediate precursor of the final blue dye) were identified. The results are interpreted in the context of a crystal structure of a P450 2A6-coumarin complex. The I300V change opens an additional pocket to accommodate the OBzl bulk. The N2297Q change is postulated to generate a hydrogen bond between Gln and the substrate oxygen. Thus, the substrate specificity of P450 2A6 was expanded, and new products were obtained in this study.

Construction and characterization of a bacterial artificial chromosome library of peach

A peach [Prunus persica (L.) Batch] bacterial artificial chromosome (BAC) library of var. Jingyu was constructed. Jingyu is a traditional variety, that displays many of the important agronomic characters of stone fruits. Since peach leaves are rich in polysaccharides, high-molecular-weight (HMW) DNA was extracted from leaf nuclei using a protocol adapted to peach. The HMW DNA embedded in agarose plugs was partially digested by HindIII. After size-selection by pulsed field gel electrophoresis, the selected DNA fragments were ligated to pBeloBAC11 and transformed into E. coli DH10B cells by electroporation. In total 20,736 recombinant clones were obtained. The BAC library has an average insert size of 95 kb and represents approximately 6.7 peach haploid genome equivalents. The BAC clones were stable in E. coli cell after 100 generations. The lack of hybridization to chloroplast and mitochondrial genes demonstrated that the library is predominantly composed of nuclear DNA. The library was screened with two molecular markers, W4 and P20, that are linked to white flesh and nectarine genes of peach, respectively. Ten positive clones were detected. Their fingerprints will be used to determine clone relationships and assemble contigs. This library should be well-suited for the map-based cloning of peach genes and genome physical mapping.

Identification and expression of the Pseudomonas syringae pv. aptata hrpZ(Psa) gene which encodes an harpin elicitor.

A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv. syringae hrpZ gene was identified in Pseudomonas syringae pv. aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants. and in E. coli DH10B (pCCP1069) containing the P. syringae pv. aptata hrp gene cluster. PCR with oligonucleotides, based on the hrpZ(Pss) gene and used as primers with the total genomic DNA of P. syringae pv. aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions. The amplicon was cloned into the pGEM-T plasmid vector, amplified in E. coli DH5alpha and sequenced. The sequence showed 95%, 83% and 61% identity with those of hrpZ(Pss), hrpZ(Psg and hrpZ(Pst) genes encoding the harpins of the P. syringae pv. syringae, glycinea and tomato, respectively. The amplicon was cloned into the pMAL expression system. The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography. On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P. syringae pv. aptata harpin.