REGULATION OF THE EXPRESSION OF NAR OPERON ENCODING NITRATE REDUCTASE IN ESCHERICHIA COLI

The nar operon, which encodes the nitrate reductase in Escherichia coli, can be induced under anaerobic conditions without nitrate to a low level and with nitrate to a maximum level. The anaerobic formation of nitrate reductase is dependent upon the fnr gene product while the narL gene product is required for further induction by nitrate. The sequence was determined across the entire promoter and regulatory region of the nar operon. The translational start site of the first structural gene of the nar operon, narG gene, was established by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. The transcriptional start site and the level of the transcript was determined by S1 mapping procedure. One major transcript was identified which was initiated 50 base pair (bp) upstream from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, fully induced by nitrate anaerobically, and greatly reduced in a ${\rm Fnr\sp-}$ mutant. Deletions were created in the 5$\sp\prime$ nar regulatory sequence with either an intact nar operon or a nar::lacZ fusion. The expression of the plasmids with deletions were determined in a strain with wild type fnr and narL loci, a Fnr- mutant strain and a NarL- mutant strain. These experiments demonstrated that the $5\sp\prime$ limit of the nar operon lies at about $-210$ bp from the transcription start site. The region required for anaerobic induction by the fnr gene product is located around $-60$ bp. Two putative narL recognition sites were identified, one of which is around $-200$ and another immediately adjacent to the fnr recognition region. The deletion of the sequences around $-200$ rendered the remaining narL complex repressive and thus decreased the expression of nar operon, suggesting that the two potential narL sites interact with each other over a significant length of DNA. ^

Occurrence and Genetic Characteristics of Third-Generation Cephalosporin-Resistant Escherichia coli in Swiss Retail Meat

Prevalence and genetic relatedness were determined for third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) detected in Swiss beef, veal, pork, and poultry retail meat. Samples from meat-packing plants (MPPs) processing 70% of the slaughtered animals in Switzerland were purchased at different intervals between April and June 2013 and analyzed. Sixty-nine 3GC-R-Ec isolates were obtained and characterized by microarray, PCR/DNA sequencing, Multi Locus Sequence Typing (MLST), and plasmid replicon typing. Plasmids of selected strains were transformed by electroporation into E. coli TOP10 cells and analyzed by plasmid MLST. The prevalence of 3GC-R-Ec was 73.3% in chicken and 2% in beef meat. No 3GC-R-Ec were found in pork and veal. Overall, the blaCTX-M-1 (79.4%), blaCMY-2 (17.6%), blaCMY-4 (1.5%), and blaSHV-12 (1.5%) β-lactamase genes were detected, as well as other genes conferring resistance to chloramphenicol (cmlA1-like), sulfonamides (sul), tetracycline (tet), and trimethoprim (dfrA). The 3GC-R-Ec from chicken meat often harbored virulence genes associated with avian pathogens. Plasmid incompatibility (Inc) groups IncI1, IncFIB, IncFII, and IncB/O were the most frequent. A high rate of clonality (e.g., ST1304, ST38, and ST93) among isolates from the same MPPs suggests that strains persist at the plant and spread to meat at the carcass-processing stage. Additionally, the presence of the blaCTX-M-1 gene on an IncI1 plasmid sequence type 3 (IncI1/pST3) in genetically diverse strains indicates interstrain spread of an epidemic plasmid. The blaCMY-2 and blaCMY-4 genes were located on IncB/O plasmids. This study represents the first comprehensive assessment of 3GC-R-Ec in meat in Switzerland. It demonstrates the need for monitoring contaminants and for the adaptation of the Hazard Analysis and Critical Control Point concept to avoid the spread of multidrug-resistant bacteria through the food chain.

A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland

A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized. Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E. coli. Two copies of the insertion element IS15Delta/26 flanked the region containing sul3. The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E. coli isolates from pigs in Switzerland.

Mdt(A), a new efflux protein conferring multiple antibiotic resistance in Lactococcus lactis and Escherichia coli

The mdt(A) gene, previously designated mef214, from Lactococcus lactis subsp. lactis plasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactis and Escherichia coli, but not in Enterococcus faecalis or in Staphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactis and in E. coli.

High colonization rates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Swiss Travellers to South Asia- a prospective observational multicentre cohort study looking at epidemiology, microbiology and risk factors.

BACKGROUND International travel contributes to the worldwide spread of multidrug resistant Gram-negative bacteria. Rates of travel-related faecal colonization with extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae vary for different destinations. Especially travellers returning from the Indian subcontinent show high colonization rates. So far, nothing is known about region-specific risk factors for becoming colonized. METHODS An observational prospective multicentre cohort study investigated travellers to South Asia. Before and after travelling, rectal swabs were screened for third-generation cephalosporin- and carbapenem-resistant Enterobacteriaceae. Participants completed questionnaires to identify risk factors for becoming colonized. Covariates were assessed univariately, followed by a multivariate regression. RESULTS Hundred and seventy persons were enrolled, the largest data set on travellers to the Indian subcontinent so far. The acquired colonization rate with ESBL-producing Escherichia coli overall was 69.4% (95% CI 62.1-75.9%), being highest in travellers returning from India (86.8%; 95% CI 78.5-95.0%) and lowest in travellers returning from Sri Lanka (34.7%; 95% CI 22.9-48.7%). Associated risk factors were travel destination, length of stay, visiting friends and relatives, and eating ice cream and pastry. CONCLUSIONS High colonization rates with ESBL-producing Enterobacteriaceae were found in travellers returning from South Asia. Though risk factors were identified, a more common source, i.e. environmental, appears to better explain the high colonization rates.

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.

Comparative methodology to investigate the presence of Escherichia coli K-12 strains in environmental and human stool samples

This study was undertaken to evaluate the specificity and efficiency of different methods to detect Escherichia coli K-12 strains. Another aim was to determine the frequency of E. coli K-12 strains among wild-type E. coli isolates from different sources. The detection of K-12 strains was performed both genotypically by K-12 specific polymerase chain reaction (PCR) and on the basis of phenotypical tests. In addition, the genome structures of E. coli strains were characterized by pulsed-field gel electrophoresis (PFGE). The most specific results could be obtained by the genotypical tests PCR and PFGE as well as by the K-12 specific phage assay. In total, 131 stool and 95 water isolates as well as 14 K-12 derivatives were examined by the different methods. No E. coli K-12 strains were detected among the wild-type isolates.

Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors.

Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adRFA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adRPA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adRPA receptor is masked by the F4adRFA. The hypothesis that F4adRFA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy-Weinberg equilibrium statistics. The F4adRPA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abRPA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abRFA/F4acR+/F4adRFA); A2 (F4abRFA/F4acR+/F4adRPA); B (F4abRFA/F4acR+/F4adR-); C1 (F4abRPA/F4acR-/F4adRFA); C2 (F4abRPA/F4acR-/F4adRPA); D1 (F4abR-/F4acR-/F4adRFA); D2 (F4abR-/F4acR-/F4adRPA); E (F4abR-/F4acR-/F4adR-).

Rapid and accurate identification of Escherichia coli K-12 strains

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.

New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Escherichia coli are susceptible to thiazolides if the tolc efflux system is inhibited

Nitazoxanide (NTZ) and other thiazolides are effective against intracellular protozoa’s, anaerobic or micro aerophilic bacteria, viruses and tumour cells. Concerning their potential effects against Escherichia coli, the published results are scarce and conflicting. In order to investigate whether thiazolides are effective against aerobically growing E. coli, we examined mutants of the TolC efflux system for their sensitivity to nitro thiazolides, including NTZ, and bromothiazolides. We determined the susceptibilities of tolC mutants to various thiazolides and found that tolC mutants of E. coli were susceptible to both nitro thiazolides and bromothiazolides indicating a mechanism of action different from nitro reduction. Moreover, we showed that thiazolides induced a spy:lacZ transcriptional fusion indicating that thiazolides generate stress in the bacterial envelope. Moreover, wild type strains became susceptible to thiazolides if the tolC efflux system was inhibited. Taken together, our results show that thiazolides are effective against E. coli if their export from the cells is impaired.

In vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli ST131: Characterization of an Acquired Extended-Spectrum AmpC (ESAC) Conferring Resistance to Cefepime.

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance are reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical E. coli isolates obtained from a patient. The CMY-2-producing E. coli (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli (CMY-33-Ec) was detected in bronchoalveolar lavage. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime.CMY-33-Ec and CMY-2-Ec were identical by rep-PCR, being of hyperepidemic ST131, but showed different β-lactam MICs (e.g., cefepime 16 vs. ≤0.5 μg/ml). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but cefepime MICs were higher for CMY-33 than CMY-2 (8 vs. 0.25 μg/ml). The kcat/Km or kinact/KI (μM(-1) s(-1)) indicated that CMY-33 possesses an ESBL-like spectrum compared to CMY-2 (cefoxitin: 0.2 vs. 0.4; ceftazidime: 0.2 vs. not measurable; cefepime: 0.2 vs. not measurable; tazobactam 0.0018 vs. 0.0009). Using molecular modeling, we show that a widened active site (∼4 Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide, therefore awareness that cefepime treatment may select for resistant isolates is critical.

2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli.

The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IIC(glc)) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IIC(glc) was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IIC(glc) into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IIC(glc), which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IIC(glc) lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.