Prevalence of β-lactam and fluoroquinolone resistance, and virulence factors in Escherichia coli isolated from chickens in Slovakia

The incidence of infections due to organisms resistant to β-lactam antibiotics has increased sharply in recent years. The goal of this study was to investigate the β-lactam resistance in 151 Escherichia coli strains isolated from chickens over a two-year period. Extended spectrum β-lactamases (ESBLs) were present in 24 strains (16%), ESBL phenotype was identified by interpretative reading of minimal inhibitory concentration values of ceftriaxon (CRT ≥ 7.1 mg/L), ceftazidime (CAZ ≥ 3.4 mg/L) and ceftiofur (CFF ≥ 8.7 mg/L). PCR detection revealed the presence of the bla _CMY-2 gene and CTX-M-1 group. We detected high resistance to ampicillin (92%), streptomycin (63%), tetracyclin (70%), ceftiofur (40%), floroquinolones (enrofloxacin 68%, ciprofloxacin 62%), florfenicol (18%), chloramphenicol (21%) and cotrimoxazol (43%). We also investigated the presence of virulence factors and mobile genetic elements, and performed plasmid replicon typing in 24 selected strains. The most prevalent integrase among the isolates was the integrase 1 with gene cassettes dfrA, aadA and genes sul1 and sul2. Plasmid mediated quinolone resistances (qnrS) were also detected in two strains. Plasmid typing showed that the Y and IncI1 were dominant plasmid replicons. The genes iss, kpsII, tsh, iutA were the most frequently detected virulence genes in ESBL-positive strains. These results demonstrate that broilers in Slovakian food markets and farms could be the source of ESBL-producing E. coli, as well as virulent and resistant strains representing a potential risk for the human population.     

Antibiotic resistance and detection of β-lactamase in bacterial strains of Staphylococci and Escherichia coli isolated from foodstuffs

Seventy strains of Staphylococcus spp. and Escherichia coli (35 each) were isolated from various foodstuffs and identified on the basis of cultural, morphological and biochemical characteristics and were further tested for their antibiotic susceptibility with commonly used antibiotics/drugs. 69.2% of the strains of Staphylococci were resistant to co-trimazine and 34.6% were resistant to penicillin-G. 19.2% of the staphylococcal isolates exhibited resistance to cloxacillin, nalidixic acid, methicillin and tetracycline whereas 15.3% of the staphylococcal isolates were resistant to amoxycillin and nitrofurantoin. The isolated E. coli strains exhibited sharp peaks of resistance to antimicrobial agents such as tetracycline (72%), doxycycline (60%) and nalidixic acid (48%). Forty-four percent of the E. coli strains were resistant to nitrofurantoin and penicillin-G respectively. Among the 13 antibiotics/drugs tested for resistance, six different resistance patterns were observed in staphylococcal isolates and seven different resistance patterns were observed in the E. coli isolates from various foodstuffs. Bacterial strains exhibiting MIC values 100 g/ml for ampicillin and cloxacillin were screened for -lactamase activity and out of 10 staphylococcal isolates, seven were found to be positive for -lactamase, whereas out of 13 E. coli isolates tested for -lactamase production, only three were found to be positive.     

Virulence properties and antimicrobial susceptibility of Shiga toxin-producing Escherichia coli strains isolated from healthy cattle from Paraná State, Brazil

The presence of Shiga toxin-producing Escherichia coli (STEC) strains in feces samples of cattle was determined using the cytotoxicity assay on Vero cells and a screening PCR system to detect stx genes. The STEC isolates were serotyped, tested for antimicrobial susceptibility, and analyzed for virulence genes using multiplex PCR. The verocytotoxin-producing E. coli - reverse passive latex agglutination (VTEC-RPLA) assay was also used to detect Shiga toxin production. The frequency of cattle shedding STEC was 36%. The isolates belonged to 33 different serotypes, of which O10:H42, O98:H41, and O159:H21 had not previously been associated with STEC. The most frequent serotypes were ONT:H7 (10%), O22:H8 (7%), O22:H16 (7%), and ONT:H21 (7%). Most of the strains (96%) were susceptible to all antimicrobial agents tested. Shiga toxin was detected by the VTEC-RPLA assay in most (89%) of the STEC strains. The frequency of virulence markers was as follows: stx1, 10%; stx2, 43%; stx1 plus stx2, 47%; ehxA, 44%; eae, 1%; and saa, 38%. Several strains belong to serotypes associated with human disease, and most of them carried a stx2-type gene, suggesting that they represent a risk to human health. The screening PCR assay showed fewer false-negative results for STEC than the Vero-cell assay and is suitable for laboratory routine.     

Phenotypic and genotypic traits of Shiga toxin-producing Escherichia coli strains isolated from beef cattle from Paraná State, southern Brazil

AIMS: To investigate the prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil. METHODS AND RESULTS: One hundred and seven faeces cattle samples were cultured on Sorbitol-MacConkey agar. Escherichia coli colonies were tested for production of Shiga toxin using Vero-cell assay. A high prevalence (57%) of STEC was found. Sixty-four STEC were serotyped and examined for the presence of stx(1), stx(2), eae, ehxA and saa genes and stx(2) variants. The isolates belonged to 31 different serotypes, of which three (O152:H8, O175:H21 and O176:H18) had not previously been associated with STEC. A high prevalence of stx(2)-type genes was found (62 strains, 97%). Variant forms found were stx(2), stx(2c), stx(2vhb), stx(2vO111v/OX393) and a form nonclassifiable by PCR-RFLP. The commonest genotypes were stx(2)ehxA saa and stx(1)stx(2)ehxA saa. CONCLUSIONS: A high frequency of STEC was observed. Several strains belong to serotypes previously associated with human disease and carry stx(2) and other virulence factors, thus potentially representing a risk to human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of STEC in Paraná State, and its findings emphasize the need for proper cattle handling to prevent human contamination.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Antibiotic resistance and plasmid pattern of enterotoxigenic ST-a strains of Escherichia coli isolated in Puebla, México

Antibiotic susceptibilities of 22 strains of Escherichia coli isolated from children from 0 to 3 years old at the University Hospital of Puebla were determined. Almost all strains were resistant to ampicillin, tetracycline, streptomycin, and kanamycin. Gel electrophoresis of DNA from 10 clinical strains of E. coli revealed a heterogeneous plasmid population. Plasmid DNA, ranging in molecular mass from 1.8 to 120 megadaltons, was demonstrated in 10 strains. Moreover, the frequency of antibiotic transfer ranged from 1.6/10(8) to 2/10, and the simultaneous transfer of the gene encoding heat-stable enterotoxin was also determined. Six out of 10 strains tested were able to cotransfer ST-a as demonstrated by the suckling mouse test. It is possible that antibiotic selective pressure may increase the isolation of enterotoxigenic E. coli strains.     

Analysis of synonymous codon usage in Shigella flexneri 2a strain 301 and other Shigella and Escherichia coli strains

In this study, we analysed synonymous codon usage in Shigella flexneri 2a strain 301 (Sf301) and performed a comparative analysis of synonymous codon usage patterns in Sf301 and other strains of Shigella and Escherichia coli. Although there was a significant variety in codon usage bias among different Sf301 genes, there was a slight but observable codon usage bias that could primarily be attributable to mutational pressure and translational selection. In addition, the relative abundance of dinucleotides in Sf301 was observed to be independent of the overall base composition but was still caused by differential mutational pressure; this also shaped codon usage. By comparing the relative synonymous codon usage values across different Shigella and E. coli strains, we suggested that the synonymous codon usage pattern in the Shigella genomes was strain specific. This study represents a comprehensive analysis of Shigella codon usage patterns and provides a basic understanding of the mechanisms underlying codon usage bias.     

Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131

Background

The prevalence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC.

Methods

A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR.

Results

Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The bla _CTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The bla _CTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes.

Conclusions

Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 β-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.     

Epidemiologic and molecular characterization of β-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients

Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of β-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, β-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.

     

Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan

The evolution of NDM genes (bla_NDM) in E. coli is accounted for expansive multidrug resistance (MDR), causing severe infections and morbidities in the pediatric population. This study aimed to analyze the phylogeny and mutations in NDM variants of E. coli recovered from the pediatric population. Carbapenem-resistant clinical strains of E. coli were identified using microbiological phenotypic techniques. PCR technique used to amplify the bla_NDM genes, identified on agarose gel, and analyzed by DNA sequencing. The amino acid substitutions were examined for mutations after aligning with wild types. Mutational and phylogenetic analysis was performed using Lasergene, NCBI blastn, Clustal Omega, and MEGA software, whereas PHYRE2 software was used for the protein structure predictions. PCR amplification of the bla_NDM genes detected 113 clinical strains of E. coli with the contribution of bla_NDM-1 (46%), bla_NDM-4 (3.5%), and bla_NDM-5 (50%) variants. DNA sequencing of bla_NDM variants showed homology to the previously described bla_NDM-1, bla_NDM-4, and bla_NDM-5 genes available at GenBank and NCBI database. In addition, the mutational analysis revealed in frame substitutions of Pro60Ala and Pro59Ala in bla_NDM-4 and bla_NDM-5, respectively. The bla_NDM-1 was ortholog with related sequences of E. coli available at GenBank. The phylogenetic analysis indicated that the NDM gene variants resemble other microbes reported globally with some new mutational sites.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli

The β-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme β-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl β,1–4 linkage, and β(1–2), β(1–4), β(1–6) and β(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.     

Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli : A comparison of the synthesis capacities of L-form strains with an E. coli producer strain

The paper describes the synthesis of the phosphorylcholine-binding miniantibody McPC603scFvDhl x in cell-wall-less L-form strains of Escherichia coli and Proteus mirabilis. Cells of these strains were transformed with the plasmid pACK02scKan, carrying the miniantibody (miniAb) coding sequence under the control of the lac promoter. L-form transformants of both species were able to synthesize the functional miniAb as an extracellular soluble product. The highest quantities were obtained by P. mirabilis L-form strains after induction with 5 mM isopropyl β-d-thiogalactopyranoside (IPTG). Yields of 45–75 mg/l total antibody protein and of 10–18 mg/l functional miniAb were estimated in the growth medium of shaking cultures 40–80 h after induction with IPTG. About 10% of the active miniAb remained cell-bound. The yields of functional miniAb could be optimized by lowering the growth temperature from 37 °C to 26–32 °C and by supplementation of the medium with 80 mM sodium fumarate. A comparison of the specific activities revealed that the P. mirabilis L-form strains have a similar synthesis capacity (2–4 mg functional miniAb/g cell dry weight) to that of the producer strain E. coli RV308. The results show that the processes of correct folding and assembling of the miniAb molecules are possible without the periplasmic compartment. Received: 14 April 1997 / Received revision: 17 July 1997 / Accepted: 25 August 1997     

Surface Expression of ω-Transaminase in Escherichia coli

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.