The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2,4-dicarboxylic acid

Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor sigmas and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog L-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.     

A novel bioassay for the detection of siderophores containing keto-hydroxy bidentate ligands

Abstract A convenient and sensitive pour-plate Petri dish bioassay for the detection of siderophores containing monoprotic keto-hydroxy bidentate ligands (KHBL) has been developed. The bioassay is based on the fact that bacteria of the Proteus-Providencia-Morganella group (Proteeae) utilize various ferric α-hydroxy- or α-ketocarboxylate complexes very efficiently. While P. vulgaris and P. rettgeri were able to utilize virtually all iron complexes supplied, Morganella morganii SBK3 was unable to utilize trihydroxamate type siderophores and was therefore selected as an indicator strain for iron complexes containing keto-hydroxy bidentate ligands (KHBL-siderophores). Filter paper disks containing the ferric complexes of siderophores were tested on tryptone or Luria broth agar, seeded with the indicator strains and supplemented with the ferrous iron chelator 2,2-dipyridyl (300 μM) to reduce the bioavailable iron. In the presence of siderophores, growth inhibition was reversed to provide a zone of growth stimulation. Ferric complexes of α-hydroxycarboxylates, α-ketocarboxylates, salicylic acid, tropolonederivatives, α-hydroxypyridinones, cepabactin, citrate, rhizoferrin and even epihydroxymugineic acid showed significant growth stimulation. From the results with the trihydroxamate-non-utilizing strain, M. morganii SBK3 , it may be inferred that the Proteeae prossess an iron transport system which recognizes ferric α-hydroxycarboxylates, α-ketocarboxylates as well as aromatic and heteroaromatic keto-hydroxy compounds, collectively named keto-hydroxy bidentate ligands. The bioassay is especially suited for detection of new siderophores from low-iron cultures of fungi and bacteria.     

Differential inactivation of glucose- and glutamate dependent acid resistance of Escherichia coli TMW 2.497 by high-pressure treatments

The inactivation by 200–400 MPa and post-pressure survival at acid conditions of E. coli TMW 2.497 was characterized by the measurement of intracellular pH (pH_in), viable cell counts, glutamate (Glu) and arginine (Arg) consumption, and the influence of mild adaptation to mild acid stress prior to pressure treatment. Glutamate and arginine did not affect viable cell counts or the pH_in during pressure application but improved the ability to maintain a high pH_in after pressure treatment. In pH 4.0 buffer without arg and glu, a 3 log reduction of cell counts occurred after 24 h of incubation, whereas little or no loss of viability was observed after 24 h incubation in the presence of glu and arg. During post-pressure incubation at pH 4.0, 10 mM glutamate were metabolized but only 2 mM arginine were used, indicating that glutamate rather than arginine was responsible for the protective effect on pH_in and survival. In conclusion, the pressure induced, irreversible loss of the transmembrane ΔpH correlates to cell death and glu stabilizes the pH_in of E. coli during post-pressure incubation.     

大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间的关系

通过对大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间关系研究发现,10℃培养,对数期细胞有最大的压力抗性,随着培养温度的升高直到4 5℃,压力抗性呈下降的趋势;相反,10℃培养,稳定期的细胞对压力最敏感,随着培养温度的升高,压力抗性呈增加趋势,30～37℃时达到最大,之后到4 5℃有下降。对数期和稳定期细胞膜脂中不饱和脂肪酸的组成随温度的上升而下降,这与从全细胞中抽提的磷脂的熔点密切相关。因此,对数期细胞压力抗性随着膜流动性的增大而升高;但稳定期细胞,膜流动性与压力抗性之间不存在简单的对应变化关系     

Involvement of phospholipids in resistance and adaptation of Escherichia coli to acid conditions and to long-term survival

In Escherichia coli membranes, three major phospholipids are formed: phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). We report here the survival of mutants lacking either PE or both PG and CL at an acid pHo and during long-term survival experiments. Stationary phase cultures of E. coli lacking PE are much more sensitive to acid shock (pHo 3) than the wild-type strain. Moreover, in the strain lacking PE, long-term survival in stationary phase is impaired and after 5 days no viable cells are recovered. The survival of an exponential phase culture to acid shock is known to be increased if the culture is exposed to moderately acid conditions (pHo 5) prior to a shift to pHo 3. If either PE or both PG and CL are missing, the exposure to pHo 5 does not increase the survival at pHo 3.     

The contribution of tellurite resistance genes to the fitness of Escherichia coli uropathogenic strains

The presence of tellurite resistance gene operons has been reported in several human pathogens despite the fact that tellurium, as well as its soluble salts, are both rare in nature and are no longer in use as antimicrobial agents. We have introduced the cloned terWZA-F genes from an uropathogenic Escherichia coli isolate into another clinical E. coli isolate that was shown to be ter-gene free. The presence of the introduced genes increased the level of potassium tellurite resistance, as well as the level of resistance to oxidative stress mediated by hydrogen peroxide; and prolonged the ability of particular strains to survive in macrophages. We therefore propose that the contribution of tellurite resistance genes to oxidative stress resistance in bacteria is at least one reason for their presence in the genomes of a broad range of pathogenic microorganisms.     

Serotyping of clinical isolates belonging to Proteus mirabilis serogroup O36 and structural elucidation of the O36-antigen polysaccharide

The O-specific polysaccharide (OPS) isolated from the lipopolysaccharide of Proteus mirabilis O36 was found to have a pentasaccharide repeating unit of the following structure: -->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAc6Ac-(1-->4)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. The structure is unique among Proteus OPS, which is in agreement with the classification of this strain into a separate Proteus O-serogroup. Remarkably, the P. mirabilis O36-polysaccharide has the same structure as the OPS of Escherichia coli O153, except that the latter is devoid of O-acetyl groups. The cross-reaction of anti-O36 antibodies with the O-part of E. coli O153 lipopolysaccharide is observed. In the present study, two steps of serotyping Proteus strains are proposed: screening of dry mass with enzyme-linked immunosorbent assay and immunoblot with the crude lipopolysaccharides. This method allowed serotyping of 99 P. mirabilis strains infecting the human urinary tract. Three strains were classified into serogroup O36. The migration pattern of these lipopolysaccharides fraction with long O-specific PSs was similar to the standard laboratory P. mirabilis O36 (Prk 62/57) lipopolysaccharide. The relatively low number of clinical strains belonging to serogroup O36 did not correspond to the presence of anti-P. mirabilis O36 antibodies in the blood donors' sera. Twenty-five percent of tested sera contained a statistically significant elevated level of antibodies reacting with thermostable surface antigens of P. mirabilis O36. The presence and amount of antibodies correlated with Thr399Ile TLR4 polymorphism types (P=0.044).     

A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity

A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified. This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel beta-subunits in the AKR6 family. The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1. Sequences encoding putative homologues of this enzyme exist in many other bacteria. The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal. The K(m) for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM. Overexpression of the recombinant enzyme in E. coli leads to increased resistance to methylglyoxal. It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.     

The widespread occurrence of the enterohemolysin gene ehlyA among environmental strains of Escherichia coli

The putative virulence factor enterohemolysin, encoded by the ehlyA gene, has been closely associated with the pathogenic enterohemorrhagic Escherichia coli (EHEC) group. Escherichia coli isolates from effluents from seven geographically dispersed municipal wastewater treatment plants were screened for the presence of enterohemolysin. A total of 338 E. coli isolates were found to express the ehlyA gene. However, none of the isolates contained the toxin-encoding genes (stxA or stxB) associated with EHEC. Two of the 338 isolates possessed the virulence factor intimin, encoded by the eae gene. These findings suggest that the ehlyA gene may be widely distributed among non-EHEC isolates in the environment.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Detection of ampicillin and tetracycline resistance genes in uropathogenic Escherichia coli strains by affinity-based nucleic acid hybrid collection

Abstract Uropathogenic Escherichia coli strains were analyzed for the presence of the ampicillin (Ap) and tetracycline (Tc) resistance genes by a novel nucleic acid solution hybridization technique. In this method probe pairs subcloned from the resistance genes are utilized. DNA from single colonies of 20 E. coli strains was released and analyzed without purification by the 3 h hybridization test. The respective resistance genes were easily identified by the test.     

Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions

AIM: To determine the rate of antibiotic resistance transmission between commensal and pathogenic representatives of the Enterobacteriaceae. METHODS AND RESULTS: Through the use of a validated in vitro simulation of the porcine ileum, the transmission of antibiotic resistance was detected between commensal Escherichia coli, E. coli O157 and Salmonella spp. Countable transconjugant populations arose readily and, in one example, proved capable of indefinite persistence. CONCLUSIONS: Genetic material conferring antibiotic resistance is readily transmissible between members of the Enterobacteriaceae under ileal conditions. Recipient phenotype influences the persistence of multi-resistant transconjugants. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that the conjugal transmission of antibiotic resistance is commonplace under ileal conditions impacts primarily on the risk of food contamination by multi-resistant bacteria. The establishment of a multi-resistant transconjugant population as a dominant member of the microflora maintains a genetic reservoir of antimicrobial resistance.     

A novel DNA microarray design for accurate and straightforward identification of Escherichia coli safety and laboratory strains

Escherichia coli K-12, B, C and W strains and their derivates are declared in biological safety guidelines as risk group 1 organisms as they are unable to colonise the human gut.

Differentiation and identification of these safety strains is mainly based on pulsed-field gel electrophoresis (PFGE), phage sensitivity tests or PCR-based methods. However, these methods are either tedious and time consuming (phage sensitivity, PFGE) or based on single specific fragments (PCR) or patterns (PFGE) lacking additional information for further differentiation of the strains.

In the current study, subtractive hybridisation techniques were applied to detect specific DNA fragments which were used to design a microarray (chip) for accurate and simple identification of these organisms, and to differentiate them from other E. coli strains. The chip can be used to identify E. coli safety strains and monitor them during ongoing experiments for changes in their genome and culture purity. The hybridisation layout of the microarray was arranged in such a way that the respective lineages of safety strains could be easily identified as distinct letters (K, B, C or W). Differentiation of single strains or subtyping was possible with further probes. In addition, a set of probes targeting genes coding for common virulence factors has been included, both to differentiate safety strains from pathogenic variants and to make sure that no transfer of these genes happens during handling or storage. The reliability of the approach has been tested on a comprehensive selection of E. coli laboratory strains and pathogenic representatives.     

新生儿病区产ESBLs大肠埃希菌整合子及其耐药性分析

目的 了解新生儿病区产ESBLs大肠埃希菌整合子的携带情况及其耐药性.方法 采用K-B琼脂扩散法对56株产ESBLs大肠埃希菌进行药敏试验;应用PCR法检测Ⅰ类、Ⅱ类和Ⅲ类整合子;以肠杆菌科重复序列-聚合酶链式反应(ERIC-PCR)进行基因分型.结果 56株产ESBLs大肠埃希菌的Ⅰ类整合子检出率为60.7％,未检出Ⅱ类和Ⅲ类整合子;菌株对庆大霉素、环丙沙星、左氧氟沙星、复方新诺明、头孢唑林、氨曲南、头孢他啶的耐药率差异有统计学意义(P＜0.05),阳性菌株的耐药率高于阴性菌株;56株大肠埃希菌分为45种基因型.结论 Ⅰ类整合子广泛存在于新生儿病区产ESBLs大肠埃希菌并与其耐药性相关.     

Production of Vero cytotoxin by strains of Escherichia coli as related to the availability of iron

Abstract Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli , there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.     

Escherichia coli genes involved in resistance to pyrazinoic acid,the active component of the tuberculosis drug pyrazinamide

The natural resistance of Escherichia coli to pyrazinoic acid (POA), the active derivative of pyrazinamide, was investigated. The TolC mutant was found to be more susceptible to POA and other weak acids than the wild-type strain. Mutation in EmrB but not AcrB efflux protein slightly increased POA susceptibility. Two transposon mutants with increased susceptibility to POA were found to harbor mutations in acnA encoding aconitase-1 and ygiY encoding a putative two-component sensor protein. Complementation of the AcnA and YgiY mutants conferred resistance to POA, whereas the complemented TolC mutant became resistant to POA and other weak acids.     

Expression of CS fimbriae in Escherichia coli strains of human and animal origin belonging to O-serovar 6, K-serovar 15 or H-serovar 16

Abstract A non-conjugative CS-fimbriae-associated plasmid pCS001 was mobilized into rifampicin-resistant mutants of strains of Escherichia coli of O-serovar 6 or K-serovar 15 or H-serovar 16. By a variety of serological procedures only the production of CS3 fimbriae was detected in transoconjugants. The finding extend previous observations that only strains of E. coli of serotype O6:K15:H16 or H− and of appropriate biotype are able to express either CS1 or CS2 fimbriae, as even a recipient of serotype O6:K15:H31 possessing a rhamnose-positive fermentation phenotype did not express either of these two fimbriae. The results indicate that expression of CS1 or CS2 fimbriae probably involves chromosomal determinants only found in strains of serotype O6:K15:H16 or H−.     

Addition of a surfactant to tryptic soy broth allows growth of a lactic acid bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica

Aims: This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture. Methods and Results: Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l^?1 Tween 80^® (TSB‐T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB‐T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB‐T80 but was significantly less in TSB. Conclusions: Supplementation of TSB with Tween 80^® allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro. Significance and Impact of the Study: This study reports the utility of TSB‐T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.