A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E. coli (ATCC 11303) rosette test

A new bacterial rosette technique for enumerating T lymphocytes is described. E. coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 μl) and after incubation at 4°C, slides are made, stained and counted. The nature of the lymphocytes forming E. coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E. coli and E, mouse E rosette and Fc receptor tests, and by mixed E. coli rosette tests and anti-Ig staining. E. coli and E rosette tests in controls and pediatric patients were also compared. The results show that T_μ and T_γ cells rosette with E. coli.     

Immunohistochemical characteristics of duodenal adenomas in familial adenomatous polyposis with special reference to cell kinetics

The duodenum is the second most frequent site of cancer in patients with familial adenomatous polyposis (FAP). The main objective of this study was to evaluate the cell kinetics in duodenal and ampullary adenomas in FAP. The endoscopic and biopsy findings of duodenal adenomas in 22 FAP subjects and 18 non-FAP subjects were compared. Adenomas in FAP included 15 ampullary adenomas and 17 nonampullary adenomas. The cell kinetics was evaluated by immunohistochemistry for Ki-67, p53, bcl-2, and cyclooxygenase 2 (COX2), and the apoptotic index (AI) as determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method. Any correlations between the indices for cell kinetics and the endoscopic findings were identified. All 50 adenomas were histologically verified to be tubular adenoma with low-grade dysplasia. Neither the expression of Ki-67, p53, bcl-2, and COX2 nor the AI differed substantially between FAP and non-FAP subjects. In patients with FAP, duodenal adenoma tended to have a higher Ki-67-labeling index than the ampullary adenoma (54.3 +/- 11.3 versus 46.8 +/- 12.7; .05 < P < .1). In addition, the Ki-67-labeling index in endoscopically normal or slightly enlarged ampullary adenoma was significantly higher than that in markedly enlarged ampullary adenoma (51.8 +/- 11.4 versus 39.4 +/- 11.3; P < .05). Duodenal adenoma in FAP subjects was not found to have a higher proliferative activity or a smaller degree of apoptosis compared with those in non-FAP subjects. The smaller proliferative activity in larger ampullary adenoma may thus be related to the static nature of ampullary adenoma in FAP.     

Orientation of the peptidoglycan chains in the sacculus of Escherichia coli

The organization of chains of oligopeptidoglycan in the saccular wall is of critical importance in the study of the mechanism and physiology of prokaryotic wall growth. The electron microphotographs of De Pedro et al, present new findings and can be used to negate or at least raise questions about the previously accepted conclusion that the glycan chains are oriented transversely to the axis of rod-shaped Escherichia coli. This suggests caution in assuming that the glycan chains in the murein structure are parallel to each other and are perpendicular to the axis of the cell.These results should reopen the question of not only the orientation of the peptidoglycan chains, but the possibility of variability in orientation. Three classes of hypotheses about wall growth are reconsidered and problems with them are presented. The new results from De Pedro's laboratory and the experimental glycan chain length distribution argue against proposed systematic models. These include models that postulate belts or hoops stretched around the circumference of the cell and mechanisms that insert new chains of the length of presumptive “docking” strands in the stress-bearing wall. They are consistent, however, with the surface stress theory that proposes that random enzyme action together with physical forces are involved in the elongation of the rod-shaped Gram-negative wall.     

Natural antimicrobial susceptibility patterns and biochemical profiles of Leclercia adecarboxylata strains

Leclercia adecarboxylata is an opportunistic human pathogen that phenotypically resembles Escherichia coli. The natural susceptibilities of 101 Leclercia strains to 70 antimicrobial agents were investigated. MICs were determined with a microdilution procedure in cation-adjusted Mueller-Hinton broth (all strains) and IsoSensitest broth (some strains). Natural susceptibility patterns were assessed using German (DIN) standards (when applicable). In addition, biochemical properties recommended for the phenotypic identification of L. adecarboxylata were evaluated, applying two commercially available identification systems for Enterobacteriaceae and seven conventional tests. L. adecarboxylata strains were naturally sensitive to tetracyclines, aminoglycosides, all but two beta-lactams, quinolones, folate pathway inhibitors, chloramphenicol, nitrofurantoin and azithromycin. They were naturally resistant to penicillin G, oxacillin, erythromycin, roxithromycin, clarithromycin, ketolides, lincosamides, streptogramins, linezolid, glycopeptides, rifampicin, fusidic acid and fosfomycin. There were only minor medium-dependent differences in susceptibility to most antibiotics. Lysine decarboxylase, malonate assimilation and acid production from arabitol and cellobiose, but not from adonitol and sorbitol, allowed definitive separation of L. adecarboxylata from E. coli. The results of this study form a database that can be applied to validate forthcoming antibiotic susceptibility tests of L. adecarboxylata, and might contribute to its reliable identification. Susceptibility patterns did not indicate obvious therapeutic difficulties for treatment of Leclercia infections. Special attention should be paid to biochemically aberrant leclerciae. Apart from biochemical features, fosfomycin susceptibility might be useful to differentiate between L. adecarboxylata and E. coli.     

Stimulation of chloramphenicol acetyltransferase synthesis by cyclic AMP in bacterial cell systems

The effect of cyclic 3,5-adenosine monophosphate (cAMP) on production of the enzyme chloramphenicol acetyltransferase (CAT) by whole bacterial cells was studied in strainsEscherichia coli CSH-2/R222 and WZ-78/R222 (cya₈₅₅). CAT synthesis in strainE. coli WZ-78/R222 was shown to have an intensity only half as great as that of strainE. coli CSH-2/R222. The production of CAT by strainE. coli CSH-2/R222 was increased only very slightly by cAMP, but its effect on the production of this enzyme in strain WZ-78/R222 was appreciable.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 10, pp. 65–66, October, 1975.     

hEGF和hbFGF的C端(78-154aa)融合蛋白的表达、纯化及活性测定

目的:将人表皮生长因子(hEGF)与人碱性成纤维细胞生长因子(hbFGF)的C端连接,构建出既可与肝素结合,又具有促进细胞生长活性的融合蛋白,并在大肠杆菌中高效表达。方法:将PCR扩增hEGF全基因与PCR扩增hbFGF5端的231bp的片段连接,克隆到表达载体pJN,构建出含融合基因的表达质粒pJRH。将pJRH转化BL21(DE3)表达菌株,获得融合蛋白的表达菌株pJRH(BL)。Westernblot检测表达产物免疫学活性。表达产物上肝素亲和柱和分子筛进行纯化。结果:融合蛋白表达产量为30%,融合蛋白不仅能与肝素结合,而且具有促细胞增殖的活性。Westernblot检测结果显示融合蛋白有EGF的免疫活性。融合蛋白的等电点为5.2。结论:本研究构建了一个hEGF和hbFGF的C端的融合蛋白,该蛋白基本保持了两者原有的生物活性。作为一个新的活性因子,本研究为探讨活性因子蛋白结构与功能的关系奠定基础。     

大肠埃希菌中氟喹诺酮耐药机制功能分析

目的 分析拓扑异构酶的突变和外排泵系统在大肠埃希菌(Escherichia coli)氟喹诺酮类药物耐药机制中的作用.方法 本研究通过基因重组技术对大肠埃希菌中拓扑异构酶不同点突变的功能进行了准确测定,同时也对大肠埃希菌中不同外排泵及膜蛋白的功能进行了分析.结果 在不同的菌株中,acrAB或tolC的切除所引起细菌耐药性的变化不同.对拓扑异构酶点突变的功能分析显示,gyrA中的点突变(S83和D87)在喹诺酮耐药机制中起主要作用,没有gyrA上的点突变,parC上的点突变(S80和A108)对细菌的耐药性不产生影响,但单独gyrA上的点突变(S83和D87)也仅导致敏感菌株对萘啶酸耐药,而对其他氟喹诺酮类药物仍表现为敏感.当对喹诺酮敏感的大肠埃希菌K-12同时具备gyrA(S83L和D87N)和parC(S801和A108V)上的点突变后,重组菌株对氟喹诺酮会自然产生耐药性,而并不需要过度表达的外排泵.结论 拓扑异构酶的突变在大肠埃希菌氟喹诺酮药物的耐药机制中起主要作用,对氟喹诺酮药物耐药的菌株通常应同时具备gyrA和parC上的点突变.     

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesinencoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap⁺ and/or sfa⁺ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing a-haemolysin (Hly⁺) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesinencoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.La PCR a permis de détecter les opérons pap, sfa et afa chez 243 souches de Escherichia coli isolées d'infections de l'arbre urinaire. On observe que respectivement 54, 53 et 2% des souches sont du génotype pap, sfa et afa. Les souches pap⁺ et/ou sfa⁺ sont plus fréquentes dans les pyelonephritis aiguës (94%) que dans les cystites (67%) (P < 0,001) et dans les bactériuries asymptomatiques (57%) (P < 0,001). Les opérons pap et/ou sfa sont décelés chez 90% des souches MRHA⁺ (hémagglutination mannoserésistante) et 37% des souches MRHA⁻ (P < 0,001). La présence des opérons pap et sfa est spécialement significative chez les souches appartenant au type III de MRHA (100%) sans adhésines P, et au type IVa (97 %) exprimant la liaison Gai-Gal typique des adhésines P. Les opérons pap et sfa sont tous deux étroitement associés aux souches toxigéniques produisant l'α-hémolysine (Hly⁺) et le facteur cytotoxique nécrotique de type 1. Une corrélation apparaît entre les opérons pap et sfa et le sérogroupe: 93 % des souches appartenant aux groupes O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 et O83 possèdent ces opérons, et seulement 37 % des souches appartenant à d'autres sérogroupes (P < 0,001) les possèdent. Ces résultats confirment l'utilité de notre typage MRHA pour l'identification (présomptive) des souches pathogènes de E. coli porteuses de divers facteurs de virulence. Ainsi, 85 % des souches possédant à la fois les opérons pap et sfa codant les adhésines sont du type III ou IVa (de MRHA) en relation avec la virulence des souches responsables d'infections urinaires et de bactériémies.     

Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.     

A simple and reliable method to screen isolates of Escherichia coli and Klebsiella pneumoniae for the production of TEM- and SHV-derived extended-spectrum β-lactamases

Objective: To evaluate which of 24 β-lactams used in susceptibility tests best discriminated between strains of Klebsiella pneumoniae and Escherichia coli that produce extended spectrum β-lactamases (ESBLs) from strains that produce older, more familiar, plasmid-mediated β-lactamases such as TEM-1 and SHV-1.
Methods: Susceptibility to the 24 β-lactam agents was determined by agar dilution and disk diffusion methodologies, using 27 strains of K. pneumoniae and E. coli that produced 22 different older plasmid-mediated β-lactamases and 28 strains that produced 17 different ESBLs.
Results: In general, strains that produced ESBLs were intermediate or resistant to cefpodoxime, whereas those that produced other β-lactamases were susceptible to this agent. The agar dilution test exhibited 96% sensitivity and 100% specificity in discriminating these two groups of organisms. The disk diffusion test exhibited 100% sensitivity and 96% specificity. All other β-lactam agents tested were inferior discriminators between the two groups of organisms.
Conclusions: Agar dilution and disk diffusion tests with cefpodoxime can be used to discriminate strains of K. pneumoniae and E. coli that produce ESBLs from those that produce older, plasmid-mediated β-lactamases.