Ability of rosetting or non-rosetting individual control and inflammatory macrophages to kill Escherichia coli X43 intracellularly

An autoradiographic method combined with a rosette technique was used to assess the bactericidal activity of individual control and inflammatory peritoneal macrophages (PM phi) in the presence or absence of expression of Fc receptor for IgG (FcR). There was a lack of FcR reactivity in a certain percentage of both categories of PM phi exposed to E. coli X43, a bacterium which is readily phagocytosed in the presence of specific antibody. Both rosetting and non-rosetting PM phi were capable of phagocytosing E. coli X43, but inflammatory PM phi showed a marked reduction in their capacity to ingest these bacteria compared with control PM phi. Once ingested the E. coli X43 were killed equally well by non-rosetting and rosetting control and inflammatory PM phi.     

膀胱上皮细胞清除胞内大肠杆菌

目的:了解大肠杆菌与膀胱上皮细胞的相互关系,观察大肠杆菌在人膀胱上皮细胞株T24中的动态变化。方法:采用大肠杆菌标准株K12和大肠杆菌临床分离株299侵袭T24细胞,并用庆大霉素杀死细胞外的细菌,分别于细菌侵袭细胞后的4、24及48 h用TritonX-100裂解细胞,释放出细胞内的活细菌,用平板菌落计数法计数胞内活菌数。结果:两株大肠杆菌在T24细胞内都不能生长,随着时间的推移,细胞内大肠杆菌活菌数量呈不断减少趋势。24 h细胞内活菌数下降为4 h活菌数的15%。48h细胞内活菌数进一步减少。结论:膀胱上皮细胞清除进入细胞内的大肠杆菌,这可能是泌尿道天然免疫防御的一个重要防御机     

Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin.

A model for ascending unobstructed urinary tract infection was developed in mice to study the pathogenesis of urinary tract infection induced by Escherichia coli associated with urinary tract infection in humans. Specifically, the model was designed to monitor the initial stages of the infectious process, e.g., bacterial adhesion. Mice were selected since the specificity and intensity of bacterial attachment of pyelonephritogenic E. coli strains to human and mouse uroepithelial cells were similar. Female mice were infected by urethral catheterization and installation of bacteria in the urinary bladder. To maximize clearance of unattached bacteria, no obstructive manipulations were performed. After sacrifice, the persistence of bacteria in kidneys and bladder was determined by viable counts on homogenized tissues. The experimental infection was standardized by using one pyelonephritis (HU734) and one normal fecal (414) E. coli isolate. With both strains all of the bladders became infected, but E. coli 414 was eliminated more rapidly than HU734. The percentage of positive kidney cultures increased with the bacterial inoculum concentration and volume. An inoculum of 0.05 ml containing 10(10) bacteria per ml was selected, giving the highest percentage of positive kidney cultures without detectable bacterial spread to the blood stream. The variation in the percentage of positive kidney cultures possibly depended on the degree of vesicoureteric reflux in the individual animals. Both in the kidneys and in the urinary bladders, strain HU734 yielded higher numbers of bacteria at 24 h and persisted longer than did strain 414. Several E. coli pyelonephritis isolates with properties associated with virulence in the human urinary tract consistently were recovered from mouse kidneys and bladders in higher numbers than E. coli strains of human fecal origin lacking those properties. The role of bacterial adhesion per se is the topic of the accompanying paper.     

Pivotal advance: characterization of mouse liver phagocytic B cells in innate immunity

Although B cells in vertebrates have been thought to lack phagocytic activity, there has been a recent report of such ability by the B cells of early vertebrates such as fish and frogs. Here, we show for the first time that mouse liver IgM(+) B cells actively phagocytose microsphere beads and Escherichia coli and that they effectively kill bacterial cells. Such phagocytic activity is not observed in other liver MNCs, except for F4/80(+) Kupffer cells. In the presence of fresh mouse serum (but not heat-inactivated serum), the heat-killed E. coli phagocytic activity of liver B cells increased significantly but was inhibited significantly by anticomplement component C3 antibody, suggesting E. coli opsonization by serum factors, including complement components. Upon i.v. injection of FITC-labeled E. coli into mice, a substantial proportion of liver B cells phagocytosed the bacteria, as compared with spleen B cells. Functional phagolysosome formation in liver B cells was supported by several reagents showing an acidic change and lysosomes in the phagocytosed vacuoles. Indeed, mouse liver B cells killed viable E. coli more efficiently than did spleen B cells in vitro. Further, E. coli-phagocytic liver B cells produced a substantial amount of IL-12. These results indicate that liver B cells have phagocytic and bactericidal activities similar to those of dedicated phagocytes and may contribute to bacterial clearance.     

Rapid microassays of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro

Simple, rapid microassays for simultaneous measurement of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro are described. All assays employ 96-well flat bottom tissue culture plates which were incubated on a microtitre plate shaker at 37 degrees C. The separate evaluation of ingestion and intracellular killing of E. coli and S. aureus was based on the incorporation of [3H]uridine into viable extracellular bacteria. There was good correlation between plate counts of viable bacteria and amount of radiolabel incorporation. Phagocytosis and killing can be measured in a maximum of 100 microliter reaction mixture, requiring only 2.5 X 10(5) neutrophils per test and the assay is complete within 60 min. Assay of superoxide production by stimulated neutrophils was based on superoxide-dependent reduction of ferricytochrome c as measured spectrophotometrically at 550 nm in wells of tissue culture plates containing 150 microliter of reaction mixture. The assay requires only 1.25 X 10(5) neutrophils per test and is complete within 50 min. Quantitation of hydrogen peroxide was based on horseradish peroxidase-dependent oxidation of phenol red. The technique is as for superoxide detection except that the reaction must be terminated by the addition of 1 M NaOH at the desired time intervals. None of the assays require sampling during the incubation period. The principal advantages of the described techniques are increased simplicity and speed, requirement of low numbers of neutrophils and applicability to analysis of large number of samples in parallel.     

Role of resident macrophages, peripheral neutrophils, and translymphatic absorption in bacterial clearance from the peritoneal cavity.

Microbial pathogens within the peritoneal cavity are thought to encounter three categories of host defense mechanisms: (i) removal mechanisms, which occur via diaphragmatic lymphatic absorption; (ii) killing mechanisms, in which host phagocytes act as effector cells; and (iii) sequestration mechanisms due to fibrin trapping and the formation of adhesions between visceral surfaces. We sought to define and quantitate the relative role of the first two components in an experimental rat model of Escherichia coli peritonitis in which fibrinous adhesions do not form. Intraperitoneal challenge with greater than or equal to 2 X 10(8) CFU of viable E. coli led to an initial decline in bacterial numbers followed by ongoing proliferation and greater than 50% mortality. With inocula of less than or equal to 5 X 10(7) CFU, elimination of bacteria occurred after moderate initial proliferation, and no mortality ensued. Nonviable, radiolabeled E. coli organisms were utilized to examine bacterial clearance via translymphatic absorption and phagocytosis. Both processes were extremely rapid, serving to eliminate free bacteria rapidly within the peritoneal cavity. Although macrophages and polymorphonuclear leukocytes within the peritoneal cavity demonstrated similar phagocytic capacities, the predominance of macrophages at the time of the initial bacterial insult led to the conclusion that these cells, in addition to translymphatic absorption, represent the first line of host defenses, acting to eliminate bacteria in the incipient stages of infection.     

Invasion of HEp-2 cells by fecal isolates of Aeromonas hydrophila

Human diarrheal isolates of Aeromonas hydrophila were found to be invasive in HEp-2 cell monolayers. Microscopic examination of two isolates from patients with symptoms of dysentery revealed that infected HEp-2 cells contained up to 50 bacteria localized within areas of the cytoplasm. A quantitative invasion assay was developed, using viable counts of total intracellular bacteria. Five fecal isolates of A. hydrophila were classified as invasive, with an average of 0.1 up to 2 bacteria per HEp-2 cell, as compared to 7 for an invasive Escherichia coli strain. 3 other fecal isolates, 1 reference strain, and 10 water isolates of A. hydrophila were similar to a noninvasive E. coli strain, with less than 0.008 bacteria per HEp-2 cell. All isolates were screened for plasmid DNA; no common plasmid was found in the invasive strains, and the loss of a 6.0-megadalton plasmid from one of these strains had no observable effect on invasiveness. Thus some A. hydrophila isolates are capable of cellular penetration and replication, and this may be an important chromosomally determined virulence property of the organism.     

Attachment of enterotoxigenic Escherichia coli to human intestinal cells.

An adherence test was developed with human ileal cells isolated from patients with a long-standing ileostomy by saline lavage through the stoma. Enterotoxigenic Escherichia coli isolated from humans bound to the human ileal cells to a greater extent (1.2 x 10(6) bacteria bound) than did control E. coli strains, including K99 pili antigen-producing strains (whether originally isolated from pigs or calves), the rabbit pathogen RDEC-1, the laboratory-derived nontoxigenic strain 334LL, or human normal fecal strains. However, K88 strains, either K88ab or K88ac, bound to the human ileal cells as well as did enterotoxigenic E. coli. Ileal cells isolated from two donors with different blood types behaved similarly. These cells remained viable and retained their binding ability for at least 3 days when stored in tissue culture medium at 4 degrees C.     

Interactions of Escherichia coli and Proteus mirabilis with mouse mononuclear phagocytes

Five strains of enterobacteria (three of Escherichia coli and two of Proteus mirabilis) were studied to assess and compare their phagocytic uptake and intracellular killing by mouse macrophages. Each strain was injected intraperitoneally into separate groups of mice and peritoneal exudate cells were harvested after 3 min for phagocytosis to occur in vivo. Acridine orange staining showed that there were approximately 10-fold fewer intracellular P. mirabilis than E. coli cells. The average numbers of viable intracellular bacteria per leucocyte were 0.03 and 0.02 for P. mirabilis strains M13 and H1, respectively, and 0.48, 0.45, and 0.28 for E. coli strains M14, A-D M5 and H40. Thus, both P. mirabilis strains were ingested less readily than any of the three E. coli strains (p less than 0.01). The rates of in-vitro intracellular killing were similar for all five strains of bacteria. The intracellular killing constants (Kk) for the three mouse isolates were 0.017, 0.016 and 0.020 min for E. coli M14 and A-D M5, and P. mirabilis M13, respectively; the Kks for the two human isolates were 0.026 and 0.029/min for E. coli H40 and P. mirabilis H1, respectively. The Kks for all five strains were not significantly different. Assuming that the numbers of viable intracellular bacteria at the beginning of the assay represented 100% viability, 6-17% of the intracellular bacteria remained viable after 2 h, reflecting log10 3.9-5.6 bacteria (6-8) x 10(6) peritoneal exudate cells. Intravenous injection of these five strains into separate groups of mice demonstrated that the P. mirabilis strains were more virulent than the E. coli strains. Injection of each P. mirabilis strain was associated with ruffled fur and death, whereas mice given any of the three E. coli strains remained visibly healthy and none died. Consistent with these observations, quantitation of viable bacteria in the liver and spleen showed that greater numbers of P. mirabilis M13 than of E. coli M14 or A-D M5 persisted in these organs; similarly greater numbers of P. mirabilis H1 than of E. coli H40 persisted in the liver and spleen. Because the rates of intracellular killing of these five strains were similar, the relative virulence of both strains of P. mirabilis appeared to be associated with decreased phagocytic uptake rather than differences in intracellular survival.     

Comparative study of intramacrophagic penetration and action on phagocytosis of a macrolide (spiramycin) and a fluoroquinolone (pefloxacin)

Antibiotic-phagocyte interaction is an important parameter involved in the elimination process of intracellular bacteria. The aim of the present study was to compare, using the same model, the phagocytic uptake and the intracellular activity of a macrolide and a quinolone. Accumulation of spiramycin and pefloxacin by guinea pig peritoneal macrophages (GPpM) was studied by means of a velocity-gradient centrifugation technique and expression of the ratio of the cellular concentration of antibiotic to the extracellular concentration (IC-EC). Three aspects of Staphylococcus aureus (209-P) phagocytosis were studied: 1) the phagocytic capacity (PC), mean number of ingested cocci by GpPM; 2) the phagocytic activity (PA), percentage of phagocyting GpPM with at least one bacterium; 3) the number of intracellular viable bacteria (IVB). Phagocytic capacity and phagocytic activity were determined by fluorescence microscopy using S. aureus stained with acridine orange. Intracellular viable bacteria were quantified by standard colony counts (CFU). The ratios of intracellular to extracellular concentration of pefloxacin and spiramycin are respectively 9 and 23. Pretreatment of guinea-pig peritoneal macrophages with 10 mg/l of each antibiotic does not modify phagocytic capacity and phagocytic activity, but lead to a decrease of intracellular viable bacteria. S. aureus pretreatment with 1/4 the MIC of each antibiotic increased phagocytic capacity and phagocytic activity and decrease intracellular viable bacteria (especially spiramycin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic effect of an alpha-hemolytic Escherichia coli strain on human blood monocytes and granulocytes in vitro.

The alpha-hemolytic Escherichia coli strain C134-73 Hly+ was primarily cytocidal to human blood monocytes and granulocytes in vitro in the presence of fresh autologous plasma. Monocytes and granulocytes underwent marked morphological changes during incubation with the bacteria, and the percentages of intact phagocytes decreased progressively with the time of incubation. The cytotoxic effect increased with the number of bacteria per phagocyte and was produced by log-phase microorganisms only. Neither free hemolysin nor free cytotoxic activity to leukocytes could be detected in the incubation medium if the bacteria were removed from the test system. Bacteria-free culture supernatants containing alpha-hemolysin were cytotoxic to monocytes, granulocytes, and lymphocytes, monocytes and granulocytes being the most sensitive. However, this effect was abolished by fresh autologous plasma. Two nonhemolytic strains, a mutant derivative of C134-73 Hly+ and strain X43, were not cytotoxic. These observations suggest that alpha-hemolysin associated with the bacterial cells may enhance the virulence of E. coli by injuring phagocytes, whereas free alpha-hemolysin may be of minor importance because its cytotoxic effect is neutralized by host plasma.     

RIP2对大肠杆菌的抗菌作用研究

目的: 探讨受体相互作用蛋白2(RIP2)对人肠细胞清除大肠杆菌的作用。方法: 使用阳离子聚合物JetPei^TM介导人 RIP2 基因(pEGFP-C2-PIP2)转染人结肠癌SW480细胞株,以转染空质粒及未转染的SW480细胞株作为对照,应用RT-PCR检测外源性RIP2mRNA水平的变化、Western blotting法检测细胞RIP2蛋白的表达;并利用大肠杆菌ATCC25922感染转染和未转染的SW480细胞24 h, 用平板培养菌落计数活菌数。应用p38 MAPK通路抑制剂SB203580作用于3组细胞,计数活菌数。结果: 与对照组相比,转染RIP2组其mRNA和蛋白表达水平均有明显升高(P<0.01, P<0.05),表明RIP2表达质粒成功转染SW480细胞。转染RIP2细胞能清除胞内大肠杆菌;而阻断p38 MAPK信号通路后,RIP2清除胞内大肠杆菌的作用被阻断。结论: RIP2转染细胞可有效清除胞内大肠杆菌,这种胞内细菌的清除作用可能与p38 MAPK信号通路有关。这些结果提示RIP2在细菌感染性疾病治疗中具有广阔的临床应用前景。     

Antigen shared by human hemopoietic precursor cells and T and B lineage cells

In this study, we investigated antigens present at the surface of acute myeloblastic leukemia (AML) cells by using the monoclonal antibody (MAb) approach. The MAb AGF43 reacted with acute myeloid and lymphoid leukemia cells and chronic lymphocytic leukemia cells and was unreactive against chronic myeloid leukemia cells. A large proportion of AML blasts showing minimal or no differentiation (AML-M1) were intensely labeled by AGF43 in contrast to a smaller percentage of blasts showing partial differentiation (AML-M2 and acute myelomonocytic leukemia). The AGF43 antigen is expressed by bone marrow lymphoid (TdT+) and myeloid (CFU-GM) progenitor cells, 95% of B cells and 65% of T cells in the blood and absent from monocytes. Only 17% of normal myeloblasts were weakly stained by AGF43. Sections of tonsil and spleen were used to confirm that, unlike antibodies to MHC class II antigens, AGF43 stained a majority of T cells and macrophages were unreactive. In conclusion, the MAb AGF43 identifies a new precursor cell antigen. The distribution of this antigen during normal myelopoiesis and on AML cells support the suggestion that acute myeloid leukemias originate in pluripotent or closely related myeloid stem cells.     

Enzyme-linked immunosorbent assay for adherence of bacteria to animal cells.

Epithelial cells scraped from human oral mucosa and from pig intestines were immobilized onto the flat bottom surfaces of microtiter plates to study the adherence of various bacterial species to host cells. Bacterial adherence was quantitated either by an enzyme-linked immunosorbent assay technique with specific antibacterial serum as the first antibody followed by peroxidase-conjugated second antibody or by using biotinylated bacteria and avidin-peroxidase as the detecting agent. Unlabeled Escherichia coli and purified E. coli 987P fimbriae inhibited the adherence of biotinylated E. coli to immobilized enterocytes. The adherence of a mannose-sensitive strain of E. coli to immobilized oral epithelial cells was inhibited by mannose derivatives. The adherence of fimbriated E. coli 987P to immobilized enterocytes was approximately four times higher than the adherence of a nonfimbriated variant of the same strain. The adherence of Streptococcus pyogenes to oral cells was detected in the range of 10 to 150 bacteria per cell and was inhibited by lipoteichoic acid and albumin. The data suggest that the putative receptors which bind bacteria on the immobilized cells retain a functional form similar to that of native cells in suspension. The proposed adherence assay is easy to perform, allows the detection of specific adherence of test bacteria, and provides objective quantitation of adherence with a sensitivity of 10 bacteria per cell. Most importantly, the assay allows the testing of many variables in the same day.     

Intra-abdominal abscess formation in mice: quantitative studies on bacteria and abscess-potentiating agents

A model of intra-abdominal (IA) abscess formation has been developed in mice. Intraperitoneal (i.p.) injection of a mixture of a potentiating agent (autoclaved colonic and caecal contents (ACC), 0.2 mg dry wt/mouse or sterile bran, 1 mg dry wt/mouse), Escherichia coli (1 X 10(6) colony forming units (cfu)/mouse) and Bacteroides fragilis (5 X 10(8) cfu/mouse) induced abscesses in 98% of mice inoculated. The abscesses persisted for at least 4 weeks in 60% of inoculated animals, and for 10 weeks in 36%. From 1 to 5 abscesses per mouse were found. Abscess formation was quantified by weighing the dissected abscesses and by culturing bacteria from them. Histologically, the abscesses were characterized by a central region of polymorphonuclear leucocytes, often with a thin mononuclear phagocyte infiltrate surrounding it, and an outer wall of vascularized connective tissue. Fluorescent antibody studies demonstrated that antigens from both bacterial species were distributed throughout the abscess. At the concentrations used, neither ACC nor sterile bran induced formation in the absence of viable bacteria.     

Intra-abdominal abscess formation in mice: quantitative studies on bacteria and abscess-potentiating agents.

A model of intra-abdominal (IA) abscess formation has been developed in mice. Intraperitoneal (i.p.) injection of a mixture of a potentiating agent (autoclaved colonic and caecal contents (ACC), 0.2 mg dry wt/mouse or sterile bran, 1 mg dry wt/mouse), Escherichia coli (1 X 10(6) colony forming units (cfu)/mouse) and Bacteroides fragilis (5 X 10(8) cfu/mouse) induced abscesses in 98% of mice inoculated. The abscesses persisted for at least 4 weeks in 60% of inoculated animals, and for 10 weeks in 36%. From 1 to 5 abscesses per mouse were found. Abscess formation was quantified by weighing the dissected abscesses and by culturing bacteria from them. Histologically, the abscesses were characterized by a central region of polymorphonuclear leucocytes, often with a thin mononuclear phagocyte infiltrate surrounding it, and an outer wall of vascularized connective tissue. Fluorescent antibody studies demonstrated that antigens from both bacterial species were distributed throughout the abscess. At the concentrations used, neither ACC nor sterile bran induced formation in the absence of viable bacteria.     

Enzyme immunoassay for detection of antibody-coated bacteria.

To quantitatively evaluate factors potentially affecting antibody coating of bacteria in urine, we developed an assay with enzyme-linked rather than fluorescein-conjugated immunoglobulin. Using the enzyme immunoassay (EIA) in an in vitro system in which concentrations of serotype O44 Escherichia coli and antibody titer to E. coli Orr O44 O antigen were known, we compared specimens run in parallel with a fluorescent antibody (FA) assay. At greater than or equal to 10(5) bacteria per ml, antibody titer to homologous O antigen correlated directly with absorbance in the EIA. Both tests had sensitivities exceeding 95% in specimens containing greater than or equal to 10(5) bacteria per ml, but the FA test detected 23 of 27 positive specimens with less than 10(5) bacteria per ml compared with 21 of 43 detected by EIA (P = 0.002). However, nonspecific fluorescence caused false positives in 8% of negative tests run by FA compared with 1% of simultaneous EIA tests (P = 0.05). pH alterations and pretreatment of bacteria with antibiotics did not affect either test. Heterologous E. coli strains showed no cross-reactivity with O44 antiserum, but all Staphylococcus aureus isolates tested caused false positives in both assays, and one Klebsiella strain repeatedly caused a false-positive FA assay. The EIA appears to be a simple, quantitative, and specific technique for detection of antibody-coated bacteria in this experimental system.     

Development of VH81X transgene-bearing B cells in fetus and adult: sites for expansion and deletion in conventional and CD5/B1 cells

The most D-proximal functional VH gene, VH81X, is preferentially expressed in the mouse fetal B cell repertoire; however, it is expressed in few B cells in the adult. To determine when VH81X gene expression affects size and phenotype of particular stages in B cell differentiation, transgenic mice have been developed expressing a germline fetal liver-derived VH81X-mu rearrangement. Comparative analysis of B lymphopoiesis reveals similarities and differences between fetal liver and adult bone marrow which pinpoint developmental stages in mice during which VH81X-expressing B cell progenitors expand or deplete compartment sizes. These include a similar reduction in c- kitR+ and establishment of a predominant CD43low/HSAhigh phenotype within the B220+ CD43+ compartment which is dependent on the association of the transgene with lambda 5. In contrast, the CD43- pre- B and immature B cell compartments are expanded in the fetus but not in the adult. In addition, there are other factors that later disfavor the survival of VH81X-expressing B1 and B2 cells. Thus the failure to detect VH81X-bearing B cells in the adult is the result of a multistep selection process occurring at all stages during B repertoire expansion.      

Role of endotoxemia in cardiovascular dysfunction and lethality: virulent and nonvirulent Escherichia coli challenges in a canine model of septic shock.

We investigated whether the severity of septic shock is determined by virulence factors associated with or the levels of endotoxemia produced by two Escherichia coli strains. Canines were challenged intraperitoneally with an E. coli strain (O6:H1:K2) that has virulence factors associated with human disease or with an equal dose of a nonvirulent strain (O86:H8) that lacks these factors. Both strains were administered in viable, heat-killed, and purified endotoxin forms. Median survival times with the virulent strain compared with the nonvirulent strain were shorter with viable bacteria (5 x 10(10) CFU/kg) (144 h versus > 672 h; Wilcoxon, P = 0.03), longer with heat-killed bacteria (5 x 10(9) CFU/kg) ( > 676 h versus 26 h; P = 0.03), and similar with purified endotoxin (15 mg/kg) (28 h versus 48 h; P = 0.71). However, whether the challenge contained viable bacteria, heat-killed bacteria, or purified endotoxin, the virulent strain produced less endotoxemia (P = 0.001). Hence, the changing outcomes with differing forms of the two strains cannot be attributed solely to endotoxin levels. The viable virulent strain caused less endotoxemia but more harm, and this does not appear to be explained by a more potent endotoxin or other heat-stable component. This study suggests that circulating endotoxin levels per se are less important in the outcome of septic shock than virulence factors associated with E. coli strains. Furthermore, the data call into question the significance of the endotoxin concentration in the blood in predicting the severity of shock and the lethality of gram-negative infections.     

Pharmacodynamic effects of amikacin, ciprofloxacin and imipenem on growing and non-growing Escherichia coli and Pseudomonas aeruginosa

Objective: To compare control-related effective regrowth times (CERTs) and postantibiotic effects (PAEs) of amikacin, ciprofloxacin and imipenem on growing and non-growing Escherichia coli and Pseudomonas aeruginosa .
Methods: CERTs and PAEs of amikacin, ciprofloxacin and imipenem were determined with bioluminescence assay of bacteriai ATP and viable counts.
Results: Negative viable count PAEs of amikacin and imipenem occurred on growing bacteria, but bioluminescence PAEs were positive. CERTs were equal with both methods. Amikacin and ciprofloxacin induced long, concentration-dependent CERTs on growing and non-growing cultures. Amikacin (32 mg/L) prevented regrowth of E. coli and induced a CERT of 6.0 h on P. aeruginosa ; corresponding CERTs on non-growing bacteria were 3.4 h and 3.3 h, respectively. Ciprofloxacin (8 mg/L) prevented regrowth of both strains in growing cultures and induced CERTs of 5.1 h on non-growing E. coli and 13.3 h on P. aeruginosa . Imipenem induced a concentration-dependent CERT on growing bacteria and no CERT on non-growing cultures. Imipenem (16 mg/L) induced a CERT of 5.3 h on growing P. aeruginosa and 3.2 h on E. coli.
Conclusion: Amikacin and ciprofloxacin induced strong pharmacodynamic effects on growing and non-growing E. coli and P. aeruginosa , while imipenem was only effective on growing cultures.