A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics.     

Bactericidal antibiotics do not appear to cause oxidative stress in Listeria monocytogenes

Oxidative stress can be an important contributor to the lethal effect of bactericidal antibiotics in some bacteria, such as Escherichia coli and Staphylococcus aureus. Thus, despite the different target-specific actions of bactericidal antibiotics, they have a common mechanism leading to bacterial self-destruction by internal production of hydroxyl radicals. The purpose of the present study was to determine if a similar mechanism is involved in antibiotic killing of the infectious human pathogen, Listeria monocytogenes. We treated wild-type L. monocytogenes and oxidative stress mutants (Δsod and Δfri) with three different bactericidal antibiotics and found no difference in killing kinetics. In contrast, wild-type E. coli and an oxidative stress mutant (ΔsodA ΔsodB) differed significantly in their sensitivity to bactericidal antibiotics. We conclude that bactericidal antibiotics did not appear to cause oxidative stress in L. monocytogenes and propose that this is caused by its noncyclic tricarboxylic acid (TCA) pathway. Hence, in this noncyclic metabolism, there is a decoupling between the antibiotic-mediated cellular requirement for NADH and the induction of TCA enzyme activity, which is believed to mediate the oxidative stress reaction.     

6-formylpterin intracellularly generates hydrogen peroxide and restores the impaired bactericidal activity of human neutrophils.

The effects of 6-formylpterin on the impaired bactericidal activity of human neutrophils were examined ex vivo. When neutrophils isolated from fresh blood were incubated with 6-formylpterin, the intracellular production of hydrogen peroxide (H(2)O(2)) occurred. The H(2)O(2) generation by 6-formylpterin in neutrophils occurred in the presence of diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. When neutrophils were incubated with DPI, the killing rate of catalase-positive bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), significantly decreased. This impaired bactericidal activity of the DPI-treated neutrophils was a mimic for chronic granulomatous disease (CGD). However, the killing rate of the DPI-treated neutrophils against E. coli and S. aureus significantly increased when 6-formylpterin was administered. Since 6-formylpterin intracellularly generates H(2)O(2) independent from the NADPH-oxidase, it was considered to improve the impaired bactericidal activity of the DPI-treated neutrophils. The use of 6-formylpterin may serve as an option of therapy for CGD.     

Biochemical and genetical approaches to the mechanism of action of penicillin

Since the discovery in 1965 that penicillin inhibits the transpeptidation reaction in peptidoglycan synthesis, a considerable effort has been put into the purification of enzymes that catalyse this reaction. This has resulted in the recognition that bacteria possess multiple forms of these penicillin-sensitive enzymes and has made it difficult to identify the precise target that penicillin inactivates to kill the organism. Recently penicillin-sensitive enzymes have been detected and studies as penicillin-binding proteins on sodium dodecyl sulphate polyacrylamide gels. The availability of this convenient method for identifying penicillin-sensitive enzymes has allowed biochemical and genetical approaches to be used to dissect their roles in the lethal effects of penicillin and other beta-lactam antibiotics. Three penicillin-binding proteins (1 B, 2 and 3) have been identified as killing targets for penicillin in Escherichia coli, whereas four other binding proteins are not implicated in the mechanism of action of the antibiotic. The complex biological effects that beta-lactam antibiotics produce on the growth of E. coli can be explained by their interaction with the three killing targets. Progress in the correlation of penicillin-binding proteins with penicillin-sensitive enzymes and in the development of strains of E. coli that overproduce penicillin-binding proteins is discussed.     

Physiological studies of the regulation of beta-lactamase expression in Pseudomonas maltophilia.

The kinetics of beta-lactamase induction in Pseudomonas maltophilia IID1275/873 were investigated. Upon induction with beta-lactam antibiotics, a correlation was seen between the increase in specific beta-lactamase activity and the generation time, as well as the concentration of inducer in the medium. The specific beta-lactamase activity increased slowly within the first 0.5 generation and then more rapidly; it decreased regularly after about 2 generations of growth in the presence of inducer. This decrease could presumably be attributed to the continuous breakdown of inducer by beta-lactamases in the culture medium. In a chemostat culture with continuous supply of fresh inducer-containing medium, the specific beta-lactamase activity could be stabilized at a high level over several generations. Removal of the beta-lactam after a certain induction time showed that a short exposure of the bacteria to inducer caused induction kinetics comparable to those resulting from continuous exposure of the cells to inducer. The two beta-lactamases of P. maltophilia, L1 and L2, were induced simultaneously under various experimental conditions.     

Bactericidal activity of catechin-copper (II) complexes against Staphylococcus aureus compared with Escherichia coli

The bactericidal activity of catechin-copper (II) complexes against Staphylococcus aureus compared with Escherichia coli was investigated in relation to the generation of hydrogen peroxide and the binding of Cu(II) ion onto the bacteria. The bactericidal activity of catechin-Cu(II) complexes against Staph. aureus (Gram-positive) was much lower than that against E. coli (Gram-negative), suggesting that the binding of copper ions to the surface of bacterial cells plays an important role in the bactericidal activity of catechin-Cu(II) complexes.     

Membrane damage to Escherichia coli and bactericidal kinetics by the alternative complement pathway of channel catfish

1. Increased permeability of cytoplasmic membranes in Escherichia coli was a consequence of alternative complement pathway (ACP) activity of serum of channel catfish, Ictalurus punctatus. Evidence was provided by beta-galactosidase activity extracellularly when E. coli was incubated with catfish serum. 2. Lesions were detected on outer membranes of E. coli following exposure to catfish serum. 3. Catfish ACP induced a temporal sequence of pre-killing and killing phases. 4. Loss of cell viability, killing rate and cytoplasmic enzyme release increased with increasing serum concentrations. 5. By incubating E. coli with sera treated to remove complement, both release of cytoplasmic enzyme and bactericidal activity were eliminated. 6. Lethal activity associated with channel catfish ACP against Gram-negative bacteria was functionally comparable to that seen in mammalian and reptilian systems.     

Antibacterial properties of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide-specific monoclonal antibody (MAb) in a murine thigh infection model: combined effects of MAb and ceftazidime

A murine monoclonal antibody (MAb) specific for the Pseudomonas aeruginosa immunotype 1 (It-1) lipopolysaccharide (LPS) O-side chain was evaluated in terms of its in vitro bactericidal opsonophagocytic activity and in vivo bacterial killing in a mouse thigh infection model. An immunoglobulin (Ig) G2a MAb Ld3-2F2, specific for It-1 LPS, mediated in vitro complement-dependent opsonophagocytic killing at a concentration of 10 microg/ml. MAb-mediated, complement-dependent killing also occurred in the absence of neutrophils at serum concentrations in excess of 20%. A remarkable synergy was observed in opsonophagocytic assays between MAb Ld3-2F2 (0.5 microg/ml) and ceftazidime (1/4 MIC). The administration of MAb Ld3-2F2 at a level of 1 microg resulted in a significant decrease in the number of bacteria in the thigh muscles of normal mice, while 100 microg of the same MAb was required for one log of reduction in the number of bacteria at the same site in neutropenic mice. The combined therapy with MAb Ld3-2F2 and ceftazidime provided a significant reduction in the density of bacteria in the thigh muscle at 9 hr post-infection in normal and neutropenic mice as compared with those after treatment alone or with no treatment (P< 0.01). These favorable in vitro and in vivo interactions of an LPS-specific IgG MAb and ceftazidime strongly support their potential for use in therapy, combined with an LPS-reactive MAb and parenteral antipseudomonas beta-lactam antibiotics in the therapy of systemic Pseudomonas infections in normal and neutropenic hosts.     

Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics

Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli chi2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42 degrees C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli chi1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of chi1776 or in chi2452 grown at 32 degrees C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of chi2452 grown at 42 degrees C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of chi2452 cells grown at 42 degrees C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of chi2452 grown either at 42 or 32 degrees C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings.     

High level expression of His-tagged colicin 5 in E. coli and characterization of its narrow-spectrum bactericidal activity and pore-forming action

Since antibiotics with a broad spectrum of activity would select for resistance among the normal flora, colicins having a narrow spectrum of activity can potentially be developed as novel antibiotics. Colicin-based bactericidal proteins with modified spectra of activity might also be developed by further gene fusion or gene modification. To achieve these goals, it is necessary to first build an efficient system to produce large amounts of colicin. In the presence of an immunity gene, we successfully constructed an expression vector pQE30-cfa-cfi producing high levels of His-tagged colicin 5 (60-80 mg/L). We found that the purified His-tagged colicin 5 possesses narrow-spectrum bactericidal activity against nonimmune Escherichia coli cells. It is highly toxic to sensitive E. coli cells at a low concentration of 0.01 microg/ml, while it is nontoxic to other tested gram-negative bacteria, gram-positive bacteria and yeast at a high concentration of 1000 microg/ml. His-tagged colicin 5 kills sensitive cells by permeabilizing their cell membranes. It is not hemolytic to rabbit erythrocytes and has no obvious cytotoxicity to other nucleated mammalian cells at a high concentration of 500 microg/ml. The His-tagged colicin 5 is similar to wild-type colicin 5 in spectrum and bactericidal activity against E. coli. It is a potential novel antibiotic particularly for treating human and animal infections caused by pathogenic E. coli. Besides producing high level of colicin 5, the highly efficient expression vector constructed here might also be a useful tool to develop colicin-based artificial bactericidal proteins.     

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12.Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.     

The threat of antibiotic resistance in Gram-negative pathogenic bacteria: beta-lactams in peril!

Beta-lactam antibiotics are the cornerstone of our antibiotic armamentarium. By inhibiting bacterial cell wall synthesis, they are highly effective against Gram-positive and Gram-negative bacteria. Unfortunately, bacteria have evolved sophisticated resistance mechanisms to combat the lethal effects of beta-lactam antibiotics. Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae are all able to evade killing by penicillins, cephalosporins and carbapenems. This multi-drug resistant phenotype that challenges health care workers worldwide is caused by an array of resistance determinants. These include altered expression of outer membrane proteins and efflux pumps, along with an increasing arsenal of beta-lactamases. Future strategies in beta-lactam design must take into account the complex nature of resistance in Gram-negative pathogens.     

Sensitivity of bacteria exposed to subinhibitory concentrations of antibiotics to the action of serum and liver phagocytes

Incubation of E.coli and S. aureus with subinhibitory concentration (1/5 MIC) of cefamandole modified bacterial morphology and resistance to host defence mechanisms. In fact, cefamandole induced filamentous forms of E. coli, when added to the perfusing medium of the isolated rat liver system, were phagocytized at a much fortes rate then control bacteria, but appeared less sensitive to serum bactericidal activity. In contrast, S.aureus, after exposure to the antibiotic, was more sensitive to the bactericidal activity of serum then controls, while treated and untreated cells were phagocytized at the some rate. The data suggest that even at low doses some antibiotics may alter bacterial structure and increase their susceptibility to host factors.     

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4(-/-) mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4(-/-) mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4(-/-) mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4(-/-) neutrophils. The bacterial killing and clearance capabilities observed in OLFM4(-/-) mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.     

Susceptibility of clinical isolates of Staphylococcus aureus to killing by oxacillin.

Sixty clinical isolates of Staphylococcus aureus have been screened for their relative susceptibility to the killing action of oxacillin. Only one of these strains was found to be exceptionally resistant to the bactericidal effect of this and other beta-lactam antibiotics. This ability to survive oxacillin inhibition of cell wall synthesis has been called "tolerance". The characteristics of the tolerant organism, which has been designated the Evans strain, in comparison with other isolates of S. aureus indicate that this form of resistance is not apparent from the minimal inhibitory concentration, is not related to an abnormal growth rate, and can be enhanced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine.     

Synergistic interactions between cefalexin and kanamycin in Mueller–Hinton broth medium and in milk

Aims:  This study investigated the in vitro bactericidal activity of an intramammary drug product by comparing the kill kinetics of cefalexin and kanamycin, alone and in fixed ratio combination, against Streptococcus uberis , Staphylococcus aureus and Escherichia coli strains isolated from field cases of bovine mastitis. The effect of milk as a diluent on the rate of bacterial killing was also assessed.
Methods and Results:  Antibacterial kill kinetics was determined against each bacterial strain in Mueller–Hinton broth (MHB) and in milk. In MHB, the fixed cefalexin : kanamycin combination (1·5 : 1 w/w) exhibited a clear synergistic bactericidal activity against the strains tested. The combination also showed an enhanced killing activity in milk, as compared to either agent alone.
Conclusions:  The data show the occurrence of synergistic interactions between cefalexin and kanamycin, resulting in a faster and enhanced bactericidal activity against major mastitis pathogens.
Significance and Impact of the Study:  The study demonstrated that the combination exhibited a larger and faster rate of kill of S. aureus , S. uberis and E. coli compared to either cefalexin or kanamycin alone, while using a lower total amount of antibiotic. Synergistic and additive effects were also observed when milk was used as a medium. The results support the use of this combination of narrow spectrum antibiotics to treat clinical mastitis via the intramammary route and provide data on its killing kinetics.     

Bactericidal activities of rat defensins and synthetic rabbit defensins on Staphylococci, Klebsiella pneumoniae (Chedid, 277, and 8N3), Pseudomonas aeruginosa (mucoid and nonmucoid strains), Salmonella typhimurium (Ra, Rc, Rd, and Re of LPS mutants) and Escherichia coli.

Rat defensins were purified and tested for in vitro bactericidal assay against gram-positive and gram-negative bacteria. Staphylococcus aureus (209P, Cowan I, Smith diffuse and Smith compact) were resistant to defensins, whereas Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus lysodeikticus and Bacillus subtilis were less sensitive. Gram-negative bacteria, such as Pseudomonas aeruginosa (mucoid and K) and Klebsiella pneumoniae (Chedid, 277, and 8N3 which were heavily capsulated, moderately capsulated and noncapsulated, respectively) were all very sensitive to defensins and killed within 20 min. Escherichia coli was moderately sensitive and the rough mutants of lipopolysaccharide (LPS) of Salmonella typhimurium LT2, such as Ra, Rc, Rd, and Re were equally sensitive to defensins, being killed within 40 min. Lysozyme did not show any bactericidal activity except against M. lysodeikticus and B. subtilis, whereas it enhanced the bactericidal activity of defensins against P. aeruginosa, E. coli, and K. pneumoniae and suppressed the killing activity of defensins against S. typhimurium and S. aureus. With regard to the three synthetic rabbit defensins, NP1, NP4, and NP5, NP1 showed strong bactericidal activity against K. pneumoniae 277, comparable to that of rat defensins. Neither NP4 nor NP5 showed any bactericidal activity, while NP5 rather enhanced the bactericidal activity of NP1 against K. pneumoniae 277.     

临床分离的革兰阴性细菌的耐药谱及耐药机制的研究

目的 了解前临床上分离G^－细菌的药敏状况和耐药机制及提供合理使用抗生素的依据。方法 主要使用MICROSCAN WALKAWAY／－40全自动微生物分析仪对1999年3月－2000年3月全院住院病人的尿、痰、腹水、脓液、创面、前列腺液、血液等培养呈阳性的标本进行细菌鉴定和药敏试验,结果共检出G^-菌1152株包括27个菌属80个菌种,觉细菌是大肠埃希菌（16.1％）、铜绿假单胞菌（6.5％）、肺炎克雷伯菌（5.3％）等。G^-杆菌（除不动杆菌外）对第三代头孢霉素敏感率已降到（3.0%-76.1%)、对亚胺培南（80.7%-92%)、头孢哌酮／舒巴坦（58.8％－100％）、阿米卡星（41.4%-93.2%)、环丙沙星（30.5％－67.3％）较敏感;对第三代头孢霉素产生超广谱β－内酰胺酶（ESBLs）,肺炎克雷伯菌高达35.0%-36.9%,大肠5埃希菌达21.8％－23％。对常用β-内酰胺类抗生素产诱导酶（IB）,铜绿假单胸菌高达51％－60.9％,弗劳地枸橼酸菌达4.5％－63.6％,阴沟肠杆菌达8.7%-35.3％。结论 目前G^－杆菌对β-内酰胺类抗生素药的主要机制是产生ESBLs和IB0G^-杆菌引起的感染首选亚胺培南单用或第三代浆孢霉素复合制剂（头孢哌酮／舒巴坦）联合阿米卡星或氟喹酮类,第三代头孢霉素除非药敏提示否则不宜选用。     

Do beta-lactam antibiotics permeabilize the outer membrane of gram-negative bacteria? An electrochemical investigation

The effects of cefotaxime and EDTA on the reducing activity of Escherichia coli and Staphylococcus aureus cultures growing in the presence of lipoic acid (LA) were investigated by potential-time measurements. The potentiometric responses of E. coli cultures exposed to EDTA indicated enhanced transmembrane transport of LA as a consequence of the outer membrane permeabilization by the chelator, whereas EDTA exerted no effect on the reducing activity of S. aureus cultures. In the same way, cefotaxime stimulated the reducing activity of E. coli, but not that of S. aureus. These results suggest that cefotaxime, and, more generally, a great variety of beta-lactam antibiotics, are able to permeabilize the outer membrane of Gram-negative bacteria.     

Comparison of the bactericidal activity of different vertebrate sera

Schwab, G. E. (University of Adelaide, Adelaide, South Australia), and P. R. Reeves. Comparison of the bactericidal activity of different vertebrate sera. J. Bacteriol. 91:106-112. 1966.-The bactericidal activity for gram-negative bacteria of normal sera from eight species of vertebrates was investigated to compare complement-mediated killing in sera from animals representing various classes of vertebrates. Although all the sera were bactericidal, there was considerable variation in the range of bacterial species killed and in the bactericidal titers. The temperature dependence of the bactericidal and hemolytic complement activities was also studied. The curves relating activity to temperature were similar in shape for sera from a homeotherm and poikilotherms, but those for the homeotherm reached their maximum at temperatures of 5 to 10 C higher than the poikilotherms. The role of lysozyme and complement in killing rough gram-negative bacteria was examined, and the results suggest that, as for smooth organisms, killing is due to antibody plus complement. These natural antibodies, like those for smooth strains, were shown to be specific.