Effects of low, subinhibitory concentrations of antibiotics on expression of a virulence gene cluster of pathogenic Escherichia coli by using a wild-type gene fusion

S fimbrial adhesins (Sfa) represent virulence factors of E. coli wild-type strains causing urinary tract infections and meningitis of the new born. In order to determine the influence of subinhibitory concentration of antibiotics on the expression of the sfa gene cluster, a wild-type strain carrying the lacZ gene, coding for the enzyme β-galactosidase fused to the sfa determinant was used. The expression of lacZ which was under the control of the sfa wild-type promoters, was now equivalent to the sfa gene expression of wild-type strain 536. With this strain the influence of subinhibitory concentrations of 28 antibiotics on the expression of the sfa determinant was studied. The expression was strongly suppressed by a treatment of the wild-type fusion strain by aztreonam, gentamicin, clindamycin and trimethoprim; the latter had a dramatic effect on sfa expression. It was further shown for clindamycin and trimethoprim that the reduction of sfa gene expression was dependent on the concentration of the antibiotics. In contrast imipinem, amphotericin B and rifampicin weakly stimulated sfa expression. We conclude that gene fusions between virulence-associated loci and indicator genes in wild-type pathogens are useful to study virulence modulation due to subinhibitory concentration of antibiotics on the genetic level.     

亚抑菌浓度抗生素对细菌耐药性和毒力影响的研究进展

细菌的耐药性和毒力是决定抗生素治疗成败的关键。近年来研究表明亚抑菌浓度抗生素可以诱导细菌耐药并影响细菌的毒力。本文介绍了亚抑菌浓度抗生素产生的来源；阐述了亚抑菌浓度抗生素对细菌耐药性和毒力的影响。亚抑菌浓度抗生素对细菌耐药性的影响涉及了细菌的耐药选择性、生物被膜和持留菌的形成、基因突变、水平基因转移和基因表达；对细菌毒力的影响主要包括细菌的黏附性、运动性和其它毒力因子。以期为畜牧生产中减少和避免亚抑菌浓度抗菌药的产生，减缓和克服细菌耐药性，降低致病菌的毒力提供参考。     

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections

Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous icaC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.     

Effects of subinhibitory concentrations of amikacin and ciprofloxacin on the hydrophobicity and adherence to epithelial cells of uropathogenic Escherichia coli strains

The effect of subinhibitory concentrations of amikacin and ciprofloxacin on the hydrophobicity and adherence to uroepithelial cells of Escherichia coli strains was investigated. The hydrophobicity of the tested strains was evaluated by the bacterial adherence to hydrocarbon–xylene test and by the salt aggregation test of ammonium sulphate. The hydrophobic character of strains exposed to 1/2 to 1/8 minimal inhibitory concentration (MIC) of amikacin and 1/2 to 1/16 MIC of ciprofloxacin was altered to a hydrophilic state. Results of the SAT also correlated with these data. Moreover, comparisons were made between the number of bacteria attached to the epithelial cells before and after exposure to 1/2, 1/4 and 1/8 MIC of antibiotics. The greatest loss of adherence capability occurred at 1/2 MIC of ciprofloxacin. In conclusion, antibiotics are often present at sub-MICs and may still be effective in reducing bacterial virulence by interfering with bacterial cell functions.     

Subinhibitory concentrations of beta-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes

In the present study we tested the ability of beta-lactam antibiotics and other cell-wall antibiotics to inhibit the production of listeriolysin which is the main virulence factor of L. monocytogenes. The amount of listeriolysin produced was measured in the supernatants with a sensitive hemolysin assay and as beta-galactosidase expression from the listeriolysin promoter. When tested in concentrations that did not affect growth of the bacteria, all beta-lactam antibiotics and all other cell-wall antibiotics tested were able to reduce the listeriolysin production in at least one of the tests used. Yet, no change in beta-galactosidase activity was detected in a strain harboring a control beta-galactosidase fusion. We therefore demonstrate for the first time that subinhibitory concentrations of antibiotics specifically reduce the production of a virulence factor of L. monocytogenes.     

Characterization of a novel gene related to antibiotic susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a human pathogen with increased intrinsic resistance to a large number of antibiotics used in clinical therapy. Therefore, understanding the mechanisms of resistance and developing therapy alternatives for P. aeruginosa are of profound importance. Previous work from our laboratory demonstrated that several mutants have isolated with altered expression of the phzA1B1C1D1E1F1G1 (phzA1) operon in the presence of sub-inhibitory concentrations (SICs) of tetracycline (TET). The present study investigates the roles of the PA0011 gene in mediating phzA1 expression at SIC of TET. The PA0011 gene encodes 2-OH-lauroytransferase by controlling the synthesis of the cell envelope and the outer membrane. We found that the PA0011 mutant strain was susceptible to several different antibiotics and environmental stresses. Complementation in the PA0011 mutant restored these phenotypes to wild-type levels. In addition, expression of the PA0011 gene, as monitored through a luciferase reporter, is increased at SICs of antibiotics. Indeed, the expression of the PA0011 gene increased about threefold in pqsR and pqsH mutants compared with the wild-type PAO1. However, the PA0011 gene negatively regulates the quorum sensing (QS) system. Taken together, these data suggest that PA0011 is involved in susceptibility to antimicrobial agents in P. aeruginosa, and that its susceptibility effect maybe partly dependent on increased QS expression.     

Susceptibility of bacteria to serum lysis or phagocytosis following growth in subinhibitory levels of lincosaminide or spectinomycin related antibiotics

The effects of subinhibitory concentrations of antibiotics on polymorphonuclear leukocyte (PML) and serum killing of Staphylococcus aureus 502A (UC 9116) and Escherichia coli UC 9451 were studied. Exposure of these bacteria to subinhibitory levels of certain lincosaminides, spectinomycin, or 6'-n-propylspectinomycin altered their susceptibility to these host defense mechanisms, while exposure to gentamicin had no effect. However, each organism responded differently to treatment with the antibiotics. S. aureus pretreated during log phase growth with subinhibitory concentrations of clindamycin, lincomycin, or pirlimycin was more susceptible to killing by PMLs than untreated bacteria. No effect on phagocytic killing was found when S. aureus was pretreated with spectinomycin, 6'-n-propylspectinomycin, or gentamicin. The S. aureus remained resistant to serum lysis despite antibiotic treatment. In contrast, spectinomycin and 6'-n-propylspectinomycin as well as clindamycin dramatically increased the susceptibility of E. coli to serum lysis (greater than 99% destroyed). Moderate killing of E. coli by PMLs was also found.     

Monobactam antibiotics in subinhibitory concentrations enhance opsonophagocytosis and serum bacteriolysis in certain Escherichia coli strains

Capsular polysaccharides are known to protect Gram-negative bacteria from complement-mediated killing and opsonophagocytosis. Monobactam antibiotics selectively inhibit penicillin-binding protein 3 (PBP3), resulting in abnormally structured peptidoglycan, causing defective cell surface structures. The authors studied the influence of subinhibitory concentrations of the monobactam antibiotics aztreonam and carumonam on serum bacteriolysis and opsonophagocytosis of four K-encapsulated and five non-K-encapsulated Escherichia coli strains. It was observed that monobactam antibiotics in subinhibitory concentrations enhanced opsonophagocytosis of the four K-encapsulated and one non-K-encapsulated E. coli strains tested. Opsonophagocytosis of the other four non-K-encapsulated E. coli strains was not enhanced. Serum bacteriolysis studies revealed that of the four K-encapsulated strains tested only one strain showed a significant enhancement of bacteriolysis after treatment with subinhibitory concentrations of monobactam antibiotics. None of the unencapsulated strains showed a significant change in percentage lysis after treatment with either aztreonam or carumonam.     

The effect of disinfectants on Plesiomonas shigelloides

The effects of ten commercially available disinfectants on virulence associated properties of Plesiomonas shigelloides were tested. All the disinfectants tested contained quaternary ammonium salts. The majority of the disinfectants when used at subinhibitory concentrations increased surface hydrophobicity as evaluated by bacterial adherence to xylene and decreased bacterial motility in a concentration dependent manner. Disinfectants did not significantly affect lipase activity. However, more than half of the antimicrobials tested increased the resistance of bacteria to hydrogen peroxide. The disinfectants, in a similar manner to antibiotics at concentrations below MIC, interfered with potential virulence factors of Plesiomonas shigelloides.     

Molecular characterisation of Hungarian macrolide-resistant Streptococcus pneumoniae isolates, including three highly resistant strains with the mef gene

The macrolide resistance of 304 Hungarian Streptococcus pneumoniae isolates was investigated. Antibiotic sensitivity testing was performed in air and in 5% CO₂. More erythromycin resistance was noted when growth was in CO₂. A resistance determinant was found in almost all isolates: erm(B) gene (87.4%), mef genes (9.2%) and one strain with the erm(TR) gene. This indicates that screening for carriage of resistance determinants should always be done in the presence of 5% CO₂. We found three isolates with mef(E), which were highly resistant to erythromycin. These contained multiple and some novel, ribosomal mutations. The most prevalent serogroups were 6, 19 and 14. Based on the PFGE pattern, we found identity between the Hungarian isolates and two PMEN clones.     

Postantibiotic effects and postantibiotic sub-MIC effects of erythromycin, roxithromycin, tilmicosin, and tylosin on Pasteurella multocida

When intermittent dosing is used during treatment, the concentrations of antibiotics fluctuate and subinhibitory concentrations may occur between doses. Postantibiotic effects (PAEs) and postantibiotic subinhibitory effects (PA SMEs) on bacteria may provide additional, valuable information for the rational use of a drug in clinical practice. In this study tilmicosin was the most active antibiotic tested against P. multocida type D with MICs ranging from 4–16 mg/l. Roxithromycin and tilmicosin induced a statistically significantly longer PAE than did tylosin against P. multocida types A and D (P<0.05). The duration of PAEs and PA SMEs were proportional to the concentrations of drugs used for exposure. The PA SMEs were substantially longer than PAEs on P. multocida. Tilmicosin had a longer PA SME compared with erythromycin, roxithromycin and tylosin for P. multocida. The computerized incubator used in the present study provided an efficient and convenient determination of PAE and PA SME, allowing frequent measurements of the bacterial growth.     

Influence of the cell wall on ciprofloxacin susceptibility in selected wild-type Gram-negative and Gram-positive bacteria

The susceptibility of several wild-type bacteria to ciprofloxacin and accumulation of the drug in these bacteria were evaluated. Species studied included Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Bacillus cereus. Ciprofloxacin susceptibility was measured for each strain using two different methods: the minimal inhibitory concentration and the bactericidal index. Significant differences were observed between the results derived from these two methods. Whereas the minimal inhibitory concentration was low in all strains tested, ciprofloxacin’s bactericidal activity, as indicated by the bactericidal index, varied with the species studied. To determine whether this finding was due to variations in cell envelope permeability to ciprofloxacin (i.e. to combined cell uptake and efflux), we studied ciprofloxacin accumulation using spectrofluorometry. In Gram-negative bacteria, differences in permeability can lead to altered susceptibility to antibiotics. In fact, the combination of slow uptake and efficient efflux seems to be crucial to the characteristic poor susceptibility of P. aeruginosa to ciprofloxacin. However, the low level of activity of ciprofloxacin against S. aureus and two Bacillus species may have resulted from the drug’s interaction with its target enzymes (i.e. topoisomerase IV in S. aureus and DNA gyrase in Bacillus spp.) rather than diminished permeability.     

Pharmacodynamic effects of subinhibitory antibiotic concentrations

The pharmacodynamics of antibiotics have become increasingly important for the determination of optimal dosing regimens. Studies over the past decade have demonstrated marked differences in the time course of antimicrobial activity for different classes of antibiotics both in vitro, in animals and in human trials. One of the explanations for the success of intermittent dosing regimens has been the delay in regrowth after the concentration has fallen under the MIC, the so called postantibiotic effect (PAE). In addition to the PAE, the success of discontinuous dosing regimens may be attributed to both the function of a normal host defence and to the effects of subinhibitory antibiotic concentrations (sub-MICs). It has been shown that there is a difference between the effects of sub-MICs following a suprainhibitory dose (postantibiotic sub-MIC effect; PA SME) and the effects of sub-MICs (SME) alone. It seems that the PA SME is more clinically relevant compared with the PAE, since exposure to suprainhibitory concentrations will always be followed by sub-MICs in vivo. A long PA SME could indicate that longer dosing intervals may be used for that antibiotic /bacterial combination and together with the known effects of sub-MICs on bacterial virulence and the influence of the immune system, it may explain the efficacy of antibiotics with short half-lives even of they are given infrequently.     

亚洲地区肺炎克雷伯菌耐药基因和毒力基因分析

目的:分析亚洲地区肺炎克雷伯菌毒力基因和耐药基因的携带情况,为制定菌株防治措施提供依据。方法:从GenBank中收集并分析了4 549株肺炎克雷伯菌的完整基因组(下载日期:2020年12月31日)。通过MLST、Kleborate和ABRicate等生物信息学软件分析肺炎克雷伯菌的MLST分型、KL分型、毒力基因以及耐药基因。结果:MLST分型结果显示,ST11是亚洲地区肺炎克雷伯菌的主要序列类型,共检测到1 519(33.39%)株,其中ST11-KL64、ST11-KL47,分别占51.15%(777/1 519)、37.20%(565/1 519)。毒力基因检测发现,ST11-KL64型菌株中,有36.04%(280/777)的菌株同时携带4种铁载体基因,有33.33%(259/777)的菌株同时携带rmpA和rmpA2基因。在ST11-KL47型菌株中,有13.52%(76/565)的菌株同时携带4种铁载体基因,有5.49%(31/565)的菌株同时携带rmpA和rmpA2基因。耐药基因检测发现,有97.25%(4 424/4 549)的菌株检测出β-内酰胺类耐药基因,有3.06%(139/4 549)的菌株携带粘菌素耐药基因。结论:亚洲地区肺炎克雷伯菌MLST分型以ST11为主、血清荚膜型以KL64、KL47为主。ST11-KL64型菌株的高毒力表型基因携带率高于ST11-KL47。应规范抗菌药物使用,加强对肺炎克雷伯菌的监测和医院内感染的防控工作,防止肺炎克雷伯菌的大规模流行。     

Effect of a trifluoromethyl ketone on the motility of proton pump-deleted mutant of Escherichia coli strain and its wild-type

We have recently found that 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone [TF18] exhibited the most potent antibacterial activity among 30 trifluoromethyl ketones against various prokaryotes, such as Escherichia coli (E. coli). In the present study, the inhibition of E. coli motility by TF18 was investigated. TF18 showed the lowest minimum inhibitory concentration (MIC) and highest inhibitory effect on the motility of E. coli strains. The wild-type E. coli was more sensitive to inhibition of motility than its proton pump-deleted mutant strain at subinhibitory concentrations. These data suggest that one of the targets of the antibacterial effect of the trifluoromethyl ketone is the proton pump system.     

Polymorphic variants of the multidrug resistance gene Mdr1a and response to antiepileptic drug treatment in the kindling model of epilepsy

Allelic variants of the human P-glycoprotein encoding gene MDR1 (ABCB1) are discussed to be associated with different clinical conditions including pharmacoresistance of epilepsy. However, conflicting data have been reported with regard to the functional relevance of MDR1 allelic variants for the response to antiepileptic drugs. To our knowledge, it is not known whether functionally relevant genetic polymorphisms also occur in the two genes (Mdr1a/Abcb1a, Mdr1b/Abcb1b) coding for P-glycoprotein in the brain of rodents. Therefore, we have started to search for polymorphisms in the Mdr1a gene, which governs the expression of P-glycoprotein in brain capillary endothelial cells in rats. In the kindling model of temporal lobe epilepsy, subgroups of phenytoin-sensitive and phenytoin-resistant rats were selected in repeated drug trials. Sequencing of the Mdr1a gene coding sequence in the subgroups revealed no general differences between drug-resistant and drug-sensitive rats of the Wistar outbred strain. A comparison between different inbred and outbred rat strains also gave no evidence for polymorphisms in the Mdr1a coding sequence. However, in exon-flanking intron sequences, four genetic variants were identified by comparison between these rats strains.

In conclusion, the finding that Wistar rats vary in their response to phenytoin, while having the same genetic background, argues against a major impact of Mdr1a genetics on pharmacosensitivity to antiepileptic drugs in the amygdala kindling model.     

次抑菌浓度的药物诱导细菌耐药与交叉耐药

铜绿假单胞菌接种于含次抑菌浓度的哌拉西林和阿米卡星的营养肉汤中，葡萄球菌接种于次抑菌浓度的氧氟沙星和环丙沙星的营养肉汤中，经传代培养可获得相对应的耐药菌株。哌拉西林诱导耐药的铜绿假单胞菌对头孢他啶亦耐药，但对阿米卡星、环丙沙星、亚胺培南／西司他丁不耐药；阿米卡星诱导耐药的铜绿假单胞菌对哌拉西林亦耐药，但对头孢他啶、环丙沙星、亚胺培南／西司他丁没有交叉耐药；氧氟沙星或环丙沙星诱导耐药的葡萄球菌对氟喹诺酮类药物都有交叉耐药，但对苯唑西林、阿米卡星、亚胺培南／西司他丁没有显著的交叉耐药性     

Inhibitory action of a new proton pump inhibitor, trifluoromethyl ketone derivative, against the motility of clarithromycin-susceptible and-resistant Helicobacter pylori

We previously reported that a trifluoromethyl ketone derivative, 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18), exhibited the potent antibacterial activity against Helicobacter pylori, but had no urease activity. In order to clarify the mechanism of anti-H. pylori action of TF18, we evaluated the growth and motility of TF18 on clarithromycin-susceptible H. pylori (CSHP) and -resistant H. pylori (CRHP). An effective proton pump inhibitor (TF18) had remarkable dose-dependent antibacterial activity and was able to inhibit the flagellar motor of both CSHP and CRHP isolates. The antimotility effect of TF18 was more pronounced at subinhibitory concentration in CRHP than in CSHP. The swimming (the forward motion) was more sensitive to the inhibition than the tumbling. Based on the results, it is supposed that TF18 works as an uncoupler similar to the ‘clutch’ in a biological motor, in which counterclockwise rotation is more sensitive to the effect of TF18 than the clockwise rotation.     

临床JK棒状杆菌抗生素敏感试验及耐药基因的监测

目的 揭示临床分离的JK棒状杆菌耐药现状,并做其耐药基因的分析.方法 常规鉴定并采用API Coryne鉴定临床标本中分离的棒状杆菌,分离的JK棒状杆菌采用琼脂稀释法测定最低抑菌浓度,根据美国NCCLS判断"敏感"、"耐药"和"中介".采用PCR方法进行基因扩增.结果 共分离出6株JK棒状杆菌,对临床常用的抗生素:青霉素、头孢唑啉、头孢美唑、头孢吡肟、亚胺培南、阿米卡星、克林霉素、环丙沙星、红霉素、米诺环素均表现高的最低抑菌浓度(MICs)值,对糖肽类的替考拉宁、万古霉素表现低的MICs值.采用自动测序仪对临床分离的多耐菌株进行基因测序(applied biosystems califo,USA).结论 临床分离的JK棒状杆菌呈现出多耐现象并存在耐药基因.