Comparison of charcoal- and starch-based media for testing susceptibilities of Legionella species to macrolides, azalides, and fluoroquinolones.

We compared growth characteristics of 46 Legionella strains grown on buffered charcoal yeast extract alpha (BCYE alpha) agar and buffered starch yeast extract (BSYE) agar and MICs of macrolides, azalides, and fluoroquinolones for these organisms. Growth was poor and not reproducible on BSYE agar. Growth was excellent on BCYE alpha, and MICs were easy to interpret. BCYE alpha is superior to BSYE for testing susceptibilities of Legionella species by agar dilution.     

Intracellular distribution and activity of antibiotics

Intracellular penetration, accumulation and disposition are important parameters governing the activity of antibiotics against intracellular bacteria. Beta-lactams diffuse into but do not accumulate in phagocytes, probably because of their acidic character. Aminoglycosides are too polar to pass across membranes and are therefore only taken up slowly by endocytosis, which results in an exclusively lysosomal localization. Lincosaminides, macrolides and fluoroquinolones all accumulate in phagocytes, the two former classes of drugs showing both a cytosolic and a lysosomal localization. Fluoroquinolones appear to be entirely soluble in cells. Analysis of their activity in a model ofStaphylococcus aureus-infected J774 macrophages has revealed low activity of clindamycin, whereas macrolides, and even more so fluoroquinolones, easily reduce the original inoculum.     

Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method.

We investigated the potential of a rapid colorimetric microassay based on the reduction of dimethylthiazol-diphenyltetrazolium bromide (MTT) for determining the growth of Mycobacterium avium-M. intracellulare complex (MAC) and MICs of clofazimine, resorcinomycin A, and the quinolone PD 127391 against MAC. The reduction of MTT was directly proportional to the number of viable bacteria. A comparison of the MTT reduction test with the [3H]glycerol uptake assay showed the former to possess higher analytical sensitivity for detecting MAC growth in microtiter plates. The MIT reduction test avoids the use of radioisotopes and costly material and equipment; it is reliable, reproducible, and convenient for rapid routine susceptibility testing of MAC.     

Use of MTT colorimetric assay to measure cell activation

The MTT tetrazolium salt colorimetric assay previously described by Mosmann (1983, J. Immunol. Methods 65, 55) to measure cytotoxicity and cell proliferation was further explored to extend its application to the measurement of cell activation. The level of MTT cleavage by viable cells of various origins was found to be directly proportional to the number of cells but to increase as a non-linear function of time. This non-linear relationship was related to a time-linear cell death during MTT incubation. The cleavage of MTT by viable cells was found to follow first order kinetics and could be fitted to Michaelis' kinetics. Different cell types exhibited similar apparent Km values (1949 microM) and different apparent maximal velocities (V). The apparent V values determined for a given cell type under different experimental conditions were rigorously similar. This analysis of MTT cleavage by viable cells suggests that the colorimetric MTT test can be useful to quantify the activation level of cells, independently of proliferation.     

Difference in Legionella pneumophila growth permissiveness between J774.1 murine macrophage-like JA-4 cells and lipopolysaccharide (LPS)-resistant mutant cells, LPS1916, after stimulation with LPS.

To elucidate the role of the oxidative burst in macrophage resistance to Legionella infection, we examined a murine macrophage-like cell line, J774.1, for permissiveness to Legionella growth, using a mutant that has a selective defect in the oxidative burst after lipopolysaccharide (LPS) stimulation. Legionella pneumophila serogroup 1 was infected into J774.1 monolayers, and then the extent of bacterial growth was estimated by a CFU assay. Both the parental cell line, JA-4, and the LPS-resistant mutant, LPS1916, were permissive for Legionella growth but became nonpermissive after pretreatment with gamma interferon. However, pretreatment of LPS1916 cells with LPS failed to inhibit bacterial growth, although LPS-treated JA-4 cells exhibited inhibited multiplication of the bacteria. The bacterial growth inhibition in JA-4 and mutant LPS1916 cells was correlated with the extent of the oxidative burst in the cells, as judged by cytochrome c reduction but not nitrite production. Neither transferrin receptor expression nor the iron content in JA-4 and LPS1916 cells, with or without LPS treatment, was correlated with suppression of Legionella growth. These results suggest that the restriction of Legionella growth in J774.1 cells is due to a bactericidal effect of the oxidative burst rather than reduction of the iron supply to the intracellular bacteria and that the effectors are reactive oxygen intermediates and not reactive nitrogen intermediates.     

In vitro activity,postantibiotic effect and human monocyte activity of grepafloxacin against Legionella species

ObjectiveTo determine the in vitro antimicrobial activity, postantibiotic effect (PAE) and human monocyte activity of grepafloxacin compared with sparfloxacin, ciprofloxacin, clarithromycin, erythromycin and rifampicin against 181 strains of Legionella pneumophila, nine strains of L. micdadei, 10 strains of L. dumoffii, seven strains of L. longbeachae and seven other Legionella strains.MethodsMICs were determined by standard agar dilution using buffered yeast extract (BYE) agar. PAE and human monocyte activity were determined by standard culture techniques.ResultsGrepafloxacin, sparfloxacin and rifampicin were the most active agents against L. pneumophila (MIC₉₀ ≥0.016 mg/L for most strains tested). Grepafloxacin was more active than erythromycin against L. dumoffii and L. longbeachae and more active than both erythromycin and clarithromycin against L. micdadei and isolates of other Legionella spp. The PAE of grepafloxacin against erythromycin-susceptible L. pneumophila (2.62 h) was higher than that of sparfloxacin (0.88 h), erythromycin (0.93 hours) and clarithromycin (0.72 h). Against erythromycin-resistant L. pneumophila, the PAE of grepafloxacin (4.18 h) was higher than those of all the other antibiotics tested. Grepafloxacin, sparfloxacin, ciprofloxacin and clarithromycin inhibited the growth of all L. pneumophila strains and other erythromycin-resistant Legionella spp. in human monocytes. However, only grepafloxacin and ciprofloxacin prevented regrowth or killed L. pneumophila after removal of extracellular antibiotic.ConclusionsGrepafloxacin showed effective antibacterial activity against the Legionella spp. tested, and has a PAE and activity within human monocytes that suggest it may be useful in the treatment of lower respiratory tract infections caused by Legionella spp.     

Induction of Rapid Cell Death by an Environmental Isolate of Legionella pneumophila in Mouse Macrophages

Legionella pneumophila, the etiological agent for Legionnaires'' disease, is ubiquitous in the aqueous environment, where it replicates as an intracellular parasite of free-living protozoa. Our understanding of L. pneumophila pathogenicity is obtained mostly from study of derivatives of several clinical isolates, which employ almost identical virulent determinants to exploit host functions. To determine whether environmental L. pneumophila isolates interact similarly with the model host systems, we analyzed intracellular replication of several recently isolated such strains and found that these strains cannot productively grow in bone marrow-derived macrophages of A/J mice, which are permissive for all examined laboratory strains. By focusing on one strain called LPE509, we found that its deficiency in intracellular replication in primary A/J macrophages is not caused by the lack of important pathogenic determinants because this strain replicates proficiently in two protozoan hosts and the human macrophage U937 cell. We also found that in the early phase of infection, the trafficking of this strain in A/J macrophages is similar to that of JR32, a derivative of strain Philadelphia 1. Furthermore, infection of these cells by LPE509 caused extensive cell death in a process that requires the Dot/Icm type IV secretion system. Finally, we showed that the cell death is caused neither by the activation of the NAIP5/NLRC4 inflammasome nor by the recently described caspase 11-dependent pathway. Our results revealed that some environmental L. pneumophila strains are unable to overcome the defense conferred by primary macrophages from mice known to be permissive for laboratory L. pneumophila strains. These results also suggest the existence of a host immune surveillance mechanism differing from those currently known in responding to L. pneumophila infection.     

测定淋巴细胞转化和鼠白细胞间素2活性的新方法——MTT比色分析法

细胞增殖通常用~3H-Tdr掺入法或活细胞计数法进行测定。由于前者使用放射性同位素易污染环境并且需要昂贵的仪器,而后者易受主观因素的影响误差较大,因此两者的应用都有一定局限性。最近Mosmann根据活的增殖细胞能代谢MTT[3-(4,5-di-methylthiazol-2-y1)2,5-diphenyl te-trazollum bromide]产生MTT甲 (for-     

Secreted phospholipases of the lung pathogen Legionella pneumophila

Legionella pneumophila is an intracellular pathogen and the main causative agent of Legionnaires’ disease, a potentially fatal pneumonia. The bacteria infect both mammalian cells and environmental hosts, such as amoeba. Inside host cells, the bacteria withstand the multifaceted defenses of the phagocyte and replicate within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). For establishment and maintenance of the infection, L. pneumophila secretes many proteins including effector proteins by means of different secretion systems and outer membrane vesicles. Among these are a large variety of lipolytic enzymes which possess phospholipase/lysophospholipase and/or glycerophospholipid:cholesterol acyltransferase activities. Secreted lipolytic activities may contribute to bacterial virulence, for example via modification of eukaryotic membranes, such as the LCV. In this review, we describe the secretion systems of L. pneumophila, introduce the classification of phospholipases, and summarize the state of the art on secreted L. pneumophila phospholipases. We especially highlight those enzymes secreted via the type II secretion system Lsp, via the type IVB secretion system Dot/Icm, via outer membrane vesicles, and such where the mode of secretion has not yet been defined. We also give an overview on the complexity of their activities, activation mechanisms, localization, growth-phase dependent abundance, and their role in infection.     

Loxapine,an antipsychotic drug,suppresses intracellular multiple-antibiotic-resistant Salmonella enterica serovar Typhimurium in macrophages

BackgroundThe emergence of multiple-antibiotic-resistant (MAR) Salmonella has been a serious threat worldwide. Salmonella can invade into host cells and evade the attacks of host humoral defenses and antibiotics. Thus, a new antibacterial agent capable of inhibiting intracellular Salmonella is highly needed.MethodsThe anti-intracellular activity and cytotoxicity of drugs on intracellular bacteria and macrophages were assayed using intracellular CFU assay and MTT cell viability assay, respectively. The uptake of gentamicin into macrophage and the effect of autophagy inhibitor on loxapine's anti-intracellular Salmonella activity were assessed by using image-based high-content system. The expression of bacterial genes was measured by real-time PCR. The efflux pump activity of bacteria was measured by Hoechst accumulation assays.ResultsWith our efforts, an antipsychotic drug, loxapine, was identified to exhibit high potency in suppressing intracellular MAR S. Typhimurium, Staphylococcus aureus, Shigella flexneri or Yersinia enterocolitica. Subsequent investigations indicated that loxapine's anti-intracellular bacteria activity was not associated with increased penetration of gentamicin into bacteria and macrophages. Loxapine didn't inhibit bacterial growth in broth at concentration up to 500 μM and has no effect on Salmonella's type III secretion system genes' expression. Blockage of autophagy also didn't reverse loxapine's anti-intracellular activity. Lastly, loxapine suppressed bacterial efflux pump activity in all bacteria tested.ConclusionAltogether, our data suggested that loxapine might suppress intracellular bacteria through inhibiting of bacterial efflux pumps. In light of its unique activity, loxapine represents a promising lead compound with translational potential for the development of a new antibacterial agent against intracellular bacteria.     

Molecular Pathogenesis of Infections Caused by Legionella pneumophila

Summary: The genus Legionella contains more than 50 species, of which at least 24 have been associated with human infection. The best-characterized member of the genus, Legionella pneumophila, is the major causative agent of Legionnaires'' disease, a severe form of acute pneumonia. L. pneumophila is an intracellular pathogen, and as part of its pathogenesis, the bacteria avoid phagolysosome fusion and replicate within alveolar macrophages and epithelial cells in a vacuole that exhibits many characteristics of the endoplasmic reticulum (ER). The formation of the unusual L. pneumophila vacuole is a feature of its interaction with the host, yet the mechanisms by which the bacteria avoid classical endosome fusion and recruit markers of the ER are incompletely understood. Here we review the factors that contribute to the ability of L. pneumophila to infect and replicate in human cells and amoebae with an emphasis on proteins that are secreted by the bacteria into the Legionella vacuole and/or the host cell. Many of these factors undermine eukaryotic trafficking and signaling pathways by acting as functional and, in some cases, structural mimics of eukaryotic proteins. We discuss the consequences of this mimicry for the biology of the infected cell and also for immune responses to L. pneumophila infection.     

Comparative assessment of antibiotic potency loss with time and its impact on antibiotic resistance

A wide range of diverse antibiotics is available in the current world’s drug market, which is mostly being used for the treatment of infectious diseases in humans as well as in veterinary. A large number of new antibiotics are continuously being added to the existing list. However, at the same time, most of the pathogens have tended to overcome antibiotic stress due to their specific mutations and adaptations with time. Hence, several antibiotics turn to be non-effective in the long run. Moreover, antibiotics also lose their effectiveness with time as they approach their expiry period. The goal of this study is to compare and elucidate the changes in the potency and effectiveness of antibiotic with time. The present study uses three different antibiotics known as ciprofloxacin, erythromycin, and tetracycline of fluoroquinolones, macrolides, and tetracyclines groups, respectively, which are collected in two forms, brand new and recently expired. Escherichia coli, gram-negative intestinal bacteria, and Staphylococcus aureus, gram-positive bacteria, have been used as test microorganisms. The obtained results show a significant loss in the potency and effectiveness of antibiotic as they progressively decompose with time. However, there is a huge possibility to recycle it as the present study suggests.     

Isolation of Legionella pneumophila by Centrifugation of Shell Vial Cell Cultures from Multiple Liver and Lung Abscesses

A 7-year-old girl was admitted to the hospital with acute lymphoblastic leukemia and was treated with allogenic cord blood transplantation. At day 30 after graft, she developed a fever and multiple nodular lesions disseminated in the liver and lungs. All bacterial cultures attempted on liver and lung biopsy specimens and blood remained sterile on standard axenic media. However, inoculation of liver and lung biopsy specimens on eukaryotic cell monolayers by the centrifugation-shell vial technique (M. Marrero and D. Raoult, Am. J. Trop. Med. Hyg. 40:197–199, 1989) led to the recovery of a strain of Legionella pneumophila serogroup 1, identified by 16S rRNA gene amplification and sequencing and serotyping. Our findings demonstrate that the centrifugation-cell culture method, which has previously been useful for the isolation of other strictly or facultatively intracellular bacteria, can also serve as a method for the recovery of L. pneumophila from clinical material.     

Role of Coptis chinensis in antibiotic susceptibility of carbapenem-resistant Klebsiella pneumoniae

BackgroundThe incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has rapidly increased. This study aimed to assess the effect of Coptis chinensis and its compounds on the minimal inhibitory concentrations (MICs) of eight antibiotics against CRKP.MethodsCell cultures were used to investigate the effects of C. chinensis and its compounds on the MICs of eight antibiotics against CRKP. The MICs for antibiotics alone and antibiotics with C. chinensis or compounds were measured and compared. Furthermore, the effects of C. chinensis on cell membrane injury and intracellular adenosine triphosphate (ATP) CRKP concentration were also measured. The Mann–Whitney rank-sum test was used to analyze the differences between means.ResultsC. chinensis exhibits a notable MIC bacteriostatic effect at 5 mg/mL on CRKP. A significant MIC reduction against CRKP exists when C. chinensis was added to colistin and colistin-containing two-antibiotic combinations. Moreover, C. chinensis could damage cell membrane integrity and decrease intracellular ATP concentration in CRKP. Thus, C. chinensis exhibits antimicrobial activity superiority with colistin against CRKP. Furthermore, the effects of identified compounds in C. chinensis on the MICs of colistin, four-to eight-, two-to four-, and one-to two-fold reductions were found in ferulic acid, magnoflorine, and jatrorrhizine hydrochloride, respectively. Among these compounds, ferulic acid destroys membrane integrity and decreases intracellular ATP concentration.ConclusionC. chinensis and ferulic acid can potentiate the antimicrobial activity of colistin and may represent a promising component of combination therapy against CRKP infections in a clinical setting.     

A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro.

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.     

Detection and susceptibility testing of Mycoplasma amphoriforme isolates from patients with respiratory tract infections

Three isolates of Mycoplasma amphoriforme, a new Mycoplasma species rarely described to date, were obtained from respiratory tract specimens from two children and one adult with respiratory tract infections. Molecular methods were required to distinguish them from Mycoplasma pneumoniae. MICs of macrolides, tetracyclines and fluoroquinolones were identical to those for M. pneumoniae, except for that of ciprofloxacin, which was slightly more potent against M. amphoriforme. M. amphoriforme could possibly have been involved in one case of severe respiratory infection with sepsis, but further studies are needed to specify its role as a potential respiratory tract pathogen.     

Surface-Associated Hsp60 Chaperonin of Legionella pneumophila Mediates Invasion in a HeLa Cell Model

HeLa cells have been previously used to demonstrate that virulent strains of Legionella pneumophila (but not salt-tolerant avirulent strains) efficiently invade nonphagocytic cells. Hsp60, a member of the GroEL family of chaperonins, is displayed on the surface of virulent L. pneumophila (R. A. Garduño et al., J. Bacteriol. 180:505–513, 1988). Because Hsp60 is largely involved in protein-protein interactions, we investigated its role in adherence-invasion in the HeLa cell model. Hsp60-specific antibodies inhibited the adherence and invasiveness of two virulent L. pneumophila strains in a dose-dependent manner but had no effect on the association of their salt-tolerant avirulent derivatives with HeLa cells. A monospecific anti-OmpS (major outer membrane protein) serum inhibited the association of both virulent and avirulent strains of L. pneumophila to HeLa cells, suggesting that while both Hsp60 and OmpS may mediate bacterial association to HeLa cells, only virulent strains selectively displayed Hsp60 on their surfaces. Furthermore, the surface-associated Hsp60 of virulent bacterial cells was susceptible to the action of trypsin, which rendered the bacteria noninvasive. Additionally, pretreatment of HeLa cells with purified Hsp60 or precoating of the plastic surface where HeLa cells attached with Hsp60 reduced the adherence and invasiveness of the two virulent strains. Finally, recombinant Hsp60 covalently bound to latex beads promoted the early association of beads with HeLa cells by a factor of 20 over bovine serum albumin (BSA)-coated beads and competed with virulent strains for association with HeLa cells. Hsp60-coated beads were internalized in large numbers by HeLa cells and remained in tight endosomes that did not fuse with other vesicles, whereas internalized BSA-coated beads, for which endocytic trafficking is well established, resided in more loose or elongated endosomes. Mature intracellular forms of L. pneumophila, which were up to 100-fold more efficient than agar-grown bacteria at associating with HeLa cells, were enriched for Hsp60 on the bacterial surface, as determined by immunolocalization techniques. Collectively, these results establish a role for surface-exposed Hsp60 in invasion of HeLa cells by L. pneumophila.     

Apoptosis in Macrophages and Alveolar Epithelial Cells during Early Stages of Infection by Legionella pneumophila and Its Role in Cytopathogenicity

The hallmark of Legionnaires’ disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.