Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10⁷-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD₅₀ of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.     

In vitro activities of 14 antibiotics against 100 human isolates of Yersinia pestis from a southern African plague focus.

A limited repertoire of antimicrobial agents is currently in use for the treatment of plague. We investigated the in vitro activities of some newer antimicrobial agents against Yersinia pestis. Among the injectable agents tested, cefotaxime was the most active, and among the oral agents, both levofloxacin and ofloxacin were highly active, with MICs at which 90% of isolates are inhibited of < 0.03 microgram/ml. the susceptibilities to the ketolide RU004 and the penem faropenem warrant attention. The enhanced activities of quinolones against Y. pestis suggest that these agents should be further investigated for the treatment of human plague in the future.     

Origins of Yersinia pestis Sensitivity to the Arylomycin Antibiotics and the Inhibition of Type I Signal Peptidase

Yersinia pestis is the etiologic agent of the plague. Reports of Y. pestis strains that are resistant to each of the currently approved first-line and prophylactic treatments point to the urgent need to develop novel antibiotics with activity against the pathogen. We previously reported that Y. pestis strain KIM6+, unlike most Enterobacteriaceae, is susceptible to the arylomycins, a novel class of natural-product lipopeptide antibiotics that inhibit signal peptidase I (SPase). In this study, we show that the arylomycin activity is conserved against a broad range of Y. pestis strains and confirm that it results from the inhibition of SPase. We next investigated the origins of this unique arylomycin sensitivity and found that it does not result from an increased affinity of the Y. pestis SPase for the antibiotic and that alterations to each component of the Y. pestis lipopolysaccharide—O antigen, core, and lipid A—make at most only a small contribution. Instead, the origins of the sensitivity can be traced to an increased dependence on SPase activity that results from high levels of protein secretion under physiological conditions. These results highlight the potential of targeting protein secretion in cases where there is a heavy reliance on this process and also have implications for the development of the arylomycins as an antibiotic with activity against Y. pestis and potentially other Gram-negative pathogens.     

In vitro activity of twenty-three antimicrobials againstYersinia pestis strains

The microdilution method was used to determine the MICs of twenty-three antimicrobials against the 22 strains ofYersinia pestis that have been stocked in the National Institute of Health, Japan. The three fluoroquinolones, ofloxacin, ciprofloxacin and sparfloxacin, were most active, with MIC_90S of 0.125 μg/ml. The cephems showed considerable differences in activity: cefdinir and cefotaxime were the most active of this group, with MIC_90S of 0.25 μg/ml; they were followed by cefminox and latamoxef with MIC_90S of 0.5 μg/ml. The two carbapenems tested, imipenem and panipenem, were potently active at MIC_90S of 0.5 μg/ml. The study obtained MIC_90S for tetracycline and doxycycline ranging from 1.0 to 2.0 μg/ml, even though these drugs are known to be clinciall active against plague. Erythromycin and clarithromycin were the least active agents.     

Comparative efficacies of candidate antibiotics against Yersinia pestis in an in vitro pharmacodynamic model

Yersinia pestis, the bacterium that causes plague, is a potential agent of bioterrorism. Streptomycin is the “gold standard” for the treatment of plague infections in humans, but the drug is not available in many countries, and resistance to this antibiotic occurs naturally and has been generated in the laboratory. Other antibiotics have been shown to be active against Y. pestis in vitro and in vivo. However, the relative efficacies of clinically prescribed regimens of these antibiotics with streptomycin and with each other for the killing of Yersinia pestis are unknown. The efficacies of simulated pharmacokinetic profiles for human 10-day clinical regimens of ampicillin, meropenem, moxifloxacin, ciprofloxacin, and gentamicin were compared with the gold standard, streptomycin, for killing of Yersinia pestis in an in vitro pharmacodynamic model. Resistance amplification with therapy was also assessed. Streptomycin killed the microbe in one trial but failed due to resistance amplification in the second trial. In two trials, the other antibiotics consistently reduced the bacterial densities within the pharmacodynamic systems from 10⁸ CFU/ml to undetectable levels (<10² CFU/ml) between 1 and 3 days of treatment. None of the comparator agents selected for resistance. The comparator antibiotics were superior to streptomycin against Y. pestis and deserve further evaluation.     

Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague

Yersinia (Y.) pestis, the causative agent of plague, is endemic in natural foci of Asia, Africa, and America. Real-time PCR assays have been described as rapid diagnostic tools, but so far none has been validated for its clinical use. In a retrospective clinical study we evaluated three real-time PCR assays in two different assay formats, 5′-nuclease and hybridization probes assays. Lymph node aspirates from 149 patients from Madagascar with the clinical diagnosis of bubonic plague were investigated for the detection of Y. pestis DNA. Results of real-time PCR assays targeting the virulence plasmids pPCP1 (pla gene), and pMT1 (caf1, Ymt genes) were compared with an F1-antigen immunochromatographic test (ICT) and cultivation of the organism. Out of the 149 samples an infection with Y. pestis was confirmed by culture in 47 patients while ICT was positive in 88 including all culture proven cases. The best real-time PCR assay was the 5′-nuclease assay targeting pla which was positive in 120 cases. In conclusion, the 5′-nuclease assay targeting pla can be recommended as diagnostic tool for establishing a presumptive diagnosis when bubonic plague is clinically suspected.     

A rapid allele variant discrimination method for Yersinia pestis strains based on high-resolution melting curve analysis

Yersinia pestis isolates were genotyped analyzing the polymorphic DNA regions named variable number tandem repeats (VNTR). Allele variants were studied by high-resolution melting analysis (HRMA) of polymerase chain reaction fragments obtained for 25 VNTR loci. After comparison with previous results, 14 loci gave distinguishable normalized melting curves and allowed to correctly assign alleles. This HRMA typing technique permits to differentiate Y. pestis isolates and turned out to be robust, reproducible, and cheap.     

CP6679, a new injectable cephalosporin with broad spectrum and potent activities against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa

 The antibacterial activities of CP6679, a new injectable cephalosporin with a broad antibacterial spectrum, were compared with those of other cephalosporins. CP6679 had stronger in-vitro activity than ceftazidime and cefpirome against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative staphylococci, and Pseudomonas aeruginosa. Its activity against MRSA was eight times stronger than that of cefpirome, and it showed high binding affinity for penicillin-binding protein 2′ of MRSA. Furthermore, the antibacterial activity of CP6679 against ceftazidime-resistant and imipenem-resistant P. aeruginosa was eight times stronger than that of ceftazidime and four times stronger than that of imipenem. In addition to its in-vitro activities, CP6679 showed the highest efficacy among all cephalosporins tested in murine models of systemic infection induced by MRSA or P. aeruginosa. It was more effective than vancomycin and cefpirome against respiratory tract infections induced by MRSA in mice. Received: October 4, 2001 / Accepted: December 12, 2001     

New Role for FDA-Approved Drugs in Combating Antibiotic-Resistant Bacteria

Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.7 murine macrophages resistant to cytotoxicity induced by the highly virulent Yersinia pestis CO92 strain. Of these compounds, we identified 94 not classified as antibiotics as being effective at preventing Y. pestis-induced cytotoxicity. A total of 17 prioritized drugs, based on efficacy in in vitro screens, were chosen for further evaluation in a murine model of pneumonic plague to delineate if in vitro efficacy could be translated in vivo. Three drugs, doxapram (DXP), amoxapine (AXPN), and trifluoperazine (TFP), increased animal survivability despite not exhibiting any direct bacteriostatic or bactericidal effect on Y. pestis and having no modulating effect on crucial Y. pestis virulence factors. These findings suggested that DXP, AXPN, and TFP may modulate host cell pathways necessary for disease pathogenesis. Finally, to further assess the broad applicability of drugs identified from in vitro screens, the therapeutic potential of TFP, the most efficacious drug in vivo, was evaluated in murine models of Salmonella enterica serovar Typhimurium and Clostridium difficile infections. In both models, TFP treatment resulted in increased survivability of infected animals. Taken together, these results demonstrate the broad applicability and potential use of nonantibiotic FDA-approved drugs to combat respiratory and gastrointestinal bacterial pathogens.     

In vitro antibacterial activity of a new oral cephalosporin S-1090

A new oral cephem antibiotic S-1090 was evaluated for its in vitro antibacterial activity in comparison with cefdinir, cefpodoxime, and cefaclor. S-1090 had fairly potent antibacterial activity when compared with cefdinir, cefpodoxime, and cefaclor. At concentrations below 0.78 μg/mL, S-1090 inhibited 90% of the strains of methicillin-susceptibleStaphylococcus aureus, most streptococci,Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Neisseria gonorrhoeae, andHaemophilus influenzae. S-1090 was minimally active against methicillin-resistantS. aureus, enterococci,Serratia marcescens, Citrobacter freundii, Enterobacter aerogenes, andEnterobacter cloacae, and inactive againstPseudomonas aeruginosa and Xanthomonas maltophilia. S-1090 was stable to hydrolysis by various penicillinases and cephalosporinases, but was rapidly hydrolyzed by penicillinase produced byMoraxella (Branhamella) catarrhalis, by plasmid-mediated OXA-1 type penicillinase, and by cephalosporinases produced byProteus vulgaris andBacteroides fragilis. S-1090 had high affinities for the “lethal target PBPs” ofS. aureus andE. coli, and showed potent bactericidal activity against these bacterial species.     

In vitro and in vivo activities of sparfloxacin and reference drugs againstChlamydia pneumoniae

The activities of sparfloxacin and reference drugs againstChlamydia pneumoniae were compared by using in vitro and in vivo methods. The minimum inhibitory concentration (MIC) (μg/ml) ranges of sparfloxacin, levofloxacin, tosufloxacin, grepafloxacin, AM-1155, DU-6859a, clarithromycin, azithromycin, and minocycline for sixC. pneumoniae strains (two standard and four clinical strains) were 0.031 to 0.063, 0.25 to 0.5, 0.063 to 0.125, 0.063 to 0.125, 0.063 to 0.125, 0.031 to 0.063, 0.016 to 0.031, 0.125 to 0.25, and 0.016 to 0.031, respectively. The in vitro potency of sparfloxacin againstC. pneumoniae was similar to that of clarithromycin, minocycline, and DU-6859a, and higher than that of the other fluoroquinolones and azithromycin. Fatal pneumonia was induced in cyclophosphamide-treated leukopenic mice by intranasal inoculation withC. pneumoniae KKpn-2. Infiltration of the lung by neutrophils and lymphocytes was confirmed by histopathologic examination. Oral treatment with the various antichlamydial agents was given for seven days; sparfloxacin and minocycline had the lowest ED₅₀ (effective treatment dose in 50% of the mice; given as mg/kg per dose) (1.11 each), followed by DU-6859a (1.92), tosufloxacin (2.09), grepafloxacin (2.41), clarithromycin and azithromycin (2.48 each), AM-1155 (2.77), and levofloxacin (>10). These results suggest that sparfloxacin may be an effective agent forC. pneumoniae infection in humans.     

Prophylaxis and therapy of plague

Plague has been a scourge of mankind for centuries, and outbreaks continue to the present day. The virulence mechanisms employed by the etiological agent Yersinia pestis are reviewed in the context of the available prophylactic and therapeutic strategies for plague. Although antibiotics are available, resistance is emerging in this dangerous pathogen. Therapeutics used in the clinic are discussed and innovative approaches to the design and development of new therapeutic compounds are reviewed. Currently there is no licensed vaccine available for prevention of plague in the USA or western Europe, although both live attenuated strains and killed whole-cell extracts have been used historically. Live strains are still approved for human use in some parts of the world, such as the former Soviet Union, but poor safety profiles render them unacceptable to many countries. The development of safe, effective next-generation vaccines, including the recombinant subunit vaccine currently used in clinical trials is discussed.     

Combined use of clarithromycin and antimycobacterial agents inMycobacterium avium complex chronic pulmonary infection

Clarithromycin, a new macrolide antibacterial agent, is effective against disseminatedMycobacterium avium complex (MAC) infection in AIDS patients. In this study, we evaluated the therapeutic effect of clarithromycin, used in combination with other antimycobacterial drugs, against chronic pulmonary MAC infections in non-AIDS patients. Patients were divided into two groups based on the antimycobacterial drugs used for treatment. Patients of group A (n=5) were recent cases diagnosed to have atypical mycobacteriosis. They were treated on diagnosis, with rifampicin (450 mg q.d.), isoniazid (400 mg q.d.), and clarithromycin (200 mg b.i.d.). Patients of group B (n=23) were treated by adding clarithromycin (200 mg b.i.d.) to an existing therapeutic regimen consisting of several antimycobacterial agents. Clinical improvement was observed in seven (25%) patients, including two (40%) from group A and five (22%) from group B. Treatment was associated with a total mycobacterial eradication rate of 100% (5/5) in group A and 22% (5/23) in group B. Clarithromycin was more effective in patients receiving the drug as the first-line therapy than when used in later therapy. Clarithromycin had a lower efficacy rate in this study compared with the reported effect of clarithromycin in AIDS patients with in disseminated MAC infection. Our results of the first-line use of clarithromycin in combination with other mycobacterial agents for the treatment of chronic pulmonary MAC infections indicate that this agent has a limited but encouraging effect on atypical pulmonary mycobacteriosis.     

Cethromycin-mediated protection against the plague pathogen Yersinia pestis in a rat model of infection and comparison with levofloxacin

The Gram-negative plague bacterium, Yersinia pestis, has historically been regarded as one of the deadliest pathogens known to mankind, having caused three major pandemics. After being transmitted by the bite of an infected flea arthropod vector, Y. pestis can cause three forms of human plague: bubonic, septicemic, and pneumonic, with the latter two having very high mortality rates. With increased threats of bioterrorism, it is likely that a multidrug-resistant Y. pestis strain would be employed, and, as such, conventional antibiotics typically used to treat Y. pestis (e.g., streptomycin, tetracycline, and gentamicin) would be ineffective. In this study, cethromycin (a ketolide antibiotic which inhibits bacterial protein synthesis and is currently in clinical trials for respiratory tract infections) was evaluated for antiplague activity in a rat model of pneumonic infection and compared with levofloxacin, which operates via inhibition of bacterial topoisomerase and DNA gyrase. Following a respiratory challenge of 24 to 30 times the 50% lethal dose of the highly virulent Y. pestis CO92 strain, 70 mg of cethromycin per kg of body weight (orally administered twice daily 24 h postinfection for a period of 7 days) provided complete protection to animals against mortality without any toxic effects. Further, no detectable plague bacilli were cultured from infected animals' blood and spleens following cethromycin treatment. The antibiotic was most effective when administered to rats 24 h postinfection, as the animals succumbed to infection if treatment was further delayed. All cethromycin-treated survivors tolerated 2 subsequent exposures to even higher lethal Y. pestis doses without further antibiotic treatment, which was related, in part, to the development of specific antibodies to the capsular and low-calcium-response V antigens of Y. pestis. These data demonstrate that cethromycin is a potent antiplague drug that can be used to treat pneumonic plague.     

鼠疫的临床诊治与预防

鼠疫是由鼠疫耶尔森菌感染引起的全身性人畜共患病,人类主要通过被跳蚤叮咬而患病。常见的临床类型为腺鼠疫、败血症型鼠疫和肺鼠疫,早期诊断和及时治疗可明显改善患者预后。治疗首选氨基糖苷类药物,次选四环素类药物。预防措施主要包括防止跳蚤叮咬和直接接触感染动物或患者,接触肺鼠疫患者时还需采取防止飞沫传播的控制措施。     

Effects of aztreonam in combination with antipseudomonal antibiotics againstPseudomonas aeruginosa isolated from patients with chronic or recurrent lower respiratory tract infection

The antipseudomonal activity of aztreonam combined with other antibiotics aas examined in vitro to suggest effective therapeutic combinations for the treatment of respiratory tract infections due toPseudomonas aeruginosa. Twenty-fiveP. aeruginosa strains were newly isolated from patients with chronic or recurrent lower respiratory tract infection. The susceptibility of these strains to aztreonam alone and to aztreonam combined with ceftazidime, cefclidin, isepamicin and imipenem was measured using the checkerboard method. The observed MICs suggested that aztreonam alone was not very potent againstP. aeruginosa associated with chronic or recurrent lower respiratory infection, but that cefclidin had strong antipseudomonal activity. The fractionary inhibitory concentration index showed synergy in 60% of theP. aeruginosa clinical isolates with aztreonam plus isepamicin, and antagonism in approximately 16% of strains with aztreonam plus imipenem. When combined with isepamcin, aztreonam may have potent activity against pseudomonal lower respiratory tract infections.     

Thrombin‐activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis

Summary. Background: Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin‐activatable fibrinolysis inhibitor (TAFI) has anti‐fibrinolytic properties as the active enzyme (TAFIa) removes C‐terminal lysine residues from fibrin, thereby attenuating accelerated plasmin formation. Results: Here, we demonstrate inactivation and cleavage of TAFI by homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show that omptin‐expressing bacteria decrease TAFI activatability by thrombin‐thrombomodulin and that the anti‐fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli expressing Pla or PgtE. The functional impairment resulted from C‐terminal cleavage of TAFI by the omptins. Conclusions: Our results indicate that TAFI is degraded directly by the omptins PgtE of S. enterica and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during infection.     

Use of an in vitro pharmacodynamic model to derive a moxifloxacin regimen that optimizes kill of Yersinia pestis and prevents emergence of resistance

Yersinia pestis, the causative agent of bubonic, septicemic, and pneumonic plague, is classified as a CDC category A bioterrorism pathogen. Streptomycin and doxycycline are the "gold standards" for the treatment of plague. However, streptomycin is not available in many countries, and Y. pestis isolates resistant to streptomycin and doxycycline occur naturally and have been generated in laboratories. Moxifloxacin is a fluoroquinolone antibiotic that demonstrates potent activity against Y. pestis in in vitro and animal infection models. However, the dose and frequency of administration of moxifloxacin that would be predicted to optimize treatment efficacy in humans while preventing the emergence of resistance are unknown. Therefore, dose range and dose fractionation studies for moxifloxacin were conducted for Y. pestis in an in vitro pharmacodynamic model in which the half-lives of moxifloxacin in human serum were simulated so as to identify the lowest drug exposure and the schedule of administration that are linked with killing of Y. pestis and with the suppression of resistance. In the dose range studies, simulated moxifloxacin regimens of ≥175 mg/day killed drug-susceptible bacteria without resistance amplification. Dose fractionation studies demonstrated that the AUC (area under the concentration-time curve)/MIC ratio predicted kill of drug-susceptible Y. pestis, while the C(max) (maximum concentration of the drug in serum)/MIC ratio was linked to resistance prevention. Monte Carlo simulations predicted that moxifloxacin at 400 mg/day would successfully treat human infection due to Y. pestis in 99.8% of subjects and would prevent resistance amplification. We conclude that in an in vitro pharmacodynamic model, the clinically prescribed moxifloxacin regimen of 400 mg/day is predicted to be highly effective for the treatment of Y. pestis infections in humans. Studies of moxifloxacin in animal models of plague are warranted.     

Antibacterial activity of epigallocatechin gallate againstHelicobacter pylori: Synergistic effect with plaunotol

The antibacterial activity of epigallocatechin gallate (EGCg), a main component of Japanese tea extract, was evaluated against 71 clinical isolates ofHelicobacter pylori recovered from patients with gastric ulcer disease at our hospital. The minimal inhibitory concentration (MIC) was distributed between 8 and 128 μg/mL, and the MIC₉₀ was 64 μg/mL. The combination efficacy of EGCg with an anti-ulcer drug, plaunotol, was evaluated for antibacterial activity againstH. pylori. With addition of 6 μg/mL of plaunotol, the MIC for EGCg decreased significantly in all strains tested. tested. These results suggested that the combination of EGCg and plaunotol could be a useful regimen in the treatment of gastric ulcer.     

In vitro and in vivo antibacterial activities of quinupristin-dalfopristin, a novel injectable streptogramin, against gram-positive cocci and gram-negative respiratory tract pathogens

Quinupristin-dalfopristin, a novel injectable streptogramin, was evaluated for both in vitro and in vivo antibacterial activities in comparison with those of erythromycin, azithromycin, clindamycin, vancomycin, ampicillin, imipenem, and ciprofloxacin. Quinupristin-dalfopristin had high activity against staphylococci and streptococci, including methicillin-resistantStaphylococcus aureus (MRSA), and penicillin-resistantStreptococcus pneumoniae, with MICs at which 90% of strains tested are inhibited (MIC₉₀) equal to 1 μg/mL or less. AgainstEnterococcus spp, quinupristin-dalfopristin was less active than vancomycin with an MIC₉₀ of 32 μg/mL, while againstHaemophilus influenzae, Moraxella (B) catarrhalis andBordetella pertussis, the MIC₉₀s of quinupristin-dalfopristin were 4.0, 1.0, and 0.1 μg/mL, respectively. The minimal bactericidal concentration (MBC) values of quinupristin-dalfopristin against methicillin-susceptible staphylococci, penicillin-resistantS. pneumoniae andH. influenzae were similar to the MIC values, while the MBCs for MRSA andE. faecalis were at least 8-fold greater than the corresponding MIC values. In a murine lung infection model using penicillin-resistantS. pneumoniae, treatment with quinupristin-dalfopristin resulted in a significant reduction in the number of organisms in the lungs compared with that in untreated animals or erythromycin-treated mice (P<0.05). The in vivo efficacy of quinupristin-dalfopristin against experimental septicemia caused by penicillin-sensitiveS. pneumoniae and MRSA was less than that using vancomycin and imipenem-cilastatin, even though it had higher in vitro activity.