Rifampin-resistant Strain Research Articles

Pulmonary Tuberculosis in the Elderly: Epidemiological, Clinical, and Evolutionary Aspects

Aims: this study aimed to investigate the epidemiological, clinical, and evolutionary aspects of pulmonary tuberculosis in the elderly and to determine the factors associated with death. 
 Study Design: This was an exhaustive, descriptive, and analytical retrospective study.
 Place and Duration of Study: Pneumo-phtisiology department of Fann National Teaching Hospital Center over three years, from January 1, 2019, to December 31, 2021.
 Methodology: This was a study using medical records of patients aged 60 and over hospitalized in the pneumo-phtisiology department. Data were collected on a pre-established from patient medical records entered with Microsoft Excel 2019 software. Data analysis was performed with Statistical Package for Sciences Socials (SPSS) version 18
 Results: we collected 620 medical records of hospitalized patients aged 60 and over, 69 of whom were diagnosed with pulmonary tuberculosis, standing for a prevalence of 11.12%. The average age was 67.13 [60;85] years, with a sex ratio of 3.92. The 60 to 65 age group was the most represented (n=36 or 52.17%). Smoking and alcoholism were frequent, 82% and 17.4% respectively. A previous history of TB infection was found in 23.19% of cases and TB contagion in 20.3% of cases. An underlying condition was at least observed in 50.72% of patients, dominated by diabetes (29%) and hypertension (23%). Co-infection with HIV-1 and tuberculosis was found in 2 patients (3%). The time to consultation was 75 days [3-180 days]. Symptomatology was polymorphic, dominated by cough (n=65; 94.20%), altered general condition (n=63; 91.30%), sputum (n=58; 84.06%), fever (n=53; 76.81%) and dyspnea (n=47; 68.12%). 46 patients (66.7%) had a BMI < 18.5 kg/m2. Physical signs were dominated by pulmonary condensation (n=52; 75.36%), and a fold of undernutrition was noted in 21.74% of cases. Microbiological evidence was 92.75% and 64 patients (92.75%) were susceptible to rifampin. A rifampin-resistant strain was detected in 5 patients (7.25%) hospitalized in the MDR-TB unit. CRP (75.36%), anemia (68.18%), hyperleukocytosis (47.7%), and hyponatremia (27.54%) were the most frequent biological signs.
 Alveolar syndrome (n=48 or 70%) and cavitary images (n=34 or 50%) were most frequently found on lung radiography, and for those who benefited from thoracic CT (n=36 or 52.17%), cavitary images were found in 24 cases (66.66%).
 Pulmonary embolism (n=9 cases, i.e. 40.9%), superinfection (n=4), and excessive hemoptysis (n=3) were the most frequent complications. The mean length of hospital stay was 17 days [3-58 days]. The 2RHZE/4RH protocol was used for 87%. Two patients were placed on a second-line anti-tuberculosis treatment protocol. Treatment was not specified in 7 patients (10.14%). The progression was favorable in 48 patients (69.56% of cases), with a case fatality rate of 30.44%. Dyspnea, chest pain, anemia, hyperleukocytosis, and hyponatremia were associated with the risk of death.
 Conclusion: The elderly are particularly exposed to tuberculosis, due to physiological aging of the immune system and co-morbidities. This study highlighted the diagnostic and evolutionary particularities of pulmonary tuberculosis in the elderly, as well as the associated fatal factors.

Polyribosome-Dependent Clustering of Membrane-Anchored RNA Degradosomes To Form Sites of mRNA Degradation in Escherichia coli.

ABSTRACTThe essential endoribonuclease RNase E, which is a component of the Escherichia coli multienzyme RNA degradosome, has a global role in RNA processing and degradation. RNase E localizes to the inner cytoplasmic membrane in small, short-lived clusters (puncta). Rifampin, which arrests transcription, inhibits RNase E clustering and increases its rate of diffusion. Here, we show that inhibition of clustering is due to the arrest of transcription using a rifampin-resistant control strain. Two components of the RNA degradosome, the 3′ exoribonuclease polynucleotide phosphorylase (PNPase) and the DEAD box RNA helicase RhlB, colocalize with RNase E in puncta. Clustering of PNPase and RhlB is inhibited by rifampin, and their diffusion rates increase, as evidenced by in vivo photobleaching measurements. Results with rifampin treatment reported here show that RNA degradosome diffusion is constrained by interaction with RNA substrate. Kasugamycin, which arrests translation initiation, inhibits formation of puncta and increases RNA degradosome diffusion rates. Since kasugamycin treatment results in continued synthesis and turnover of ribosome-free mRNA but inhibits polyribosome formation, RNA degradosome clustering is therefore polyribosome dependent. Chloramphenicol, which arrests translation elongation, results in formation of large clusters (foci) of RNA degradosomes that are distinct from puncta. Since chloramphenicol-treated ribosomes are stable, the formation of RNA degradosome foci could be part of a stress response that protects inactive polyribosomes from degradation. Our results strongly suggest that puncta are sites where translationally active polyribosomes are captured by membrane-associated RNA degradosomes. These sites could be part of a scanning process that is an initial step in mRNA degradation.

Outbreak of an Uncommon Rifampin-resistant blaNDM-1Citrobacter amalonaticus Strain in a Digestive Rehabilitation Center: The Putative Role of Rifaximin.

We describe a sudden 2-week outbreak due to a blaNDM-1Citrobacter amalonaticus strain in a 22-bed digestive rehabilitation center. Three of the 5 colonized patients received long-term rifaximin treatment to prevent hepatic encephalopathy. The strains were genotypically identical, phenotypically resistant to rifampin, and harbored arr-3, a rifampin adenosine diphosphate-ribosyl transferase.

Effect of Macrolide and Rifampin Resistance on Fitness of Rhodococcus equi during Intramacrophage Replication and In Vivo.

The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a facultative intracellular pathogen of macrophages and causes severe bronchopneumonia when inhaled by susceptible foals. Standard treatment for R. equi disease is dual-antimicrobial therapy with a macrolide and rifampin. Thoracic ultrasonography and early treatment with antimicrobials prior to the development of clinical signs are used as means of controlling endemic R. equi infection on many farms. Concurrently with the increased use of macrolides and rifampin for chemoprophylaxis and the treatment of subclinically affected foals, a significant increase in the incidence of macrolide- and rifampin-resistant R. equi isolates has been documented. Previously, our laboratory demonstrated decreased fitness of R. equi strains that were resistant to macrolides, rifampin, or both, resulting in impaired in vitro growth in iron-restricted media and in soil. The objective of this study was to examine the effect of macrolide and/or rifampin resistance on intracellular replication of R. equi in equine pulmonary macrophages and in an in vivo mouse infection model in the presence and absence of antibiotics. In equine macrophages, the macrolide-resistant strain did not increase in bacterial numbers over time and the dual macrolide- and rifampin-resistant strain exhibited decreased proliferation compared to the susceptible isolate. In the mouse model, in the absence of antibiotics, the susceptible R. equi isolate outcompeted the macrolide- or rifampin-resistant strains.

Influence of temperature, humidity, and diluent type on survival of Salmonella spp. on the surface of raw tomatoes

Tomatoes are an important commodity, placing fourth among most popular vegetables in the U.S. However, fresh tomatoes lack a final pathogen elimination step and have been implicated in Salmonella-related outbreaks. The purpose of the study was to evaluate survival of Salmonella post-drying in three diluents on the surface of green mature tomatoes at 12 °C or 25 °C. Additionally, low and high air relative humidity influence was evaluated at 25 °C on pathogen survival. A five Salmonella rifampin-resistant strain cocktail was double-washed in buffered peptone water (BPW) and resuspended in 0.1% peptone, BPW, or fresh tomato serum. Inoculum (0.1 mL) was allowed to dry on the surface of tomatoes. For study I, tomatoes were placed in 12 °C and 25 °C incubators with no humidity control and sampled on days 0, 1, 3, and 5. For study II, tomatoes were sampled on days 0, 1 (biosafety hood storage) and on day 5 after storage in two 25 oC incubators (low and high relative humidity). Salmonella was recovered from tomatoes (20 mL BPW) and plated (TSA-rif80, 37 °C, 48 hours). Post-drying Salmonella counts (ca. 4.5 - 5.0 log10 CFU.mL-1) remained at 4.03 and 4.40 log10 CFU.mL-1 in serum after 5 days of storage at 12 °C and 25 °C, respectively. Conversely, corresponding counts in BPW and peptone were lower at ca. 1.4 to 1.8 and 2.2 to 2.8 log units at 12 °C and 25 °C, respectively. At low humidity, post-drying Salmonella counts showed highest decline for peptone (final 1.98 log10 CFU.mL-1) compared to BPW (3.79 log10 CFU.mL-1) and tomato serum (4.75 log10 CFU.mL-1) on day 5. Counts declined rapidly to 0.03, 0.56, and 0.44 log10 CFU.mL-1 for peptone, BPW, and tomato serum, respectively, at high humidity on day 5. To summarize, it was shown that increased solutes have protective effect on Salmonella in desiccated conditions, while high humidity storage causes accelerated death of stationary culture within five days storage period.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such "differentially detectable" (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB, which encodes the canonical rifampin target, the β subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin.

Draft Genome Sequence of Highly Rifampin-Resistant Propionibacterium namnetense NTS 31307302T Isolated from a Patient with a Bone Infection.

Propionibacterium namnetense was recently described as a potential bone pathogen, which is closely related to Propionibacterium acnes, a skin commensal microorganism. Here, we report the draft genome sequence of the highly rifampin-resistant strain NTS 31307302T isolated from a patient with a tibia infection.

Highly efficient production of hyaluronic acid by Streptococcus zooepidemicus R42 derived from heterologous expression of bacterial haemoglobin and mutant selection.

During Streptococcus zooepidemicus fermentation, most carbon sources are used to synthesize lactic acid, which can inhibit strain growth and hyaluronic acid production. Here, we expressed bacterial haemoglobin (Vhb) in Strep.zooepidemicus. Due to highly efficient oxygen use, only 15·26gl(-1) lactic acid was produced, which is 0·73 times the quantity produced by the control strain. Compared with the control strain (1·61gl(-1) ), hyaluronic acid (HA) production in this strain did not substantially increase, only to 2·16gl(-1) . Next, we used a series of N-methyl-N'-nitro-N-nitroso-guanidine (NTG) treatments and selection programmes. Finally, we generated a hyaluronidase-negative and rifampin-resistant mutant strain that produces high levels of HA. The optimum carbon concentration for maximum hyaluronic acid production is only 30gl(-1) of sucrose, which is lower than the control strain (60gl(-1) ). The oxygen transfer rate coefficient KL a increased significantly to 372±53h(-1) from 18±4h(-1) of the control. The optimum carbon source for this strain is 21gl(-1) of sucrose, 9gl(-1) of maltose and 5gl(-1) of glutamic acid. Hyaluronic acid accumulated at 6·7gl(-1) in the culture broth. However, the molecular weight of HA decreased from 1835KDa (Control) to 429kDa. The prepared low-molecular weight HA could function as potential antiangiogenic substances, antiviral and antitumour agents to possibly be used as functional food ingredients. Hyaluronic acid (HA) has been used for a wide range of applications in health, cosmetic and clinical fields. During fermentation of Streptococcus to produce HA, 80-85% of the carbon source is used to produce lactic acid and acetic acid, and only approx. 5 and 10% of the carbon source is used to produce HA and biomass respectively. Here, we expressed bacteria haemoglobin (Vhb) in Streptococcus zooepidemicus, which can dramatically inhibit lactic acid production. After NTG treatments and selection programmes, we identified a mutant strain with highly efficient hyaluronic acid production (6·7gl(-1) ) under economic fermentation conditions.

In vitro activity of rifampin against and rpoB mutations in Chlamydia trachomatis clinical isolates

Objective To evaluate the susceptibility of Chlamydia trachomatis clinical isolates to rifampin, and assess the relationship between rpoB mutations and antibiotic resistance in them. Methods A microculture method was used to determine the minimal inhibitory concentration (MIC) of rifampin in 52 Chlamydia trachomatis clinical isolates. The rpoB gene was amplified from all the clinical isolates and a standard strain of Chlamydia trachomatis followed by single-strand conformation polymorphism (SSCP) analysis. Sequencing of PCR products was carried out for two clinical isolates. Results No rifampin-resistant strain was found among these clinical isolates. The MIC of rifampin varied from 0.004 to 0.030 mg/L. Neither SSCP analysis nor sequencing showed rpoB mutations. Conclusions No rpoB mutations were found in Chlamydia trachomatis isolates from patients unresponsive to rifampin. The unresponsiveness to rifampin may be attributed to multiple factors. Key words: Chlamydia trachomatis; Rifampin; Microbial sensitivity tests; Drug resistance, microbial; DNA mutational analysis; Genes, rpoB

Pseudomonas stutzeri Strain Possessing a Self-Transmissible TOL-Like Plasmid Degrades Phenol and Promotes Maize Growth in Contaminated Environments

Phenol is volatile organic pollutant that plants can little degrade. For complete degradation of volatile pollutants, we introduced Pseudomonas stutzeri strain P7 to phenol-contaminated soils. The strain effectively degraded phenol and even promoted plant growth. A TOL-like plasmid was detected in the strain and found to be responsible for phenol degradation and self-transmissible. In addition, phenol degradation by strain P7 was more rapid in the contaminated soils with than without plants over the full course of the experiment; especially by 5days, the phenol concentration was reduced by about 30% in soil without plants and reduced by about 50-65% in soil with plants. This situation also occurred when inoculated with different transconjugants. Furthermore, transfer frequencies of TOL-like plasmid were significantly higher in soil with than without plants. Populations of rifampin-resistant P7 strain remained relatively constant for 20days, while the number of rhizosphere bacteria that contained the degradative plasmids gradually increased at the later stages, suggesting that plants might stimulate plasmid transfer from strain P7 to indigenous bacteria, one possible reason for plant enhancing microbial degradation. This is attractive for implementation of combinations of phytoremediation and bioaugmentation in degradation of volatile pollutants that plants can little degrade.

Design, synthesis and evaluation of acridine and fused-quinoline derivatives as potential anti-tuberculosis agents

The synthesis of twelve acridine and polycyclic acridine derivatives prepared via the Friedländer reaction is described. The one-pot reactions of 2-amino-5-chloro or 5-nitro-benzophenones and a variety of cyclanones and indanones were carried out in a MW oven under TFA catalysis in good yields. The products were designed according natural antituberculosis products and were evaluated for growth inhibitory activity towards Mycobacterium tuberculosis H37Rv (Mtb) through the National Institute of Allergy and Infectious Diseases (NIAID, USA). Three of them underwent additional testings. The cyclopenta[b]quinoline derivative 9 and the acridine derivative 13 showed remarkable MIC values against the rifampin resistant strain. The former exhibited bactericidal activity at 50 μg/mL, its intracellular activity is similar to rifampin and it was not cytotoxic at low concentrations so it can be considered a new lead compound.

Rifampin-ResistantMycobacterium bovisBCG–Induced Disease in HIV-Infected Infant, Vietnam

To the Editor: Guidelines for the diagnosis and management of Mycobacterium bovis BCG disease in HIV-infected children are lacking. BCG strains are intrinsically resistant to pyrazinamide and in some cases have low-level resistance to isoniazid (6). However, data on acquired drug resistance in M. bovis BCG are limited. We describe a case of BCG disease caused by a rifampin-resistant strain of M. bovis BCG in an HIV-infected infant in Vietnam. The daughter of a known HIV-infected woman, who did not fully adhere to antiretroviral therapy (ART) during pregnancy, received the M. bovis intradermal BCG (Pasteur strain) vaccine at birth. HIV infection was diagnosed in the infant by PCR when she was 8 weeks of age. At 9 months of age, she was admitted to the Pediatric Infectious Diseases Department of the Pham Ngoc Thach Hospital (Ho Chi Minh City, Vietnam) because of a voluminous ipsilateral axillary mass at the site of the vaccination, fever, weight loss, and hepatosplenomegaly. The percentage of CD4+ T cells was 27% (1,620 cells/mm3). Regional BCG disease was clinically diagnosed without microbiological investigation, and a broad antimycobacterial therapy targeting M. tuberculosis complex species was started with 5 mg/kg isoniazid, 10 mg/kg rifampin, and 25 mg/kg pyrazinamide. After 6 weeks of antimycobacterial therapy, ART was initiated with lamivudine, stavudine, and abacavir. After 6 months of antimycobacterial treatment, the infant was hospitalized again for recurrent inflammation and fistulization of the axillary lymph nodes associated with fever. Fluid from the axillary mass was collected by fine-needle aspiration for bacteriologic investigations. Direct microscopic examination showed acid-fast bacilli, and the mycobacterial infection was confirmed by culture. By using conventional biochemical methods, the mycobacterial isolate was assigned to the M. bovis species. Pyrazinamide was discontinued, and antimycobacterial therapy was continued for 4 supplementary months with rifampin (15 mg/kg) and isoniazid (10 mg/kg). After 2 months, drug susceptibility testing results confirmed pyrazinamide intrinsic resistance and isoniazid and ethambutol susceptibility and showed rifampin resistance. The late inflammatory reaction after introduction of ART was evocative of immune reconstitution inflammatory syndrome. Nevertheless, drug resistance may have contributed. Despite the rifampin resistance, the patient showed clinical improvement, and the rifampin/isoniazid treatment was continued for 2 more months. The child’s BCG disease was cured on completion of 10 months of antituberculous treatment. Retrospective molecular investigations using the GenoType MTBC Kit (Hain Lifescience, Nehren, Germany) enabled identification of the isolate stored at −80°C as M. bovis BCG strain. A mutation in the rpoB gene (codon 531, Ser531Tyr) associated with rifampin resistance was detected by using the GenoType MTBDRplus Kit (Hain Lifescience) and partial sequencing of the rpoB gene (5,7). No mutation in the katG and inhA genes, frequently associated with isoniazid resistance, was detected. To our knowledge, this case is the second report of rifampin-resistant M. bovis BCG disease in HIV-infected children. The first report involved a child in South Africa who was vaccinated with the Danish BCG strain (4); this strain shows low-level resistance to isoniazid and therefore has a high risk of evolving to multidrug resistance in instances of suboptimal isoniazid levels. The M. bovis BCG Pasteur strain (American Type Culture Collection 35734) used for vaccination in Vietnam is isoniazid and rifampin susceptible and pyrazinamide resistant (9). Despite appropriate antimycobacterial treatment, the relatively low doses of isoniazid (5 mg/kg), poor adherence, or inadequate absorption of drugs because of HIV-related gastrointestinal disease may have resulted in subtherapeutic in vivo drug concentrations and thus in selection of a drug-resistant M. bovis BCG strain. This case should alert clinicians of the possible emergence of rifampin resistant M. bovis BCG strains. Because disseminated BCG disease in HIV-infected children presents a high risk for illness and/or death, these patients should receive optimal tuberculosis treatment (2) based on 4-drug (rifampin, isoniazid, ethambutol, and pyrazinamide) regimen doses for at least 9 months until M. tuberculosis is ruled out (3). Untreated local BCG immune reconstitution inflammatory syndrome may not necessarily progress to dissemination; therefore, treatment would not appear necessary (8). Some studies suggest that the survival of HIV-infected children with BCG disease could be attributed to early initiation of ART in combination with other treatments (1,3). In the South Africa case, the child died, and the authors suggested that this outcome was related to the severity of the clinical features, the severe HIV-related immune suppression, and the absence of ART (4). In the case in Vietnam, despite the emergence of drug resistance, the early initiation of ART in a child with a localized disease, the persistent efficacy of isoniazid, and the spontaneous fistulization of the abscess probably contributed to the good outcome for the infant. In conclusion, this case highlights the challenges in management of BCG disease in children. It also emphasizes the possible risk for emergence of acquired drug resistance in M. bovis BCG strains, complicating the medical management of such cases.

Effect of chemical sanitizers on Salmonella enterica serovar Poona on the surface of cantaloupe and pathogen contamination of internal tissues as a function of cutting procedure.

Survival of Salmonella enterica subsp. enterica serovar Poona on surface and stem scar portions of inoculated cantaloupe following sanitizer application, transfer of pathogen from the rind to the flesh during cutting, and growth of Salmonella Poona on cantaloupe cubes over 15 days of refrigerated storage were investigated. Cantaloupes inoculated with a rifampin-resistant strain of Salmonella Poona (10(7) CFU/ml) for 3 min and dried for 12 h were washed with chlorine (200 mg free chlorine per liter, 3 min), lactic acid (2%, 2 min), or ozone (30 mg/liter, 5 min). Fresh-cut cantaloupe cubes were prepared by (i) cutting the cantaloupe and then removing the rind or by (ii) peeling the rind and then cutting the flesh into pieces. The numbers of Salmonella bacteria recovered were higher in the stem scar portion (6.3 ± 0.3 log CFU/cm(2)) than the surface (4.8 ± 0.2 log CFU/cm(2)). Surface treatment with tap water or chlorine did not reduce Salmonella numbers, while treatment with lactic acid or ozone reduced Salmonella by 2.5 or 2.3 log CFU/cm(2), respectively. The use of lactic acid to sanitize the cantaloupes resulted in less Salmonella transfer to flesh during cutting; Salmonella numbers decreased to below detectable levels over 9 days of refrigerated (4°C) storage. Cutting cantaloupes after peeling the rind was more effective at reducing transfer of Salmonella to the internal tissue than cutting of cantaloupes prior to rind removal. These data suggest that treatment of cantaloupe rinds with lactic acid or ozone may be effective at reducing Salmonella numbers, while lactic acid application resulted in reduction of Salmonella transfer to cantaloupe flesh.

Vibrio vulnificus rpoS Expression Is Repressed by Direct Binding of cAMP-cAMP Receptor Protein Complex to Its Two Promoter Regions

Vibrio vulnificus, a septicemia-causing pathogenic bacterium, acquires resistance against various stresses and expresses virulence factors via an rpoS gene product. In this study, we investigated the transcriptional characteristics of this global regulator. Two distinct transcriptional initiation sites for the rpoS gene, the proximal promoter (P(p)) and the distal promoter (P(d)), were defined by primer extension experiments. Various rpoS::luxAB transcriptional fusions indicated that P(d) is a major promoter of rpoS expression. Western blot analysis showed that RpoS levels were inversely correlated with intracellular levels of 3',5'-cyclic AMP (cAMP). The expressions of both P(d) and P(p) were increased in cya and crp mutants. The exogenous addition of cAMP to the cya mutant resulted in repressed expression of rpoS. In addition, rpoS expression was significantly lowered in the cpdA mutant, in which the level of cAMP was elevated because of the absence of 3',5'-cAMP phosphodiesterase. In vitro transcription assays using the V. vulnificus RNA polymerase showed that the transcripts from both promoters were reduced by addition of the cAMP-cAMP receptor protein (CRP). The cAMP-CRP was shown to bind to two rpoS promoters by electrophoretic mobility shift assays. The alteration of the putative CRP-binding site on each rpoS promoter, via site-directed mutagenesis, abolished the binding of cAMP-CRP as well as regulation by cAMP-CRP. Therefore, this study shows a relationship between the level of intracellular cAMP and the degree of rpoS expression and further demonstrates, for the first time, the direct binding of the cAMP-CRP complex to rpoS upstream regions, which results in repression of rpoS gene expression.

Efficacy of Novel Rifamycin Derivatives against Rifamycin-Sensitive and -Resistant Staphylococcus aureus Isolates in Murine Models of Infection

Novel rifamycins (new chemical entities [NCEs]) having MICs of 0.002 to 0.03 microg/ml against Staphylococcus aureus and retaining some activity against rifampin-resistant mutants were tested for in vivo efficacy against susceptible and rifampin-resistant strains of S. aureus. Rifalazil and rifampin had a 50% effective dose (ED50) of 0.06 mg/kg of body weight when administered as a single intravenous (i.v.) dose in a murine septicemia model against a susceptible strain of S. aureus. The majority of NCEs showed efficacy at a lower i.v. dose (0.003 to 0.06 mg/kg). In addition, half of the NCEs tested for oral efficacy had ED50s in the range of 0.015 to 0.13 mg/kg, i.e., lower or equivalent to the oral ED50s of rifampin and rifalazil. NCEs were also tested in the septicemia model against a rifampin-resistant strain of S. aureus. Twenty-four of 169 NCEs were efficacious when administered as a single oral dose of 80 mg/kg. These NCEs were examined in the murine thigh infection model against a susceptible strain of S. aureus. Several NCEs dosed by intraperitoneal injection at 0.06 mg/kg caused a significant difference in bacterial titer compared with placebo-treated animals. No NCEs showed efficacy in the thigh model against a highly rifampin-resistant strain. However, several NCEs showed an effect when tested against a partially rifampin-resistant strain. The NCEs having a 25-hydroxyl moiety were more effective as a group than their 25-O-acetyl counterparts. These model systems defined candidate NCEs as components of potential combination therapies to treat systemic infections or as monotherapeutic agents for topical applications.

Improving the Microbiological Quality and Safety of Fresh-Cut Tomatoes by Low-Dose Electron Beam Irradiation

The effect of electron beam irradiation on microbiological quality and safety of fresh-cut tomatoes was studied. Fresh tomatoes were obtained from a local supplier and then cut into cubes that were separated from the stem scars. Both cubes and stem scars were inoculated with a rifampin-resistant strain of either Salmonella Montevideo or Salmonella Agona, separated into treatment groups, and treated by electron beam irradiation at 0.0 (control), 0.7, or 0.95 kGy. The effect of electron beam irradiation on Salmonella, lactic acid bacteria, yeast, and mold counts and pH of tomato cubes and stem scars was determined over a 15-day storage period at 4°C. Results indicated that although irradiation treatment significantly reduced most microbial populations on tomato samples, there were no differences in the reduction of microbial populations between treatments of 0.7 and 0.95 kGy. Irradiation at either dose resulted in a significant reduction in Salmonella when compared with the control (P < 0.05). Lactic acid bacteria, yeasts, and molds were more resistant to irradiation than were Salmonella. No differences were detected between the two Salmonella serotypes in response to irradiation treatment. These results indicate that irradiation at doses of at least 0.7 kGy can be used for pathogen reduction in fresh-cut tomatoes. If the use of doses greater than 1 kGy were approved, this technology might be very effective for use in fresh-cut tomatoes to eliminate significant populations of pathogens and to ensure the microbial quality of the product.

Optimization of Ferrioxamine E Concentration as Effective Supplementation for Selective Isolation of Salmonella Enteritidis in Egg White

Utilization of ferrioxamine E (FE) as a sole source of iron distinguishes Salmonella from a number of related species, including Escherichia coli. FE is not able to serve as a source of iron for E. coli or the Proteus-Providencia-Morganella group. This confers a selective advantage on Salmonella Enteritidis in egg white supplemented with FE. The optimum concentration of FE that promoted a selective advantage for Salmonella in egg white was determined. Four supplementation concentrations were evaluated (25, 50, 200, and 500 μg/ml) in egg white artificially inoculated with proportionally mixed cultures of a rifampin-resistant strain of Salmonella Enteritidis (0.1 ml of 102 CFU/ml) and E. coli K-12 (0.1 ml of 101 through 108 CFU/ml). After a 24-h incubation at 37°C, Salmonella and E. coli populations were enumerated. At higher concentrations of FE (>50 μg/ml), both Salmonella and E. coli were able to use the iron supplement (1 to 8.5 log CFU/ml and 1.8 to 8 log CFU/ml, respectively); however, lower FE concentrations (≤50 μg/ml) exclusively promoted Salmonella growth. Salmonella was unrecoverable without supplementation. This study indicates that optimum levels of FE supplementation in egg can improve the selective detection for Salmonella Enteritidis among other competitive organisms.

Anti‐HIV Natural Product (+)‐Calanolide A Is Active Against Both Drug‐Susceptible and Drug‐Resistant Strains of Mycobacterium tuberculosis.

Naturally occurring anti-HIV-1 agent (+)-calanolide A was found to be active against all of the strains of Mycobacterium tuberculosis tested, including those resistant to the standard antitubercular drugs. Efficacy evaluations in macrophages revealed that (+)-calanolide A significantly inhibited intracellular replication of M. tuberculosis H37Rv at concentrations below the MIC observed in vitro. Preliminary mechanistic studies indicated that (+)-calanolide A rapidly inhibits RNA and DNA synthesis followed by an inhibition of protein synthesis. Compared with known inhibitors, this scenario is more similar to effects observed with rifampin, an inhibitor of RNA synthesis. Since (+)-calanolide A was active against a rifampin-resistant strain, it is believed that these two agents may involve different targets. (+)-Calanolide A and its related pyranocoumarins are the first class of compounds identified to possess antimycobacterial and antiretroviral activities, representing a new pharmacophore for anti-TB activity.

Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis

Naturally occurring anti-HIV-1 agent (+)-calanolide A was found to be active against all of the strains of Mycobacterium tuberculosis tested, including those resistant to the standard antitubercular drugs. Efficacy evaluations in macrophages revealed that (+)-calanolide A significantly inhibited intracellular replication of M. tuberculosis H37Rv at concentrations below the MIC observed in vitro. Preliminary mechanistic studies indicated that (+)-calanolide A rapidly inhibits RNA and DNA synthesis followed by an inhibition of protein synthesis. Compared with known inhibitors, this scenario is more similar to effects observed with rifampin, an inhibitor of RNA synthesis. Since (+)-calanolide A was active against a rifampin-resistant strain, it is believed that these two agents may involve different targets. (+)-Calanolide A and its related pyranocoumarins are the first class of compounds identified to possess antimycobacterial and antiretroviral activities, representing a new pharmacophore for anti-TB activity.

Emergence of rifampin-resistant Rhodococcus equi with several types of mutations in the rpoB gene among AIDS patients in northern Thailand.

The antimicrobial susceptibilities of 30 Rhodococcus equi isolates obtained from 30 patients between 1993 and 2001 in northern Thailand were investigated. The MICs showed a tendency toward resistance to various antibiotics but sensitivity to imipenem, minocycline, vancomycin, and teicoplanin (MICs, </=0.5 micro g/ml) and relative sensitivity to meropenem, clarithromycin, and ciprofloxacin (MICs, </=2 micro g/ml). Of the 30 isolates, 26 were susceptible (MICs, </=1 micro g/ml), 1 showed low-level resistance (MIC, 8 micro g/ml), and 3 showed high-level resistance (MICs, >/=64 micro g/ml) to rifampin. PCR amplification and DNA sequencing of the rpoB gene and molecular typing by pulsed-field gel electrophoresis (PFGE) were performed for eight R. equi isolates from eight AIDS patients with pneumonia or lung abscess caused by R. equi between 1998 and 2001, including one low- and three high-level rifampin-resistant isolates. As a result, two high-level rifampin-resistant strains with PFGE pattern A had a Ser531Trp (Escherichia coli numbering) mutation, and one high-level rifampin-resistant strain with PFGE pattern B had a His526Tyr mutation, whereas one low-level rifampin-resistant strain with PFGE pattern C had a Ser509Pro mutation. Four rifampin-susceptible strains with PFGE patterns D and E showed an absence of mutation in the rpoB region. Our results indicate the presence of several types of rifampin-resistant R. equi strains among AIDS patients in northern Thailand.