Difference In Bacterial Load Research Articles

Evaluation of traditional fish preservation method of Masmin from skipjack tuna (Katsuwonus pelamis) in Lakshadweep, India, with respect to nutritional and environmental perspectives

Masmin preparation is a traditional method that has been practiced for many years to preserve skipjack tuna (Katsuwonus pelamis) mainly in Lakshadweep. However, the method is not evaluated scientifically for its quality concerning nutrition and environmental contamination. Thus, in the present study tuna fillets from four stages of Masmin preparation, namely fresh, boiling, smoking, and sun-drying are evaluated for bacterial, nutritional, and heavy metal contents. The analysis revealed no significant difference in bacterial load, proximate nutrition (p = .93) and essential minerals, and heavy metals (p = .94) among all stages, suggesting the final product “Masmin” is nutritionally equivalent to the fresh tuna. However, pathogenic bacteria, such as Vibrio harveyi, Pseudomonas spp. Bacillus spp. are found, indicating poor hygiene during the processing. Heavy metals concentration like Al, Cd, Co, Cr, Mn, Ni, Zn, and Pb has increased with respect to previous reports, indicating an increase in pollution level in the catchment area that demands proper monitoring. Practical applications The findings inform the personnel engaged in Masmin preparation and consumption about the nutritional value of Masmin which is comparable to the fresh tuna even after different processing stages. Thus, the traditional fish preservation method could be applied to other economic important fishes where the cold facility is limited for fish preservation. On the basis of the microbiological analysis, we recommend the use of solar dryer and smokers in a traditional method of Masmin preparation to reduce microbial and dust contamination during processing and also it reduces the processing time that ultimately increases the production rate. Further, the findings from the heavy metal analysis bring into notice the increase in pollution level in Lakshadweep that raises health concern for the island populations and consumers.

Identificación bacteriana en teléfonos celulares de estudiantes de medicina que acuden o no, a un Hospital General en Mérida, Yucatán, México

The cell phone is an important tool for communication, consultation and information exchange; Studies carried out in hospitals have shown that cell phones of medical personnel are contaminated by nosocomial pathogens. However, the bacterial species that the cell phones house and their potential risk have not been determined. We analized the cell phones of 30 first-year medical students who do not visit the hospital in their studies vs. 30 students who perform their clerkship practices in hospitals, to determine the differences in bacterial loads. Samples were taken by sterile swabs moistened with isotonic saline. McConkey and salt-mannitol agar were used as culture media. The biochemical tests used for identification of enterobacteria were citrate, MIO, LIA, urea and TSI. Degradation tests of mannitol, coagulase, catalase and oxidase were used for the identification of staphylococci. Likewise, smears and Gram staining of the isolated colonies were performed. Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli and Pseudomona sp. were mainly found. Differences in number and bacterial species were found in the cell phones of each study group, representing a reservoir of potentially pathogenic bacteria for humans.

Surface bacterial flora of Tilapia (Oreochromis niloticus) in open markets of Yenagoa, Bayelsa state

Surface bacterial flora of Tilapia in open markets of Yenagoa, Bayelsa State was investigated in order to ascertain their pathogen content, natural toxins and other possible contaminants which is a major cause of diarrhea disease, illness and deaths worldwide. Nine (9) Tilapia fish were obtained from each of three open markets located at Swali, Kpansia and Tombia in Yenagoa. The fish were put in different plastic bags tagged and transported to the Biological science laboratory of the Niger Delta University, Amasoma, Bayelsa State. Swab specimens were then taken from each fish and a stock solution was prepared for each fish specimen. Slide samples for each stock solution were made and analyzed using an electron microscope with magnification of 3000x. Result reveals that the bacteria streptococci, staphylococci were present in all fish samples. There were significant differences in bacterial load in the three different markets in Yenagoa. Based on observation from this study, it can be concluded that fresh fish in Yenagoa pose a serious health risk if consumed without proper washing and cooking.

COBERTURA DE LA LENGUA Y CUENTAS BACTERIANAS SALIVARES EN SUJETOS SANOS Y CON GINGIVITIS Y EN PACIENTES CON PERIODONTITIS

Purpose: The purpose of this study was to investigate the discoloration and coating of the tongue in healthy/gingivitis subjects and periodontitis patients. Furthermore, to determine the relationship between the appearance of the tongue and the bacterial load in salivary samples. Material and Methods: Two groups of patients were studied, 70 healthy/gingivitis subjects and 56 periodontitis patients. After scoring of the tongue a salivary sample of each patient was taken and analyzed using a phase-contrast microscope. Results: This investigation showed that most discoloration was found on the distal part of the tongue. The mean number of bacteria per ml sample in relation to a pink, white and yellow appearance of the tongue was 948, 855 and 900 (x10 6 ) respectively. The mean number of bacteria per ml sample in relation to no, thin, and thick coating was 948, 863 and 895 (x10 6 ) respectively. Analysis did not reveal a relationship between discoloration, coating thickness and total bacterial load. The mean number of bacteria per ml in healthy/gingivitis subjects was 860 and in periodontitis patients 918 (x10 6 ). Conclusion: No relationship between the appearance of the tongue and salivary bacterial load could be detected. There was no difference in bacterial load between the healthy/gingivitis and the periodontitis group within the present study population.

Effects of antibiotic-induced differences in bacterial load on growth and shape of early larval European seabass (Dicentrarchus labraxL.)

The aim of the present study was to quantify the effects of antibiotic-induced differences in bacterial load on the size and shape of fish larvae, using Dicentrarchus labrax of day after hatching (DAH) 3 as a case study. They were split in two treatment groups and reared in 50 ml vials until DAH 14, with the control treatment (“NA”) including larvae reared in filtered autoclaved seawater without antibiotics, while the second (“A”) included larvae reared in filtered autoclaved seawater with rifampicin, ampicillin, kanamycin, trimethoprim and gentamicin, with a concentration of 10 mg/L each. They were sampled for bacterial presence on DAH 4, 7 and 14, and had their mortalities recorded, their total lengths, gut lengths, anal body depths, eye diameters, head depths, yolk sac lengths and yolk sac depths measured, and their outlines analysed on DAH 7 and 14. Treatment NA exhibited the highest mortalities on DAH 14. The antibiotics had a significant size effect, yielding larvae with larger total length on DAH 7 and 14, larger bodies on DAH 7, and on DAH 14 larger anal body depth and greater variance in body size. Their effect on outline shape was also significant in both age classes, with increasing differences from DAH 7 to DAH 14. On DAH 7, “A” specimens were more uniform in their dorso-ventral development, and on DAH 14 “NA” had more slender shapes. The beneficial total length and size effects and the witnessed shape effects might be associated with the low bacterial presence.

Amino acid metabolic signaling influences Aedes aegypti midgut microbiome variability.

The mosquito midgut microbiota has been shown to influence vector competence for multiple human pathogens. The microbiota is highly variable in the field, and the sources of this variability are not well understood, which limits our ability to understand or predict its effects on pathogen transmission. In this work, we report significant variation in female adult midgut bacterial load between strains of A. aegypti which vary in their susceptibility to dengue virus. Composition of the midgut microbiome was similar overall between the strains, with 81–92% of reads coming from the same five bacterial families, though we did detect differences in the presence of some bacterial families including Flavobacteriaceae and Entobacteriaceae. We conducted transcriptomic analysis on the two mosquito strains that showed the greatest difference in bacterial load, and found that they differ in transcript abundance of many genes implicated in amino acid metabolism, in particular the branched chain amino acid degradation pathway. We then silenced this pathway by targeting multiple genes using RNA interference, which resulted in strain-specific bacterial proliferation, thereby eliminating the difference in midgut bacterial load between the strains. This suggests that the branched chain amino acid (BCAA) degradation pathway controls midgut bacterial load, though the mechanism underlying this remains unclear. Overall, our results indicate that amino acid metabolism can act to influence the midgut microbiota. Moreover, they suggest that genetic or physiological variation in BCAA degradation pathway activity may in part explain midgut microbiota variation in the field.

Galectin-3 Is a Target for Proteases Involved in the Virulence of Staphylococcus aureus.

Staphylococcus aureus is a major cause of skin and soft tissue infection. The bacterium expresses four major proteases that are emerging as virulence factors: aureolysin (Aur), V8 protease (SspA), staphopain A (ScpA), and staphopain B (SspB). We hypothesized that human galectin-3, a β-galactoside-binding lectin involved in immune regulation and antimicrobial defense, is a target for these proteases and that proteolysis of galectin-3 is a novel immune evasion mechanism. Indeed, supernatants from laboratory strains and clinical isolates of S. aureus caused galectin-3 degradation. Similar proteolytic capacities were found in Staphylococcus epidermidis isolates but not in Staphylococcus saprophyticus Galectin-3-induced activation of the neutrophil NADPH oxidase was abrogated by bacterium-derived proteolysis of galectin-3, and SspB was identified as the major protease responsible. The impact of galectin-3 and protease expression on S. aureus virulence was studied in a murine skin infection model. In galectin-3+/+ mice, SspB-expressing S. aureus caused larger lesions and resulted in higher bacterial loads than protease-lacking bacteria. No such difference in bacterial load or lesion size was detected in galectin-3-/- mice, which overall showed smaller lesion sizes than the galectin-3+/+ animals. In conclusion, the staphylococcal protease SspB inactivates galectin-3, abrogating its stimulation of oxygen radical production in human neutrophils and increasing tissue damage during skin infection.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis.

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role.OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection.METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains.FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells.MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Changes in Host Cytokine Patterns of TB Patients with Different Bacterial Loads Detected Using 16S rRNA Analysis.

BackgroundTuberculosis (TB) has overtaken HIV as the biggest infectious disease killer, with the majority of deaths occurring in sub-Saharan Africa. However it is unknown how differences in bacterial load alter host immune profiles in the sputum and blood of TB patients.Methods16S ribosomal RNA analysis was used to determine bacterial load in sputum samples obtained from 173 patients with active TB (57 pre-treatment and 116 post-treatment). Host analyte concentrations in sputum and Mycobacterium tuberculosis (Mtb) antigen stimulated whole blood assay supernatants were analysed using multiplex cytokine arrays.ResultsMultiple logistic regression adjusting for age, sex and HIV status showed highly significant correlation of bacterial load with IL1β, IL2, IL1RA, IL4, IL6, IL8, IL9, IL15, IL17, EOTAX, FGF, IFN-γ, GCSF, MCP1, M1P1α, M1P1β, PDGF, TNFα, VEGF in sputum. With increasing time on treatment, FGF levels in sputum displayed the most significant inverse correlation with reduction in bacterial load.ConclusionsWe show that differences in bacterial load correlates with changes in several host biomarkers. These findings have implications for development of tests for TB diagnosis and treatment response.

Gut bacteria differentially affect egg production in the anautogenous mosquito Aedes aegypti and facultatively autogenous mosquito Aedes atropalpus (Diptera: Culicidae).

BackgroundAedes aegypti and A. atropalpus are related mosquitoes that differ reproductively. Aedes aegypti must blood-feed to produce eggs (anautogenous) while A. atropalpus always produces a first clutch of eggs without blood-feeding (facultatively autogenous). We recently characterized the gut microbiota of A. aegypti and A. atropalpus that were reared identically in the laboratory. Here, we assessed the effects of specific members of the gut microbiota in A. aegypti and A. atropalpus on female fitness including egg production.MethodsGnotobiotic A. aegypti and A. atropalpus larvae were colonized by specific members of the gut microbiota. Survival, development time, size and egg production for each treatment was then compared to axenic and conventionally reared larvae.ResultsMost species of bacteria we tested supported normal development and egg production by A. aegypti but only one betaproteobacterium, a Comamonas, supported development and egg production by A. atropalpus to equivalent levels as conventionally reared females. Aedes atropalpus females colonized by Comamonas contained similar stores of glycogen and protein as conventionally reared females, whereas females colonized by Aquitalea did not. Small differences in bacterial loads were detected between gnotobiotic and conventionally reared A. aegypti and A. atropalpus, but this variation did not correlate with the beneficial effects of Comamonas in A. atropalpus.ConclusionsSpecific members of the gut microbiota more strongly affected survival, size and egg production by A. atropalpus than A. aegypti.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1660-9) contains supplementary material, which is available to authorized users.

Isolation site influences virulence phenotype of serotype 14 Streptococcus pneumoniae strains belonging to multilocus sequence type 15.

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported that S. pneumoniae serotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly in in vitro and in vivo phenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally related S. pneumoniae strains.

Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs.

Mucociliary clearance (MCC) and submucosal glands are major components of airway innate immunity that have impaired function in cystic fibrosis (CF). Although both of these defense systems develop postnatally in the ferret, the lungs of newborn ferrets remain sterile in the presence of a functioning cystic fibrosis transmembrane conductance regulator gene. We evaluated several components of airway innate immunity and inflammation in the early CF ferret lung. At birth, the rates of MCC did not differ between CF and non-CF animals, but the height of the airway surface liquid was significantly reduced in CF newborn ferrets. CF ferrets had impaired MCC after 7 days of age, despite normal rates of ciliogenesis. Only non-CF ferrets eradicated Pseudomonas directly introduced into the lung after birth, whereas both genotypes could eradicate Staphylococcus. CF bronchoalveolar lavage fluid (BALF) had significantly lower antimicrobial activity selectively against Pseudomonas than non-CF BALF, which was insensitive to changes in pH and bicarbonate. Liquid chromatography-tandem mass spectrometry and cytokine analysis of BALF from sterile Caesarean-sectioned and nonsterile naturally born animals demonstrated CF-associated disturbances in IL-8, TNF-α, and IL-β, and pathways that control immunity and inflammation, including the complement system, macrophage functions, mammalian target of rapamycin signaling, and eukaryotic initiation factor 2 signaling. Interestingly, during the birth transition, IL-8 was selectively induced in CF BALF, despite no genotypic difference in bacterial load shortly after birth. These results suggest that newborn CF ferrets have defects in both innate immunity and inflammatory signaling that may be important in the early onset and progression of lung disease in these animals.

72 Potential for airborne bacterial contamination in an outpatient respiratory clinic

Objective Recent studies have shown that bacteria of clinical significance in respiratory infection, such as Pseudomonas aeruginosa , have potential for airborne dissemination. This study examined airborne bacteria at an outpatient respiratory clinic and to determine levels of antibiotic resistance among airborne isolates. Methods Air was sampled (Casella slit air sampler, 30litres/min) on two separate days at various locations and time points in a Belfast Respiratory Outpatient Clinic, starting before clinic, until clinic finished. Bronchiectasis (BE) patients attended morning clinic, and Cystic Fibrosis (CF) patients in the afternoon. Air samples were deposited onto Muller-Hinton agar, incubated for 24–48 hrs/37°C/O 2 , and individual colonies counted, Gram stained and identified by 16s rRNA sequencing. The antibiotic susceptibility of selected isolates (n = 11) was determined by E-test®. Results The majority of bacteria detected were Gram +ve (184/242; 76%). The most common were Micrococcus, Kocuria and Staphylococcus spp. Moraxella spp. and one Stenotrophomonas maltophilia isolate were also cultured. No apparent differences in bacterial load were observed between either the BE or CF clinics, or at sequential time points. Of the isolates tested, 8/11 (73%) were resistant to clindamycin. Discussion Although Moraxella, staphylococci and S. maltophilia were observed, the majority of bacteria isolated were those normally designated as skin commensals. Work is currently ongoing, using molecular methods to more fully describe the airborne microbiome and resistome, and to compare patient vs airborne isolates to gauge the potential for transmission within the clinic.

The role of Rhipicephalus sanguineus sensu lato saliva in the dissemination of Rickettsia conorii in C3H/HeJ mice.

Animal models have been developed for the study of rickettsial pathogenesis. However, to understand what occurs during the natural route of rickettsial transmission via the tick bite, the role of tick saliva should be considered in these models. To address this, we analysed the role of tick saliva in the transmission of Rickettsia conorii (Rickettsiales: Rickettsiaceae) in a murine host by intradermally (i.d.) inoculating two groups of susceptible C3H/HeJ mice with this Rickettsia, and infesting one group with nymphal Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks. Quantification of bacterial loads and mRNA levels of interleukin-1β (IL-1β), IL-10 and NF-κB was performed in C3H/HeJ lung samples by real-time quantitative polymerase chain reaction (PCR) and real-time reverse transcriptase PCR, respectively. Lung histology was examined to evaluate the pathological manifestations of infection. No statistically significant difference in bacterial load in the lungs of mice was observed between these two groups; however, a statistically significant difference was observed in levels of IL-1β and NF-κB, both of which were higher in the group inoculated with rickettsiae but not infected with ticks. Lung histology in both groups of animals revealed infiltration of inflammatory cells. Overall, this study showed that i.d. inoculation of R. conorii caused infection in the lungs of C3H/HeJ mice and tick saliva inhibited proinflammatory effects.

Macrophages modulate bacterial persistence and gastric pathology during Helicobacter pylori infection (INC4P.346)

Abstract Based on preliminary data suggesting that macrophages are critical regulators of Helicobacter pylori colonization and gastric pathology, we investigated how macrophage phenotypes shape the outcome of infection and persistence. The loss of PPARγ in the myeloid compartment favors a 5 to 10-fold decrease in bacterial loads at the expense of more severe gastric lesions. PPARγ-deficient macrophages present altered control of transcription factors such as NFκB, which results in a pro-inflammatory phenotype. Stomach lamina propria analyses at weeks 0 to 4 post-infection revealed increased levels of CD11b+ F4/80hi CD64+ CR3CR1+ macrophages in WT mice, while PPARγKO mice failed to expand and maintain such population. Macrophage depletion using clodronate liposomes resulted in a significant reduction of gastric H. pylori in WT mice, thus abrogating the differences in bacterial loads observed between WT and PPARγKO mice. In vitro co-culture of H. pylori with bone marrow derived macrophages showed that either PPARγ loss or pharmacological blockade enhances bactericidal activity, which is associated to significant differential expression of chil1, etv5, iigp1, ptger4, sqle, osm, hspa2 and rptoros levels as revealed by global transcriptome analyses. Hence, macrophages facilitate H. pylori infection by 1) serving as bacterial reservoirs and allowing intracellular replication, and 2) favoring a gastric regulatory response that favors persistence.

Overexpression of activated protein C hampers bacterial dissemination during pneumococcal pneumonia.

BackgroundDuring pneumonia, inflammation and coagulation are activated as part of anti-bacterial host defense. Activated protein C (APC) has anticoagulant and anti-inflammatory properties and until recently was a registered drug for the treatment of severe sepsis. Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia.MethodsWe aimed to investigate the effect of high APC levels during experimental pneumococcal pneumonia. Wild type (WT) and APC overexpressing (APChigh)-mice were intranasally infected with S. pneumoniae and sacrificed after 6, 24 or 48 hours, or followed in a survival study.ResultsIn comparison to WT mice, APChigh-mice showed decreased bacterial dissemination to liver and spleen, while no differences in bacterial loads were detected at the primary site of infection. Although no differences in the extent of lung histopathology were seen, APChigh-mice showed a significantly decreased recruitment of neutrophils into lung tissue and bronchoalveolar lavage fluid. Activation of coagulation was not altered in APChigh-mice. No differences in survival were observed between WT and APChigh-mice (P =0.06).ConclusionAPC overexpression improves host defense during experimental pneumococcal pneumonia. This knowledge may add to a better understanding of the regulation of the inflammatory and procoagulant responses during severe Gram-positive pneumonia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0559-3) contains supplementary material, which is available to authorized users.

Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.

Establishment of a guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen

Objective To establish a suitable guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen .Methods Two different immunization strategies by using the recombinant TB vaccine were employed to boost BCG primed guinea pigs in this study .One was for short-term evaluation with 14 weeks interval between prime and boost immunization and another was for long -term evaluation with 54 weeks interval .In the short-term evaluation group , guinea pigs were boosted twice with the recombinant TB vaccine （ AEC/BC02 ） in every two weeks , while guinea pigs in the long-term evaluation group were boosted for three times with two weeks interval between each injection .A negative con-trol group （ NS→NS） and a BCG control group （ BCG→NS） were both set up in two evaluation groups .One week after the last immunization , all guinea pigs were challenged with M.tuberculosis.Six to seven weeks after bacteria challenge , all animals were euthanized and dissected to evaluate lesion scores of liver , spleen and lung, as well as the viable bacterial load in spleen .Results In the short-term evaluation group , the le-sion scores in those boosted with vaccine （3.33±5.00） was lower than that of BCG control group （5.56± 7.27） （P〉0.05） and negative control group （47.00±28.11） （P=0.0001）.The difference between BCG control group and negative control group in lesion score was also significant .The animals in vaccine boosted group had lower bacterial loads （0.78±1.55 log10 ） in spleen than that in BCG control group （1.06±1.87） （P〉0.05） and negative control group （5.47±0.61） （P=0.0003）.In the long-term evaluation group, the lesion score in those boosted with vaccine was lower （5.0±7.6） than that in BCG control group （14.4± 13.5） （P=0.0394） and negative control group （56.9±14.1） （P〈0.0001）.The animals in vaccine boos-ted group （1.00±1.86 log10） had lower bacterial loads in spleen than that in BCG control group （1.46± 1.94） （P〉0.05） and negative control group （5.43±0.56） （P=0.01）.There was a significant difference in bacterial load between BCG control group and negative group （P=0.0089）.Conclusion The results suggest that the interval time between BCG-prime and boost immunization should be properly prolonged in the guinea pig model used for evaluating the protective effects of new TB vaccines in BCG prime -boost regimen . Key words: Tuberculosis; BCG-prime boost regimen; Animal model; Guinea pig; Recombinant TB vaccine

Inhibition of IKKβ in Enterocytes Exacerbates Sepsis-Induced Intestinal Injury and Worsens Mortality

Nuclear factor-κB is a critical regulator of cell-survival genes and the host inflammatory response. The purpose of this study was to investigate the role of enterocyte-specific NF-kB in sepsis through selective ablation of IkB kinase. Prospective, randomized controlled study. Animal laboratories in university medical centers. Mice lacking functional NF-kB in their intestinal epithelium (Vil-Cre/Ikkβ) and wild-type mice were subjected to sham laparotomy or cecal ligation and puncture. Animals were killed at 24 hours or followed 7 days for survival. Septic wild-type mice had decreased villus length compared with sham mice, whereas villus atrophy was further exacerbated in septic Vil-Cre/Ikkβ mice. Sepsis induced an increase in intestinal epithelial apoptosis compared with sham mice, which was further exacerbated in Vil-Cre/Ikkβ mice. Sepsis induced intestinal hyperpermeability in wild-type mice compared with sham mice, which was further exacerbated in septic Vil-Cre/Ikkβ mice. This was associated with increased intestinal expression of claudin-2 in septic wild-type mice, which was further increased in septic Vil-Cre/Ikkβ mice. Both, pro-inflammatory and anti-inflammatory cytokines were increased in serum following cecal ligation and puncture, and interleukin 10 and monocyte chemoattractant protein-1 levels were higher in septic Vil-Cre/Ikkβ mice than in septic wild-type mice. All septic mice were bacteremic, but no differences in bacterial load were identified between wild-type and Vil-Cre/Ikkβ mice. To determine the functional significance of these results, animals were followed for survival. Septic wild-type mice had lower mortality than septic Vil-Cre/Ikkβ mice (47% vs 80%, p<0.05). Antitumor necrosis factor administration decreased intestinal apoptosis, permeability, and mortality in wild-type septic mice, and a similar improvement in intestinal integrity and survival were seen when antitumor necrosis factor was given to Vil-Cre/Ikkβ mice. Enterocyte-specific NF-kB has a beneficial role in sepsis by partially preventing sepsis-induced increases in apoptosis and permeability, which are associated with worsening mortality.

Efficacy of Daptomycin on Experimental Methicillin-Resistant Staphylococcus aureus Keratitis in Rabbits

We aimed to compare the efficacy of topical daptomycin (DAP) with that of vancomycin (VA) in the treatment of keratitis caused by methicillin-resistant Staphylococcus aureus (MRSA). One hundred colony-forming unit MRSA bacteria were injected intrastromally into both corneas of 28 rabbits. Sixteen hours after injection, the rabbits' eyes were treated with 1 drop of topical DAP (10 or 50 mg/mL), VA (50 mg/mL), or isotonic saline for 19 doses. Their eyes were examined for clinical severity before and after treatment. The minimum inhibitory concentration values of VA and DAP against the bacterial strain were found to be 2 and 0.5 μg/mL, respectively. The mean pre- and post-treatment clinical scores of the eyes did not differ significantly among the groups. However, the mean difference between the post- and pretreatment clinical scores was significantly lower in the 50 mg/mL DAP group than in the other groups (P=0.042). A marked decrease in bacterial load was detected in all treatment groups compared to the control group (P=0.002). Although there were no significant differences in bacterial load among the treatment groups, the 50 mg/mL DAP group showed the greatest decrease. The mean % epithelial erosion rate tended to be higher in the 50 mg/mL VA group than in the other groups (P=0.31). Topical DAP significantly reduced the bacterial load and showed activity against MRSA comparable to that of fortified VA in this experimental model.