Assessment of Serum Bactericidal and Opsonophagocytic Activity of Antibodies to Gonococcal Vaccine Targets.

There is no vaccine available to prevent Neisseria gonorrhoeae infection, however there is currently a high level of interest in developing gonococcal vaccines due to the increasing number of cases and continuing emergence of antimicrobial resistance worldwide. A key aspect of vaccine development is the investigation of the functional immune response raised to the vaccine targets under investigation. Here, we describe two assays used to assess the functional immune response raised against gonococcal vaccine targets: the serum bactericidal assay (SBA) and the opsonophagocytic assay (OPA).

Safety assessment of Gram-negative bacteria associated with traditional French cheeses.

Twenty Gram-negative bacterial (GNB) strains were selected based on the biodiversity previously observed in French traditional cheeses and their safety was assessed considering various safety criteria. For the majority of tested GNB strains, only gastric stress at pH 2 (vs pH 4) resulted in low survival and no regrowth after an additional simulated gastro-intestinal stress. Presence of milk was shown to be rarely protective. The majority of strains was resistant to human serum and had a low level of adherence to Caco-2 cells. When tested for virulence in Galleria mellonella larvae, GNB strains had LD 50 values similar to that of safe controls. However, four strains, Hafnia paralvei 920, Proteus sp. (close to P. hauseri) UCMA 3780, Providencia heimbachae GR4, and Morganella morganii 3A2A were highly toxic to the larvae, which suggests the presence of potential virulent factors in these strains. Noteworthy, to our knowledge, no foodborne intoxication or outbreak has been reported so far for any of the GNB belonging to the genera/species associated with the tested strains. The role of multiple dynamic interactions between cheese microbiota and GIT barriers could be key factors explaining safe consumption of the corresponding cheeses.

Effects of ascent to high altitude on human antimycobacterial immunity.

BACKGROUND: Tuberculosis infection, disease and mortality are all less common at high than low altitude and ascent to high altitude was historically recommended for treatment. The immunological and mycobacterial mechanisms underlying the association between altitude and tuberculosis are unclear. We studied the effects of altitude on mycobacteria and antimycobacterial immunity. METHODS: Antimycobacterial immunity was assayed in 15 healthy adults residing at low altitude before and after they ascended to 3400 meters; and in 47 long-term high-altitude residents. Antimycobacterial immunity was assessed as the extent to which participants' whole blood supported or restricted growth of genetically modified luminescent Bacille Calmette-Guérin (BCG) mycobacteria during 96 hours incubation. We developed a simplified whole blood assay that could be used by a technician in a low-technology setting. We used this to compare mycobacterial growth in participants' whole blood versus positive-control culture broth and versus negative-control plasma. RESULTS: Measurements of mycobacterial luminescence predicted the number of mycobacterial colonies cultured six weeks later. At low altitude, mycobacteria grew in blood at similar rates to positive-control culture broth whereas ascent to high altitude was associated with restriction (p ≤ 0.002) of mycobacterial growth to be 4-times less than in culture broth. At low altitude, mycobacteria grew in blood 25-times more than negative-control plasma whereas ascent to high altitude was associated with restriction (p ≤ 0.01) of mycobacterial growth to be only 6-times more than in plasma. There was no evidence of differences in antimycobacterial immunity at high altitude between people who had recently ascended to high altitude versus long-term high-altitude residents. CONCLUSIONS: An assay of luminescent mycobacterial growth in whole blood was adapted and found to be feasible in low-resource settings. This demonstrated that ascent to or residence at high altitude was associated with decreased mycobacterial growth in whole blood relative to controls, consistent with altitude-related augmentation of antimycobacterial cellular immunity.

Assessment of a microplate method for detection of staphylococcal secretory inhibitor of platelet microbicidal protein.

We developed a novel microplate spectrophotometric assay for the detection of staphylococcal secretory inhibitor of platelet microbicidal protein (SIPMP) in

[Assessment of bactericidal and growth-inhibiting activity of blood serum using flow cytometry and photometry].

Method of measurement of biological fluids bactericidal activity against Staphylococcus aureus using laser flow cytometry has been developed and proposed for clinical use. Overall bactericidal activity of sera of healthy donors has been assessed by this method. Strong positive correlation between bactericidal activity measured by flow cytometry and ability of the sera of healthy donors to inhibit bacterial growth assessed by photometric method was determined. High degree of positive correlation between results of cytometry and classical microbiological method of measurement of mentioned parameters has been shown.

Construction, expression and biological assessment of BPI23-Fcgamma1 recombinant protein prokaryotic expression vector.

OBJECTIVE: To construct pBV-BPI600-Fcgamma1(700) recombinant expression vector, to transform it into Escherichia coli DH5alpha, and to induce the expression of BPI23-Fcgamma1 anti-bacterial recombinant protein. METHODS: Genes coding for BPI23 and Fcgamma1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcgamma1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5alpha and BPI23-Fcgamma1 recombinant protein was expressed by a temperature-induced method. RESULTS: (1) Expected amplified products BPI600hp and Fcgamma1(700bp) were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcgamma1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. 3) pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcgamma1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcgamma1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization. CONCLUSION: pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and BPI23-Fcgamma1 recombinant protein with double biological activity of BPI and IgGFc was expresed in Escherichia coli.

Crossover assessment of serum bactericidal activity of grepafloxacin, ofloxacin and clarithromycin against respiratory pathogens after oral administration to healthy volunteers.

Serum bactericidal activity was studied in a crossover manner in 10 volunteers, after 2-day administration of grepafloxacin 600 mg qd, ofloxacin 400 mg bid and clarithromycin 500 mg bid. Bactericidal activity against clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Klebsiella pneumoniae, was estimated using a standardized microdilution method. Grepafloxacin was highly active against Gram-negative organisms and adequate against pneumococci (mean, 1:13.3). Clarithromycin was very active against both penicillin-susceptible and penicillin-partially-resistant S. pneumoniae (1:102.5) but had poor activity against H. influenzae (1:3.1). Minor adverse effects were commonly associated with grepafloxacin.

Effect of IFN-gamma on the killing of S. aureus in human whole blood. Assessment of bacterial viability by CFU determination and by a new method using alamarBlue.

Given the increasing incidence of methicillin resistant Staphylococcus aureus (MRSA) and the recent emergence of MRSA with a reduced susceptibility to vancomycin, alternative approaches to the treatment of infection are of increasing relevance. The purpose of these studies was to evaluate the effect of IFN-gamma on the ability of white blood cells to kill S. aureus and to develop a simpler, higher throughput bacterial killing assay. Using a methicillin sensitive clinical isolate of S. aureus, a clinical isolate of MRSA, and a commercially available strain of MRSA, studies were conducted using a killing assay in which the bacteria were added directly into whole blood. The viability of the bacteria in samples harvested at various time points was then evaluated both by the classic CFU assay and by a new assay using alamarBlue. In the latter method, serially diluted samples and a standard curve containing known concentrations of bacteria were placed on 96-well plates, and alamarBlue was added. Fluorescence readings were taken, and the viability of the bacteria in the samples was calculated using the standard curve. The results of these studies demonstrated that the CFU and alamarBlue methods yielded equivalent detection of bacteria diluted in buffer. For samples incubated in whole blood, however, the alamarBlue method tended to yield lower viabilities than the CFU method due to the emergence of a slower growing subpopulation of S. aureus upon incubation in the blood matrix. A significant increase in bacterial killing was observed upon pretreatment of whole blood for 24 h with 5 or 25 ng/ml IFN-gamma. This increase in killing was detected equivalently by the CFU and alamarBlue methods. In summary, these studies describe a method that allows for the higher throughput analysis of the effects of immunomodulators on bacterial killing.

Assessment of immune response to meningococcal disease: comparison of a whole-blood assay and the serum bactericidal assay.

A whole-blood assay (WBA), which assesses the complete bactericidal activity of blood, was compared with the serum bactericidal assay (SBA), which measures antibody and complement mediated cell lysis. Twenty children infected with serogroup B strains and 25 infected with serogroup C strains were studied 8-12 weeks after disease, and 29 healthy children were used as controls. The infecting strain (convalescent children only) and two reference strains, MC58 (B:15:P1.7, 16) and NCTC 8554 (C:NT:P1.5) were used. In children previously infected with a serogroup B strain, bactericidal activity was detected in 95% and 85% to their infecting strain by the WBA (>50% killing) and the SBA (s), respectively. Bactericidal activity to the reference serogroup B and C strain was detected by WBA in 70 and 75% of children, respectively, and the SBA in 45% and 20%. In contrast bactericidal activity was detected to both serogroup C strains in >80% of children previously infected with a serogroup C strain using either assay and in 48% (WBA) and 20% (SBA) to the reference serogroup B strain. Levels of bactericidal activity were detectable in fewer control children. Children convalescing from meningococcal disease develop an immune response to their infecting strain, detectable by both the WBA and SBA, which is independent of age. However, the WBA appears to be a more sensitive measure of bactericidal activity to heterologous strains than the SBA.

Clinical and immunological assessment of a candidate Lyme disease vaccine in healthy adults: antibody persistence and effect of a booster dose at month 12.

A candidate Lyme vaccine was administered to 20 adult volunteers following a 0, 1, 2 months vaccination schedule, with a booster at 12 months. An immune response, assessed as 'LA-2 equivalent' antibody titres using an inhibition ELISA, was induced in all vaccinees which persisted until the booster. Titres were increased 25-fold following the booster and persisted through month 24. There was a good correlation between 'LA-2 equivalent' antibody titres and a bactericidal assay (r2 = 0.86). Local symptoms were mild, resolving spontaneously within 72 h, with no reports of rash, arthralgia or other systemic symptoms. This Lyme vaccine was safe, well-tolerated and elicited an antibody response in all volunteers which persisted at least 12 months after the booster.

External quality assessment of the serum bactericidal test: results of a methodology/interpretation questionnaire.

Two hundred microbiology laboratories in the UK took part in two separate experimental external quality assessment distributions related to the serum bactericidal test (SBT). In the first, Staphylococcus aureus NCTC 6571 (vancomycin MIC 1 mg/L), was tested against a human serum containing vancomycin 38 mg/L plus gentamicin 0.5 mg/L. In the second, Streptococcus oralis PAJ 112/4183 (penicillin MBC < or = 0.03 mg/L) and Streptococcus sanguis PAJ 107/4184 (penicillin MBC = 128 mg/L) were tested against human serum containing penicillin 15 mg/L. Respondents returned their laboratory results and a questionnaire on clinical interpretation and technical aspects. Most laboratories (194/199, 97.5%) recommend the use of the SBT in the management of infective endocarditis but only 48 (25.2%) often or always change therapy on the basis of the result. A wide range of interpretative criteria, definitions of bactericidal endpoints and methodologies are used. Performance in the first distribution was acceptable for 75% of laboratories but in the second only 34% could identify penicillin tolerance; 34 respondents reported an SBT result of < or = 2 for the tolerant strain, 81 laboratories reported one of > or = 16. Technical factors related to acceptable performance were: sonication of broth before counting the inoculum; knowing the inoculum size in cfu/mL; use of a 4-8 h broth culture to make the inoculum; incubation of recovery plates for > 36 h; use of a calibrated pipette to sample for surviving bacteria; use of measured volumes to add the inoculum. Use of uncalibrated pipettes or standard loops to recover survivors was related to poor performance. Microbiology departments in the UK should review the clinical need to perform the SBT in the light of their local circumstances and if they elect to continue to offer this test, revise their methodologies which could be producing misleading results when testing alpha-haemolytic streptococci.

Comparison of three different methods in the assessment of neutrophil function.

Three different methods to measure the oxidative respiratory burst of neutrophils were performed. Of the three, the chemiluminescence technique was observed to be the most sensitive among them. The strong statistical correlation and an acceptable agreement between chemiluminescence with that of the killing assay provides evidence for using the chemiluminescence assay as an alternative method of detecting gross defects of neutrophil respiratory burst killing assays.

Optimal protocol for processing high-volume and Peds Plus blood cultures by the BACTEC NR860.

The optimal incubation period for blood cultures collected in high-volume (Plus 26) and anaerobic (NR7A) media or Peds Plus medium and processed by the BACTEC NR860 was evaluated during a 12-month period. Organisms were recovered from 9% (1528 of 16,870) of blood culture vials (14% [1302 of 9118] of sets). Ninety-eight percent of positive vials were detected by day 5. Thirty-three vials (Plus 26 or NR7A) became positive on day 6 or 7; all positive Peds Plus vials were detected by day 5. Therapy did not change in response to detection of the organisms (26 "occasional" pathogens, 9 "usual" pathogens) in these vials. One usual pathogen had potential clinical significance, but the patient from whom the culture was collected died before it became positive. In the author's institution, the 5-day protocol for processing blood cultures could be adopted without compromising patient care.

Third generation cephalosporins in the parenteral to oral switch.

In the present economic climate, it is increasingly necessary to ensure the cost-effectiveness of all aspects of healthcare. The expenditure on medications in a hospital is largely determined by the workload and throughput, but efforts to rationalise the use of medications will result in benefits both in patient care and overall costs. The purpose of this report is to discuss the advantages of switching from parenteral to oral cephalosporin therapy after the initial stage of infection treatment, the potential of presently available oral cephalosporins for use in a parenteral-to-oral switch regimen, and the outcome of a parenteral-to-oral switch programme, which used parenteral cefotaxime and oral cefixime, implemented at Hillingdon Hospital.

[An assessment of the bactericidal capacity of the blood monocytes in tuberculosis patients]. / Otsenka bakteritsidnoi sposobnosti monotsitov krovi bol'nykh tuberkulezom.

The nitro-blue tetrasolum (NBT) test was used for the study of the bactericidal capacity of blood monocytes in 30 healthy subjects and 86 pulmonary tuberculosis patients. The monocyte capacity of tuberculosis patients to restore NBT in the process of phagocytosis was found to be reduced. The NPT test parameters in the absence of cell stimulation were the same in both groups of the examined subjects. Administration of 50 TU of tuberculin subcutaneously is accompanied by the lowered capacity of monocytes to restore NPT during phagocytosis in patients with minimal activity of the tuberculous process, while this capacity was increased in subjects with post-tuberculous changes in the lungs.

Lomefloxacin: microbiologic assessment and unique properties.

In comparative studies, lomefloxacin, a new difluorinated quinolone, exhibits broad antibacterial activity in vitro, similar or superior to that of other quinolones (enoxacin, ofloxacin, pipemidic acid, nalidixic acid, and norfloxacin) but less than that of ciprofloxacin. Lomefloxacin inhibited Neisseria gonorrhoeae, Moraxella (Branhamella) catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and the majority of aerobic gram-negative rods, including nosocomial isolates, at concentrations readily achievable in biologic fluids and tissues. Lomefloxacin was less active against obligate anaerobes and streptococci. Organisms resistant to methicillin, penicillin, or the aminoglycosides were susceptible to lomefloxacin. No significant lomefloxacin resistance was identified in 18 countries in which in vitro studies were conducted, with the exception of a small number of strains tested in France. The frequency with which spontaneous single-step resistance to lomefloxacin develops in vitro is low.

An assessment of the factors contributing to the killing of type 3 Streptococcus pneumoniae by human polymorphonuclear leukocytes in vitro.

To characterize the factors that contribute to the killing of type 3 S. pneumoniae, human neutrophils were obtained from healthy donors and incubated with viable organisms. In contrast to prior observations with other pneumococcal serotypes, killing was not detected when 10(6) colony forming units (cfu) were incubated at 37 degrees C for 2-4 hours with 10(6) neutrophils in the presence of 20-80% fresh autologous serum; further, pneumococcidal activity was not found when preopsonized bacteria and primed neutrophils were employed in the standard assay. However, when the bacterium to cell ratio was reduced to 1:100 and 1:1000, microbicidal action was detected; a 10-fold reduction in the number of viable bacteria was observed when 2 x 10(3) cfu were incubated with 2 x 10(6) neutrophils and 80% autologous serum at 37 degrees C for 4 hours. To assess the effects of serum factors on killing, bactericidal assays were performed in the presence of normal human serum (NHS), heat-inactivated human serum (HIHS) and absorbed human serum (AHS); heating reduced and absorption eliminated the capacity of serum to support killing. Studies performed with mutanolysin, an enzyme that lyses type 3 pneumococci, demonstrated that the effects of HIHS and AHS on bactericidal activity were highly correlated with alterations in the ability of the sera to support phagocytosis. Studies of neutrophil activation revealed changes in the production of superoxide anion that correlated well with phagocytosis and killing; however, the results of assays of leukotriene B4 generation and degranulation (beta-glucuronidase and lactoferrin release) were more variable. In mixing experiments, the capacity of HIHS to support killing was normalized with NHS; however, the ability of AHS to promote killing was not restored with HIHS or NHS. Thus, these studies demonstrate the relatively limited capacity of human serum to support the killing of type 3 pneumococci, and they emphasize the importance of killing assays in assessing interactions between the bacterium and neutrophils.

[A comprehensive assessment of the phagocytic activity of the blood neutrophils in patients with rubromycosis treated with griseofulvin and nizoral]. / Kompleksnaia otsenka fagotsitarnoi aktivnosti neitrofilov krovi u bol'nykh rubrofitiei, lechennykh grizeoful'vinom i nizoralom.

The neutrophilic phagocytic activity (absorption and enzymic bactericidal) is intensified in rubromycosis patients treated with nizoral vs. those administered griseofulvin. it is advisable to combine griseofulvin therapy with biogenic stimulants (pyrogenal, aloe, fiBS, vitreous body).