A two-part phase 1 study to establish and compare the safety and local tolerability of two nasal formulations of XF-73 for decolonisation of Staphylococcus aureus: A previously investigated 0.5mg/g viscosified gel formulation versus a modified formulation.

OBJECTIVES: Successful decolonisation of nasal Staphylococcus aureus (SA) carriage by mupirocin is limited by increasing drug resistance. This randomised, open-label, phase 1 study compared the safety and local tolerability of two nasal formulations of XF-73, a novel porphyrinic antibacterial with rapid intrinsic activity against SA. METHODS: The study was performed in 60 healthy adults. In Part 1, eight non-SA carriers were randomised to groups of four subjects each and were treated with XF-73 concentrations of 0.5mg/g 2% gel or 2.0mg/g 2% gel. In Part 2, 52 persistent SA carriers were randomised to groups of 13 subjects each and were treated with XF-73 concentrations of 0.5mg/g 2% gel, 2.0mg/g 2% gel, 0.5mg/g 4% gel or 4% viscosified placebo gel. Plasma pharmacokinetic and pharmacodynamic studies were performed. Antistaphylococcal activity was assessed as the presence/absence of SA and by quantification of colonisation using a semiquantitative scale (SA score). RESULTS: 56 subjects (8/8 from Part 1 and 48/52 from Part 2) completed the study, with 47/60 comprising the pharmacokinetic population and 48/60 the pharmacodynamic population. There was no measurable systemic absorption of XF-73. XF-73 treatment was associated with rapid reduction in SA score in all subjects. The most common treatment-emergent adverse events (TEAEs) were rhinorrhoea and nasal dryness (15.5% each in Parts 1 and 2). TEAEs were mild and resolved spontaneously. CONCLUSION: XF-73 was well tolerated with minimal side effects at doses of 0.5mg/g 2% gel and 2.0mg/g 2% gel. These findings support further development of XF-73.

Development and validation of an LC-MS/MS method for the simultaneous determination of bedaquiline and rifabutin in human plasma.

This article describes the simultaneous determination of bedaquiline fumarate (TMC-207) and rifabutin in human plasma by stable isotope dilution tandem mass spectrometry. The methodology was developed for an investigation of potential drug-drug interactions of the two anti-tuberculosis drugs when given together to healthy human volunteers. Use of the two drugs in combination to treat disease caused by Mycobacterium tuberculosis is contemplated as the bacterium becomes resistant to many currently available drugs.

How to Identify Exposed Women Who Are Infected with Neisseria gonorrhoeae.

Treatment trials of antibiotics for Neisseria gonorrhoeae infections frequently enroll primarily men with urethritis, as the diagnosis of acute gonococcal infection in men with urethritis is easily made by Gram stain of the urethral exudate, followed by confirmatory culture or nucleic acid amplification tests (NAATs). Enrolling women in treatment trials is of great importance, but N. gonorrhoeae cervical infections cause nonspecific symptoms. This makes it difficult to conduct interventional trials, as large numbers of women with nonspecific symptoms need to be screened for infection. Gram stain of cervical secretions has a strikingly low sensitivity, and culture and/or NAAT results are not available at the time of screening. This necessitates recall and delayed treatment of infected women who may not return and who may spread the infection during the interval. In this chapter we present an algorithm, derived from a comparison of women who did, or did not, become infected during exposure, which identifies those women who are highly likely to be infected before culture and/or NAAT results are available. The algorithm provides an efficient way to conduct interventional trials in women without the problem of recall and delayed treatment.

Effects of Rifamycin Coadministration on Bedaquiline Desmethylation in Healthy Adult Volunteers.

There is an urgent need to identify safe and effective combination treatments for multidrug-resistant (MDR) Mycobacterium tuberculosis infection (TB). Bedaquiline, a new diarylquinoline, is approved for the treatment of MDR pulmonary TB in combination with other drugs, which could include rifabutin, which is also used to treat drug-resistant TB. Both rifabutin and bedaquiline are metabolized via cytochrome P450 3A4, and rifabutin is an inducer of this enzyme. Bedaquiline is metabolized into its primary N-monodesmethyl metabolite, M2, and further desmethylated into an N-didesmethyl metabolite, M3. Both metabolites are cytotoxic and induce phospholipidosis. The effect of rifabutin on the generation and disposition of the 2 metabolites was investigated in healthy adult volunteers coadministered bedaquiline and either rifabutin or rifampin. Subjects received single oral doses (400 mg) of bedaquiline on days 1 and 29. Oral rifabutin (300 mg) or rifampin (600 mg) were given daily on days 20-41. In the rifabutin group maximum M2 concentrations (Cmax ) increased significantly (P < .001) from 47.59 to 79.53 ng/mL, and clearance slowed slightly (P = .01). This resulted in significantly (P < .001) increased overall exposure (area under the concentration-time curve [AUC0-τ ]). Peak concentrations of M3 increased approximately 3-fold with little decline thereafter. In rifampin recipients M2 Cmax doubled (48.44 to 101.52 ng/mL), but M2 clearance and time to Cmax significantly (P < .001) increased, and AUC0-∞ and mean residence time significantly decreased (P < .001). Peak M3 concentrations increased 4-fold and rapidly declined. Although both rifamycins accelerate desmethylation of bedaquiline and M2, differences in clearance resulted in sustained elevations of both metabolites during rifabutin, but not rifampin, treatment.

Risk of Gonococcal Infection During Vaginal Exposure is Associated With High Vaginal pH and Active Menstruation.

BACKGROUND: An understanding of the biological reasons why 25% to 35% of women resist infection during vaginal intercourse with a man infected with Neisseria gonorrhoeae could lead to novel control measures. We sought modifiable biological bases for infection resistance by comparing women in the same core-mixing group who did or did not become infected after sexual exposure. METHODS: We enrolled 61 female contacts of index men with gonorrhea seen at Baltimore City Health Department clinics from January 2008 through May 2012. Exposure and sexual practices and histories, co-infections, physical signs on exam, patient symptom report, and menstrual history were collected. RESULTS: Thirty-eight (62.3%) of the exposed women developed cervical infections. Multiple logistic regression found that a vaginal pH of 4.5 or higher at presentation to clinic was significantly associated with gonococcal infection (adjusted odds ratio, 5.5; P = 0.037) in women who presented within one menstrual cycle, 35 days. In this group of women, there was a significant association between acquiring an N. gonorrhoeae cervical infection and sexual exposure during menstruation (adjusted odds ratio 12.5; P = 0.05). CONCLUSIONS: Modification of vaginal pH could be explored as novel strategy for reducing the risk of N. gonorrhoeae infections in women.

Mycobactericidal activity of bedaquiline plus rifabutin or rifampin in ex vivo whole blood cultures of healthy volunteers: A randomized controlled trial.

BACKGROUND: Bedaquiline, an antimycobacterial agent approved for drug-resistant tuberculosis, is metabolized by CYP3A4, an hepatic enzyme strongly induced by rifampin, an essential part of drug-sensitive tuberculosis treatment. We examined the pharmacokinetic interactions of bedaquiline plus either rifampin or rifabutin in 33 healthy volunteers. This sub-study of that trial examined the mycobactericidal activity of these drugs against intracellular Mycobacterium tuberculosis using ex vivo whole blood culture. METHODS: Subjects were randomly assigned to receive two single 400 mg doses of bedaquiline, alone, and, after a 4 week washout period, in combination with steady-state daily dosing of either rifabutin 300 mg or rifampin 600 mg. Blood samples were collected prior to dosing and at multiple time points subsequently, to measure plasma drug concentrations and bactericidal activity in ex vivo M tuberculosis-infected whole blood cultures (WBA). RESULTS: Single oral doses of bedaquiline produced readily detectable WBA ex vivo, reaching a maximal effect of -0.28 log/day, with negative values indicating bacterial killing. Plasma concentrations of 355 ng/ml were sufficient for intracellular mycobacteriostasis. Combined dosing with rifampin or rifabutin produced maximal effects of -0.91 and -0.79 log/d, respectively. However, the activity of the rifabutin combination was sustained throughout the dosing interval, thereby producing a greater cumulative or total effect. At low drug concentrations, rifabutin plus bedaquiline yielded greater mycobactericidal activity than the sum of their separate effects. Neither drug metabolites nor cellular drug accumulation could account for this observation. CONCLUSIONS: The combination of rifabutin plus bedaquiline produces sustained intracellular mycobactericidal activity that is greater than the sum of their individual effects. Further studies of the treatment-shortening potential of this combination are warranted.

A Phase 1 Pharmacokinetic and Safety Study of Extended-Duration, High-dose Cefixime for Cephalosporin-resistant Neisseria gonorrhoeae in the Pharynx.

BACKGROUND: There are no fully oral recommended treatment regimens for gonorrhea. Inadequately treated pharyngeal gonococcal infections are a likely reservoir for transmission and development of antimicrobial resistance. We sought to determine an oral cefixime dosing regimen that would theoretically treat pharyngeal infections by gonococci with minimum inhibitory concentrations 0.5 µg/mL. METHODS: We conducted an open-label, nonrandomized, phase I pharmacokinetic and safety study of cefixime in 25 healthy male and female volunteers divided into 4 dosing cohorts (cohort A, 400 mg; cohort B, 800 mg; cohort C, 1200 mg; and cohort D, 800 mg every 8 hours × 3 doses [total dose 2400 mg]) with a target serum concentration of at least 2.0 µg/mL for more than 20 hours. Cefixime concentrations from serum and pharyngeal fluid were determined with use of a validated liquid chromatography-tandem mass spectrometry assay. Safety measures included laboratories, physical examinations, and symptom diaries. RESULTS: None of the single-dose regimens attained the target concentration; however, 50% of subjects in cohort D attained the target concentration. Variation in absorption and protein binding contributed to differences in concentrations. Pharyngeal fluid concentrations were negligible. The single-dose regimens were well tolerated; the multidose regimen resulted in mild to moderate gastrointestinal symptoms in 43% of subjects. CONCLUSIONS: None of the dosing regimens achieved the target concentration. However, the proposed theoretical target was extrapolated from penicillin data; there are no empirically derived pharmacokinetic/pharmacodynamic criteria for pharyngeal gonorrhea. Under alternative cephalosporin-specific therapeutic goals, the multidose regimen may be effective, although the absence of cefixime in pharyngeal fluid is concerning. A clinical trial evaluating efficacy and defining pharmacokinetic/pharmacodynamic outcomes may be warranted.

Impact of Rifabutin or Rifampin on Bedaquiline Safety, Tolerability, and Pharmacokinetics Assessed in a Randomized Clinical Trial with Healthy Adult Volunteers.

Bedaquiline is a diarylquinoline that specifically inhibits mycobacterial ATP synthase. Bedaquiline has been used to effectively treat tuberculosis (TB) caused by drug-susceptible and drug-resistant Mycobacterium tuberculosis Rifamycins are a cornerstone of combination drug regimens for the treatment of TB. This phase 1, open-label, randomized, controlled trial evaluated the effect of steady-state dosing of rifabutin or rifampin on the safety, tolerability, and pharmacokinetics of bedaquiline given as a single dose. Thirty-three healthy subjects were enrolled to receive a 400-mg single oral dose of bedaquiline at two time points, on study days 1 and 29. Subjects were randomly assigned to once daily oral doses of rifabutin (300 mg/day, n = 17) or rifampin (600 mg/day, n = 16) during period 2 from days 20 to 41. Serial blood sampling for bedaquiline measurement occurred on days 1 and 29 through 336 h after bedaquiline administration. The day 29 bedaquiline pharmacokinetic parameter estimates were compared to the corresponding day 1 estimates for each rifamycin group. Steady-state rifampin reduced bedaquiline AUC0-336 approximately 45%, from 47.69 h·µg/ml in period 1 to 26.33 h·µg/ml in period 2. Bedaquiline apparent clearance accelerated 24% in rifampin-treated subjects from 6.59 liters/h in period 1 to 8.19 liters/h in period 2. Steady-state rifabutin resulted in little quantitative impact on bedaquiline exposure but was associated with grade 3 and 4 adverse events before and after the day 29 bedaquiline dose. Dosage adjustments may therefore be necessary to ensure that bedaquiline plasma concentrations reach therapeutic levels safely when combining bedaquiline and rifamycins in TB treatment regimens. (This single-site, randomized, open-label, prospective study in healthy adult volunteers was registered at Clinicaltrials.gov under registration no. NCT01341184.).

Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

A response adaptive randomization platform trial for efficient evaluation of Ebola virus treatments: A model for pandemic response.

The outbreak of Ebola virus disease in West Africa is the largest ever recorded. Numerous treatment alternatives for Ebola have been considered, including widely available repurposed drugs, but initiation of enrollment into clinical trials has been limited. The proposed trial is an adaptive platform design. Multiple agents and combinations will be investigated simultaneously. Additionally, new agents may enter the trial as they become available, and failing agents may be removed. In order to accommodate the many possible agents and combinations, a critical feature of this design is the use of response adaptive randomization to assign treatment regimens. As the trial progresses, the randomization ratio evolves to favor the arms that are performing better, making the design also suitable for all-cause pandemic preparedness planning. The study was approved by US and Sierra Leone ethics committees, and reviewed by the US Food and Drug Administration. Additionally, data management, drug supply lines, and local sites were prepared. However, in response to the declining epidemic seen in February 2015, the trial was not initiated. Sierra Leone remains ready to rapidly activate the protocol as an emergency response trial in the event of a resurgence of Ebola. (ClinicalTrials.gov Identifier: NCT02380625.) In summary, we have designed a single controlled trial capable of efficiently identifying highly effective or failing regimens among a rapidly evolving list of proposed therapeutic alternatives for Ebola virus disease and to treat the patients within the trial effectively based on accruing data. Provision of these regimens, if found safe and effective, would have a major impact on future epidemics by providing effective treatment options.

Lack of lipid A pyrophosphorylation and functional lptA reduces inflammation by Neisseria commensals.

The interaction of the immune system with Neisseria commensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenic Neisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded by lptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensal Neisseria species confirmed that lptA was absent in 15 of 17 strains examined but was present in N. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lacking lptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensal Neisseria strains that lacked lptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of two N. lactamica commensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcal lptA deletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensal Neisseria strains by reducing the inflammatory potential of LOS.

Urethral exudates of men with Neisseria gonorrhoeae infections select a restricted lipooligosaccharide phenotype during transmission.

Neisseria gonorrhoeae lipooligosaccharides (LOSs) induce immunoglobulin G that protects men from experimental infection. This raises the possibility that an LOS vaccine might prevent gonorrhea. Gonococci make different LOS molecules, depending on whether 3 genes, lgtA, lgtC, and lgtD, are in frame (IF) or out of frame (OOF). Mispairing of polymeric guanine (polyG) tracts within each gene determines its frame during replication. We amplified lgtA, lgtC, and lgtD from diagnostic slides of urethral exudates and sequenced their polyG tracts. We found that lgtA in exudative bacteria is IF and that lgtC is OOF. The frame of lgtD varied widely: it was OOF in most but not all cases. This genotype would result in synthesis of polylactosamine α chains that could be sialylated. Polylactosamine α chains would enhance virulence, and their sialylation would enable gonococci to survive within polymorphonuclear cells; however, an active LgtD in a few bacteria could provide a survival advantage in other sites of infection.

The association between body mass index and severe biliary infections: a multivariate analysis.

BACKGROUND: Obesity has been associated with worse infectious disease outcomes. It is a risk factor for cholesterol gallstones, but little is known about associations between body mass index (BMI) and biliary infections. We studied this using factors associated with biliary infections. METHODS: A total of 427 patients with gallstones were studied. Gallstones, bile, and blood (as applicable) were cultured. Illness severity was classified as follows: none (no infection or inflammation), systemic inflammatory response syndrome (fever, leukocytosis), severe (abscess, cholangitis, empyema), or multi-organ dysfunction syndrome (bacteremia, hypotension, organ failure). Associations between BMI and biliary bacteria, bacteremia, gallstone type, and illness severity were examined using bivariate and multivariate analysis. RESULTS: BMI inversely correlated with pigment stones, biliary bacteria, bacteremia, and increased illness severity on bivariate and multivariate analysis. CONCLUSIONS: Obesity correlated with less severe biliary infections. BMI inversely correlated with pigment stones and biliary bacteria; multivariate analysis showed an independent correlation between lower BMI and illness severity. Most patients with severe biliary infections had a normal BMI, suggesting that obesity may be protective in biliary infections. This study examined the correlation between BMI and biliary infection severity.

Human lipooligosaccharide IGG that prevents endemic meningococcal disease recognizes an internal lacto-N-neotetraose structure.

Antibodies that initiate complement-mediated killing of Neisseria meningitidis as they enter the bloodstream from the oropharynx protect against disseminated disease. Human IgGs that bind the neisserial L7 lipooligosaccharide (LOS) are bactericidal for L3,7 and L2,4 meningococci in the presence of human complement. These strains share a lacto-N-neotetraose (nLc4) LOS α chain. We used a set of mutants that have successive saccharide deletions from the nLc4 α chain to characterize further the binding and bactericidal activity of nLc4 LOS IgG. We found that the nLc4 α chain conforms at least four different antigens. We separately purified IgG that required the nLc4 (non-reducing) terminal galactose (Gal) for binding and IgG that bound the truncated nLc3 α chain that lacks this Gal residue. IgG that bound the internal nLc3 α chain killed both L3,7 and L2,4 strains, whereas IgG that required the nLc4 terminal Gal residue for binding killed L2,4 stains but not L3,7 strains. These results show that the diversity of LOS antibodies in human serum is as much a function of the conformation of multiple antigens by a single glycoform as of the production of multiple glycoforms. Differences in sensitivity to killing by human nLc4 LOS IgG may account for the fact that fully two-thirds of endemic group B meningococcal disease in infants and children is caused by L3,7 strains, but only 20% is caused by L2,4 stains.

Neisseria gonorrhoeae-induced transactivation of EGFR enhances gonococcal invasion.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, adheres to and invades into genital epithelial cells. Here, we investigate host components that are used by the bacteria for their entry into epithelial cells. We found that gonococcal microcolony formation on the surface of HEC-1-B cells disrupted the polarized, basolateral distribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member, and induced their accumulation under the microcolonies at the apical membrane. Gonococcal infection increased EGFR and ErbB2 phosphorylation. The EGFR kinase inhibitor, AG1478, reduced gonococcal invasion by 80%, but had no effect on adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the mRNA levels of several ligands of EGFR. Prevention of EGFR ligand shedding by blocking matrix metalloproteinase activation reduced gonococcal invasion without altering their adherence, while the addition of the EGFR ligand, HB-EGF, was able to restore invasion to 66% of control levels. These data indicate that N. gonorrhoeae modulates the activity and cellular distribution of host EGFR, facilitating their invasion. EGFR activation does not appear to be due to direct gonococcal binding to EGFR, but instead by its transactivation by gonococcal induced increases in EGFR ligands.

Structural characterization of an oligosaccharide made by Neisseria sicca.

Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization--time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-D-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its beta-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) beta-L-rhamnose (1-3) beta-N-acetyl-D-glucosamine (1-] with an N-acetyl-D-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.

Structural requirements for monoclonal antibody 2-1-L8 recognition of neisserial lipooligosaccharides.

Monoclonal antibodies (MAbs) that bind neisserial lipooligosaccharides (LOS) have been widely used in structural studies of these glycolipids. MAb 2-1-L8 binds LOS with a lactosyl a chain (Gal beta1-4 Glc beta1-4 [Glc-NAc alpha1-2 Hep2 alpha1-3] Hep1 alpha1-KDO) and at least one phosphoethanolamine (PEA) substitution of Hep2, but the requirement for PEA substitution and/or the exact position of this substitution, cyclic or exocyclic, remains unclear. In order to clarify the exact specificity of this MAb, we engineered an isogenic family of lpt mutants that each make LOS with a lactosyl a chain, but that lacked cyclic (-3Hep2), exocyclic (-6Hep2), or any PEA residues. Mass spectrometry showed that mutants that lack either Lpt3 or Lpt6 make small amounts of LOS with two PEA substitutions. Thus, each enzyme is able to phosphoethanolaminylate the alternate site, albeit with low efficiency. LOS made by the mutant that lacked both Lpt3 and Lpt6 was devoid of PEA. LOS made by the deltalpt3 mutant did not bind MAb 2-1-L8 on Western blot analysis, whereas delta pt6 LOS did. Analysis of intact mutants by fluorescence-activated cell sorting confirmed that PEA substitution at 3Hep2, but not at 6Hep2, is needed for optimal binding of MAb 2-1-L8. These data confirm that the MAb 2-1-L8 epitope requires a -3Hep2 cyclic PEA substitution for optimal conformation and that this MAb specifies the PEA-3Hep2 lactosyl LOS structure.

Bacteria entombed in the center of cholesterol gallstones induce fewer infectious manifestations than bacteria in the matrix of pigment stones.

PURPOSE: The clinical significance of bacteria in the pigment centers of cholesterol stones is unknown. We compared the infectious manifestations and characteristics of bacteria from pigment stones and predominantly cholesterol stones. METHODS: Three hundred forty patients were studied. Bile was cultured. Gallstones were cultured and examined with scanning electron microscopy. Level of bacterial immunoglobulin G (bile, serum), complement killing, and tumor necrosis factor-alpha production were determined. RESULTS: Twenty-three percent of cholesterol stones and 68% of pigment stones contained bacteria (P < 0.0001). Stone culture correlated with scanning electron microscopy results. Pigment stone bacteria were more often present in bile and blood. Cholesterol stone bacteria caused more severe infections (19%) than sterile stones (0%), but less than pigment stone bacteria (57%) (P < 0.0001). Serum and bile from patients with cholesterol stone bacteria had less bacterial-specific immunoglobulin G. Cholesterol stone bacteria produced more slime. Pigment stone bacteria were more often killed by a patient's serum. Tumor necrosis factor-alpha production of the groups was similar. CONCLUSIONS: Bacteria are readily cultured from cholesterol stones with pigment centers, allowing for analysis of their virulence factors. Bacteria sequestered in cholesterol stones cause infectious manifestations, but less than bacteria in pigment stones. Possibly because of their isolation, cholesterol stone bacteria were less often present in bile and blood, induced less immunoglobulin G, were less often killed by a patient's serum, and demonstrated fewer infectious manifestations than pigment stone bacteria. This is the first study to analyze the clinical relevance of bacteria within cholesterol gallstones.

Gallstones containing bacteria are biofilms: bacterial slime production and ability to form pigment solids determines infection severity and bacteremia.

OBJECTIVE: Gallstone bacteria provide a reservoir for biliary infections. Slime production facilitates adherence, whereas beta-glucuronidase and phospholipase generate colonization surface. These factors facilitate gallstone formation, but their influence on infection severity is unknown. METHODS: Two hundred ninety-two patients were studied. Gallstones, bile, and blood (as applicable) were cultured. Bacteria were tested for beta-glucuronidase/phospholipase production and quantitative slime production. Infection severity was correlated with bacterial factors. RESULTS: Bacteria were present in 43% of cases, 13% with bacteremia. Severe infections correlated directly with beta-glucuronidase/phospholipase (55% with vs 13% without, P < 0.0001), but inversely with slime production (55 vs 8%, slime <75 or >75, P = 0.008). Low slime production and beta-glucuronidase/phospholipase production were additive: Severe infections were present in 76% with both, but 10% with either or none (P < 0.0001). beta-Glucuronidase/phospholipase production facilitated bactibilia (86% with vs 62% without, P = 0.03). Slime production was 19 (+/-8) vs 50 (+/-10) for bacteria that did or did not cause bacteremia (P = 0.004). No bacteria with slime >75 demonstrated bacteremia. CONCLUSIONS: Bacteria-laden gallstones are biofilms whose characteristics influence illness severity. Factors creating colonization surface (beta-glucuronidase/phospholipase) facilitated bacteremia and severe infections; but abundant slime production, while facilitating colonization, inhibited detachment and cholangiovenous reflux. This shows how properties of the gallstone biofilm determine the severity of the associated illness.

Biochemical analysis of Lpt3, a protein responsible for phosphoethanolamine addition to lipooligosaccharide of pathogenic Neisseria.

The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N. gonorrhoeae FA1090 and identified a homolog of lpt3 in N. gonorrhoeae. A PCR amplicon containing lpt3 was amplified from F62DeltaLgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62DeltaLgtA, producing strain F62DeltaLgtAlpt3::Tn5. LOS isolated from this strain lost the ability to bind monoclonal antibody (MAb) 2-1-L8. Complementation of this mutation by genetic removal of the transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry analysis of LOS isolated from the F62DeltaLgtA indicated that this strain contained two PEA modifications on its LOS. F62DeltaLgtAlpt3::Tn5 lacked a PEA modification on its LOS, a finding consistent with the hypothesis that lpt3 encodes a protein mediating PEA addition onto gonococcal LOS. The DNA encoding lpt3 was cloned into an expression vector and Lpt3 was purified. Purified Lpt3 was able to mediate the addition of PEA to LOS isolated from F62DeltaLgtAlpt3::Tn5.