High Bacterial Inoculum Research Articles

Inoculum effect of cefiderocol against NDM-1 producing Escherichia coli in vitro and in a murine model of peritonitis.

Cefiderocol is a siderophore cephalosporin active in vitro against carbapenemase-producing Enterobacterales, including New Delhi metallo-β-lactamases (NDM-1). A significant impact of the size of bacterial inoculum on its efficacy has been described in vitro, the clinical impact of which is unclear. Here, we analyse the inoculum effect of cefiderocol against E. coli-NDM-1 in vitro and in a murine peritonitis model. Escherichia coli 62-pTOPO and its isogenic variant expressing NDM-1, 62-pTOPO-NDM, were constructed from a clinical strain. MICs and bactericidal kinetics were determined at standard (105 cfu/mL) and high inoculum (107 cfu/mL). The in vivo effect was assessed in a severe murine peritonitis model, comparing low (106 cfu/mL) and high (108 cfu/mL) inoculum. Survival rates, organ sterilization and bacterial counts in spleen and peritoneal fluid were compared. Cefiderocol MICs for 62-pTOPO and 62-pTOPO-NDM at standard and high inoculum were 0.008, 2, 2 and 1024 mg/L, respectively. Bactericidal activity was not achieved in vitro for 62-pTOPO-NDM at high inoculum with high cefiderocol concentrations (16 mg/L). In vivo, for 62-pTOPO-NDM, no difference was found in survival, organ sterilization or bacterial counts between low and high inoculum. For 62-pTOPO, no difference was observed in survival, despite less organ sterilization and higher bacterial counts in organs with the high inoculum. A significant inoculum effect of cefiderocol was observed in vitro for 62-pTOPO and 62-pTOPO-NDM. However, the effectiveness of cefiderocol was not reduced in vivo with a high bacterial inoculum. In vitro inoculum effect of cefiderocol may not be clinically significant.

Inoculum effect of CTX-M-15, OXA-48, and KPC-2 producing Klebsiella pneumoniae on meropenem and ceftazidime-avibactam efficacy.

The inoculum effect, characterized by diminished antibacterial activity at high bacterial inocula, is studied in the context of beta-lactam and beta-lactamase inhibitor combinations against beta-lactamase-producing Enterobacterales. The inhibition of ESBL + OXA-48 and KPC enzymes, in combination with ceftazidime, demonstrates encouraging results. In this study, 20 Klebsiella pneumoniae isolates were tested with different inocula (1-5 × 105 and 1-5 × 107cfu/ml) using broth microdilution methods. The inoculum effect was observed in meropenem against OXA-48 + CTX-M-15- and KPC-2-producing isolates but not with ceftazidime/avibactam. Notably, meropenem exhibited inoculum effect against carbapenemase-producing strains, whereas ceftazidime-avibactam remained effective. We conclude that ceftazidime-avibactam is recommended for high-inoculum infections.

Meropenem MICs at Standard and High Inocula and Mutant Prevention Concentration Inter-Relations: Comparative Study with Non-Carbapenemase-Producing and OXA-48-, KPC- and NDM-Producing Klebsiella pneumoniae.

The minimal inhibitory concentration (MIC) is conventionally used to define in vitro levels of susceptibility or resistance of a specific bacterial strain to an antibiotic and to predict its clinical efficacy. Along with MIC, other measures of bacteria resistance exist: the MIC determined at high bacterial inocula (MICHI) that allow the estimation of the occurrence of inoculum effect (IE) and the mutant prevention concentration, MPC. Together, MIC, MICHI and MPC represent the bacterial "resistance profile". In this paper, we provide a comprehensive analysis of such profiles of K. pneumoniae strains that differ by meropenem susceptibility, ability to produce carbapenemases and specific carbapenemase types. In addition, we have analyzed inter-relations between the MIC, MICHI and MPC for each tested K. pneumoniae strain. Low IE probability was detected with carbapenemase-non-producing K. pneumoniae, and high IE probability was detected with those that were carbapenemase-producing. MICs did not correlate with the MPCs; significant correlation was observed between the MICHIs and the MPCs, indicating that these bacteria/antibiotic characteristics display similar resistance properties of a given bacterial strain. To determine the possible resistance-related risk due to a given K. pneumoniae strain, we propose determining the MICHI. This can more or less predict the MPC value of the particular strain.

199. A novel and scalable antimicrobial surface modification technology to prevent and mitigate implant related surgical site infections: an in-vivo safety and efficacy study

<h3>BACKGROUND CONTEXT</h3> Despite significant technological advancements in material science, a high level of hand-hygiene and systematic use of antiseptics and antibiotics, surgical site infections (SSI) are still to date the number one complication seen after surgery in the United States. Methicillin-resistant Staphylococcus aureus (MRSA) is the largest purveyor of health care-acquired infections (HCAI) and is responsible for significant morbidity and mortality. <h3>PURPOSE</h3> This study aimed to demonstrate the in-vivo safety and antibacterial efficacy of non-eluting titanium implants covalently bound with a novel broad-spectrum biocidal quaternary ammonium compound (DBG21) against MRSA biofilm. <h3>STUDY DESIGN/SETTING</h3> Subcutaneous MRSA infection mouse model comparing the antimicrobial efficacy of untreatedversusDBG21-treated titanium alloy discs. A separate comparative safety study was performed in the absence of infection. <h3>PATIENT SAMPLE</h3> Sixty-five 11-week-old balb/c mice. <h3>OUTCOME MEASURES</h3> Efficacy study: Number of adherent bacteria on implants (CFU) and in the surrounding tissue (CFU/g) at days 7 and 14. Safety study: systemic and subacute toxicity (clinical tolerance, blood tests and histology). <h3>METHODS</h3> Efficacy Study: TiAl6V4 discs, 6mm ø, 0.5 mm thick, were activated, dip-coated in a polymer solution, and baked to produce covalently bound DBG21-treated discs. Untreated TiAl6V4 discs were used as controls. All discs underwent subsequent sterilization by 25kGy gamma-irradiation, were implanted either untreated for control mice or DBG21-treated for treated mice, and were challenged with a high-bacterial inoculum of MRSA (ATCC43300) in a well-established subcutaneous infection mouse model (11-week-old balb/c) in the absence of antibiotics. A veterinary ethics committee validated the protocol. Sixty-five mice (33 controls, 32 treated) were included in an antibacterial efficacy study. After implantation of one disc in a posterior subcutaneous pocket and closure of the skin for each mouse, 7 log10CFU (colony forming units) of MRSA were injected through a catheter into the operating site. Mice were sacrificed after either 7 days (18 treated, 18 controls) or 14 days (15 controls, 14 treated) to determine the number of adherent bacteria (biofilm) on implants and in the peri-implant surrounding tissues. Results were expressed respectively as median log10CFU reduction on implants and median log10CFU/g reduction in tissue compared with untreated control mice. The statistical significance (p) was determined using Mann-Whitney tests. Safety Study: Ten mice implanted with either control (5 mice) or DBG-21 treated (5 mice) discs were assessed in a safety study in the absence of infection (ISO 10993-11:2017 for systemic and subacute toxicity including clinical tolerance, blood tests and local histology). <h3>RESULTS</h3> Efficacy Study: At both 7 and 14 days, DBG21-treated implants yielded a significant decrease in MRSA biofilm (respectively 3.6 median log10CFU (99.97%) reduction (p < 0.001) and 1.9 median log10CFU (98.7%) reduction (p = 0.037)) and peri-implant surrounding tissues (respectively 2.7 median log10CFU/g (99.8%) reduction (p < 0.001) and 5.6 median log10CFU/g (99.9997%) reduction (p < 0.001)). At day 7, three treated mice (16.6%) were completely sterilized on both tissue and implant. At day 14, 10 treated mice (71.4%) had sterile peri-implant tissue. Safety Study: There were no significant differences between control and treated mice in terms of clinical scores, blood tests and local histology after up to 11 days from implantation. <h3>CONCLUSIONS</h3> Preventing biofilm formation has been recognized as a key element of SSI prevention. Yet, no biofilm-resistant titanium implants are available in clinical practice to prevent and mitigate the burden of SSIs. This small animal safety and efficacy trial suggests that DBG-21 is a promising candidate for antimicrobial surface modification of titanium implants. <h3>FDA DEVICE/DRUG STATUS</h3> DBG21 antimicrobial surface modification (Not approved for this indication).

Ethanol Intoxication Impairs Respiratory Function and Bacterial Clearance and Is Associated With Neutrophil Accumulation in the Lung After Streptococcus pneumoniae Infection.

Alcohol consumption is commonplace in the United States and its prevalence has increased in recent years. Excessive alcohol use is linked to an increased risk of infections including pneumococcal pneumonia, mostly commonly caused by Streptococcus pneumoniae. In addition, pneumonia patients with prior alcohol use often require more intensive treatment and longer hospital stays due to complications of infection. The initial respiratory tract immune response to S. pneumoniae includes the production of pro-inflammatory cytokines and chemokines by resident cells in the upper and lower airways which activate and recruit leukocytes to the site of infection. However, this inflammation must be tightly regulated to avoid accumulation of toxic by-products and subsequent tissue damage. A majority of previous work on alcohol and pneumonia involve animal models utilizing high concentrations of ethanol or chronic exposure and offer conflicting results about how ethanol alters immunity to pathogens. Further, animal models often employ a high bacterial inoculum which may overwhelm the immune system and obscure results, limiting their applicability to the course of human infection. Here, we sought to determine how a more moderate ethanol exposure paradigm affects respiratory function and innate immunity in mice after intranasal infection with 104 colony forming units of S. pneumoniae. Ethanol-exposed mice displayed respiratory dysfunction and impaired bacterial clearance after infection compared to their vehicle-exposed counterparts. This altered response was associated with increased gene expression of neutrophil chemokines Cxcl1 and Cxcl2 in whole lung homogenates, elevated concentrations of circulating granulocyte-colony stimulating factor (G-CSF), and higher neutrophil numbers in the lung 24 hours after infection. Taken together, these findings suggest that even a more moderate ethanol consumption pattern can dramatically modulate the innate immune response to S. pneumoniae after only 3 days of ethanol exposure and provide insight into possible mechanisms related to the compromised respiratory immunity seen in alcohol consumers with pneumonia.

In Vitro Activities of Ceftazidime-Avibactam and Aztreonam-Avibactam at Different Inoculum Sizes of Extended-Spectrum β-Lactam-Resistant Enterobacterales Blood Isolates.

β-lactam–avibactam combinations have been proposed as carbapenem-sparing therapies, but little data exist on their in vitro activities in infections with high bacterial inocula. We investigated the in vitro efficacies and the inoculum effects of ceftazidime–avibactam and aztreonam–avibactam against extended-spectrum β-lactam-resistant Enterobacterales blood isolates. A total of 228 non-repetitive extended-spectrum β-lactam-resistant Escherichia coli and Klebsiella pneumoniae blood isolates were prospectively collected in a tertiary center. In vitro susceptibilities to ceftazidime, aztreonam, meropenem, ceftazidime–avibactam, and aztreonam–avibactam were evaluated by broth microdilution method using standard and high inocula. An inoculum effect was defined as an eightfold or greater increase in MIC when tested with the high inoculum. Of the 228 isolates, 99% were susceptible to ceftazidime–avibactam and 99% had low aztreonam–avibactam MICs (≤8 mg/L). Ceftazidime–avibactam and aztreonam–avibactam exhibited good in vitro activities; MIC50/MIC90 values were 0.5/2 mg/L, 0.125/0.5 mg/L, and ≤0.03/0.25 mg/L, respectively, and aztreonam–avibactam was more active than ceftazidime–avibactam. The frequencies of the inoculum effect with ceftazidime–avibactam and aztreonam–avibactam were lower than with meropenem (14% vs. 38%, p < 0.001 and 30% vs. 38%, p = 0.03, respectively). The β-lactam-avibactam combinations could be useful as carbapenem-sparing strategies, and aztreonam–avibactam has the better in vitro activity but is more subject to the inoculum effect than ceftazidime–avibactam.

Expression of CTX-M-15 limits the efficacy of ceftolozane/tazobactam against Escherichia coli in a high-inoculum murine peritonitis model

ObjectivesAmong carbapenem-sparing therapies, ceftolozane/tazobactam has been proposed for the treatment of infections due to CTX-M-15-producing Escherichia coli. However, few data exist on its in vivo activity in infections associated with a high bacterial inoculum. MethodsWe analysed ceftolozane/tazobactam activity against susceptible E. coli CFT073-RR and its CTX-M-15-producing transconjugant E. coli CFT073-RR Tc blaCTX-M-15, in vitro at low and high inocula, and in a high-inoculum murine model of peritonitis. ResultsAgainst E. coli CFT073-RR Tc blaCTX-M-15, ceftolozane/tazobactam bactericidal effect was impaired in vitro with only a minor inoculum effect; this translated into reduced activity compared with imipenem in the mouse peritonitis model. ConclusionsCombination of extended spectrum β-lactamase expression and high inoculum size may be a clinical situation at risk of reduced bactericidal activity of ceftolozane/tazobactam.

Auranofin, at clinically achievable dose, protects mice and prevents recurrence from Clostridioides difficile infection

Clostridioides difficile is the leading cause of nosocomial infections and a worldwide urgent public health threat. Without doubt, there is an urgent need for new effective anticlostridial agents due to the increasing incidence and severity of C. difficile infection (CDI). The aim of the present study is to investigate the in vivo efficacy of auranofin (rheumatoid arthritis FDA-approved drug) in a CDI mouse model and establish an adequate dosage for treatment. The effects of increased C. difficile inoculum, and pre-exposure to simulated gastric intestinal fluid (SGF) and simulated intestinal fluid (SIF), on the antibacterial activity of auranofin were investigated. Auranofin’s in vitro antibacterial activity was stable in the presence of high bacterial inoculum size compared to vancomycin and fidaxomicin. Moreover, it maintained its anti-C. difficile activity after being exposed to SGF and SIF. Upon testing in a CDI mouse model, auranofin at low clinically achievable doses (0.125 mg/kg and 0.25 mg/kg) significantly protected mice against CDI with 100% and 80% survival, respectively. Most importantly, auranofin (0.125 mg/kg and 0.25 mg/kg) significantly prevented CDI recurrence when compared with vancomycin. Collectively, these results indicate that auranofin could potentially provide an effective, safe and quick supplement to the current approaches for treating CDI.

Effective Inhibition of Ammonium Released from Heavily Contaminated Sediments through Selective Oxidation with Zeolite Layer

In order to study the remediation effect of heavily contaminated sediments, the experiments to repair heavily contaminated sediments were carried out under selective and nonselective oxidation conditions. Results showed that a lot of denitrifying bacteria was detected on the surface of modified zeolite by fluorescence in situ hybridization (FISH) after inoculating both nitrifying and denitrifying bacteria for 7 days. Up to 3.69 mg/g, in-situ regeneration of ammonium on zeolite loading with high ammonium (ammonium adsorption: 4.20 m/g) was obtained, which was 1.42 times that of nonselective oxidation (2.60 mg/g). This indicated that the in-situ regeneration rate of zeolite under high ammonium adsorption (5.0 mg/g) and high bacterial inoculum (80 mL/g) was enhanced. Moreover, only 1.50 mg/L total nitrogen with 84% inhibition in the overlying water under selective oxidation conditions was observed, which was 2.37 times the inhibition percentage of modified zeolite under nonselective oxidation conditions. The results illuminated that effective inhibition of ammonium released from heavily contaminated sediments can be achieved through selective oxidation with zeolite layer. At the same time, the in-situ service life of attached biofilm-modified zeolite under selective oxidation conditions was 5.87 years, which was extended by 3 years compared with nonselective oxidation conditions.

2063. Extracellular Release of β-Lactamase Is Responsible for the Cefazolin Inoculum Effect (CzIE) in Methicillin-Susceptible Staphylococcus aureus

BackgroundCefazolin is becoming first-line therapy for MSSA infections since it appears to be better tolerated than isoxazolyl penicillins with similar outcomes. An important concern when using cephalosporins as first-line therapy for MSSA is the CzIE, defined as MICs ≥ 16 µg/mL when performed at high bacterial inoculum (~107 CFU/mL) compared with standard inoculum (~105 CFU/mL). We postulated that release of BlaZ (a lipoprotein) to the extracellular milieu is the mechanism responsible for the CzIE. Confirmation of this phenomenon would permit developing a rapid test to identify this phenomenon in clinical settings.MethodsWe monitored the hydrolysis of 50 μM of nitrocefin by S. aureus supernatants after induction with ampicillin (150 µg/mL) for 1 h. A total of 150 μL of supernatants (after centrifugation) was incubated with 50 μM nitrocefin at 25°C in 20 mM HEPES, pH 7.4, 100 mM NaCl for 30 minutes. Nitrocefin hydrolysis was monitored by following the change in absorbance at 482 resulting from opening of the β-lactam ring of nitrocefin. Visual inspection to monitor color changes was also performed. We initially used 3 strains of MSSA, (i) S. aureus TX0117, a well-characterized strain that exhibits the CzIE; (ii) TX0117c, a derivative of TX0117 that harbors a mutation inactivating BlaZ and abolishing the CzIE, and (iii) ATCC 29213 a BlaZ-positive strain that lacks the CzIE. Subsequently, we validated the methodology in 10 South American isolates of different backgrounds that had been previously characterized for the CZIE.ResultsA statistically significant difference in ODs after 30 minutes was observed in TX0117 (CzIE) vs. TX0117c (no CzIE) and ATCC 29213 (no CzIE) (all P < 0.001), suggesting high BlaZ activity in supernatants of TX0117 and supporting the release of the enzyme as the main mechanism of the CzIE. All South American isolates that exhibited the CzIE were identified by the nitrocefin assay. Of note, isolates producing Type C BlaZ gave a weaker reaction, although still significantly different from isolates without the CzIE. Hydrolysis of nitrocefin was also readily detectable by visual inspection.ConclusionThe CzIE is likely due to release of BlaZ to the extracellular milieu. A rapid test that can readily identify MSSA strains exhibiting the CzIE is feasible.Disclosures W. Miller, Merck: Investigator, Research support. C. Arias, Merck & Co., Inc.: Grant Investigator, Research support; MeMed: Grant Investigator, Research support; Allergan: Grant Investigator, Research support.

New dosing strategies for an old antibiotic: pharmacodynamics of front-loaded regimens of colistin at simulated pharmacokinetics in patients with kidney or liver disease.

Increasing evidence suggests that colistin monotherapy is suboptimal at currently recommended doses. We hypothesized that front-loading provides an improved dosing strategy for polymyxin antibiotics to maximize killing and minimize total exposure. Here, we utilized an in vitro pharmacodynamic model to examine the impact of front-loaded colistin regimens against a high bacterial density (10(8) CFU/ml) of Pseudomonas aeruginosa. The pharmacokinetics were simulated for patients with hepatic (half-life [t1/2] of 3.2 h) or renal (t1/2 of 14.8 h) disease. Front-loaded regimens (n=5) demonstrated improvement in bacterial killing, with reduced overall free drug areas under the concentration-time curve (fAUC) compared to those with traditional dosing regimens (n=14) with various dosing frequencies (every 12 h [q12h] and q24h). In the renal failure simulations, front-loaded regimens at lower exposures (fAUC of 143 mg · h/liter) obtained killing activity similar to that of traditional regimens (fAUC of 268 mg · h/liter), with an ∼97% reduction in the area under the viable count curve over 48 h. In hepatic failure simulations, front-loaded regimens yielded rapid initial killing by up to 7 log10 within 2 h, but considerable regrowth occurred for both front-loaded and traditional regimens. No regimen eradicated the high bacterial inoculum of P. aeruginosa. The current study, which utilizes an in vitro pharmacodynamic infection model, demonstrates the potential benefits of front-loading strategies for polymyxins simulating differential pharmacokinetics in patients with hepatic and renal failure at a range of doses. Our findings may have important clinical implications, as front-loading polymyxins as a part of a combination regimen may be a viable strategy for aggressive treatment of high-bacterial-burden infections.

Mecillinam/clavulanate combination: a possible option for the treatment of community-acquired uncomplicated urinary tract infections caused by extended-spectrum -lactamase-producing Escherichia coli

Extended-spectrum β-lactamases (ESBLs) have emerged as an important mechanism of β-lactam resistance among community uropathogens. We characterized the ESBLs of a collection of Escherichia coli isolates recovered from outpatients with urinary tract infection during nationwide surveillance conducted from 2005 to 2006 in Greece, and evaluated the in vitro activity of mecillinam and mecillinam/clavulanate against them. ESBLs were characterized with PCR and sequencing. In vitro interactions were evaluated with agar dilution with and without clavulanate (4 mg/L) using an inoculum of 10(4) or 10(6) cfu/spot as well as with time-kill methodology. Among 48 ESBL producers, 47 (97.9%) were susceptible to mecillinam. CTX-M-type enzymes were produced by 87.2%, with CTX-M-3 being the most prevalent. SHV enzymes were found in 10.6%, VEB enzymes in 2.1%, TEM enzymes in 19.2% and OXA-type enzymes in 12.8%. Synergy with clavulanate was detected in 60.4% using the agar dilution method and in 43.8% using the time-kill methodology. An inoculum effect was detected in 64.6% of isolates, but this phenomenon was inverted and synergy was evidenced for 85.4% with clavulanate. When a high inoculum was used, 60.4% (29/48) were resistant to mecillinam, but 97.9% (47/48) were susceptible in the presence of clavulanate. CTX-M-type enzymes were the most prevalent among ESBL-producing E. coli uropathogens in Greece. Mecillinam may be useful in uncomplicated cystitis caused by ESBL producers with low MICs. The addition of the inhibitor could improve and extend the activity of mecillinam, even in the setting of infection with a high bacterial inoculum, and merits clinical evaluation.

In vitro efficiency of the piperacillin/tazobactam combination against inhibitor-resistant TEM- and complex mutant TEM-producing clinical strains of Escherichia coli

We investigated the bacteriostatic and bactericidal activities of piperacillin/tazobactam against 16 clinical Escherichia coli producing inhibitor-resistant TEM β-lactamases (IRT; 13/16) and complex mutant TEM enzymes (CMT; 3/16). Bacteriostatic activity was evaluated by three methods (disc diffusion, Vitek2 automated system, MIC determination by a microdilution method) and a time-killing study was used to investigate the bactericidal effect against standard (5 × 10(5) cfu/mL) and high inocula (5 × 10(6) cfu/mL). Piperacillin/tazobactam was bacteriostatic against most of the tested strains (15/16). Using a high inoculum, the piperacillin/tazobactam combination was not bactericidal against the 13 IRT-producing strains and one of the CMT-producing strains (1/3). A loss of bactericidal activity was still observed for seven IRT-producing strains (7/13) with a standard bacterial inoculum (<99.9% killing over 24 h). Despite usual in vitro bacteriostatic activity, the piperacillin/tazobactam combination was not bactericidal against most IRT-producing clinical strains of E. coli, especially for the treatment of a high bacterial inoculum. This possible loss of bactericidal effect should be brought to the attention of physicians and may require high dosing regimens for the treatment of severe infections.

Stérilisations retardées du liquide céphalorachidien au cours des méningites à pneumocoque de l'enfant

Delayed cerebrospinal fluid sterilization is defined by a positive second lumbar puncture, recommanded according to the guidelines from the French Consensus Conference of 1996 between the 36th and 48th hours after the beginning of antibiotics prescribed for pneumococcal meningitis. The aim of this study was to analyze specifically delayed cerebrospinal fluid sterilization, identified during the first 5 years of the French observatory of children bacterial meningitis. The Groupe de Pathologie Infectieuse Pédiatrique (GPIP) and Association Clinique et Thérapeutique Infantile du Val de Marne (ACTIV) has set up since the first of January 2001 a descriptive national multicenter network, to determine incidence, main characteristics, and prognosis of bacterial meningitis in children. A questionnaire, available in all paediatric unit taking care of bacterial meningitis, was completed by a referral doctor. It contained reasons for inclusion in the study, anamnesis, clinical examination, treatment, pneumococcal characteristics, and short term prognosis. Delayed cerebrospinal fluid sterilization were identified, and the analysis of their medical records completed the questionnaire. From 1st January 2001 to 31 December 2005, 616 pneumococcal meningitis were identified. Among them, 442 had a second lumbar puncture, and 8 had delayed cerebrospinal fluid sterilization. The analyis of their medical records were reviewed to describe their characteristics. Two had an osteomeningeal breach, one a ventriculoperitoneal valve. All received previously an antibiotic, and were treated with a curative antibiotic by cephalosporins. Vancomycin was given in 6 cases. The antibiotic was inadapted to the French guidelines for 1 patient. There are 4 vaccine type pneumococci and only 1 strain was resistant to penicillin, and intermediate to cephalosporins. The controlled lumbar puncture was made between 36.5 and 179.4hours after beginning antibiotics. One patient has received a double dose of steroids. Three were in a coma, had convulsions, and were ventilated, none died. One patient has a sequellar paired deafness, two a severe disability, four a normal psychomotor development. The delay of sterilization is a rare situation and represented only 1.8 % of pneumococcal meningitis during the first five years of the observatory. These results suggest that a second lumbar puncture to assess sterilization could be proposed only in cases of unfavourable clinical course, MIC greater than or equal 0.5mg/l to 3GC, risk factors for delayed cerebrospinal fluid sterilization and high bacterial inoculum.

Contribution of Toll-like receptors to the innate immune response to Gram-negative and Gram-positive bacteria

AbstractInnate recognition of bacteria is a key step in the activation of inflammation and coagulation, and it is dependent on pathogen-associated molecular pattern (PAMP) ligation to Toll-like receptors (TLRs) and CD14. The dominant receptors activated when cells encounter a whole bacterium, which express several PAMPs, are poorly defined. Herein, we have stimulated various human cells with prototypic Gram-negative and Gram-positive bacteria. Receptor-dependent responses to whole bacteria were assessed using both TLR-transfected cells and specific monoclonal antibodies against TLRs, MD-2, and CD14. Enterobacteria-activated leukocytes and endothelial cells in a TLR4/MD-2–dependent manner, most likely via lipopolysaccharide (LPS). TLR2 activation was observed with a high bacterial inoculum, and in epithelial cells expressing TLR2 but not TLR4. Pseudomonas aeruginosa stimulated cells by both TLR2 and TLR4/MD-2. Gram-positive bacteria activated cells only at high concentrations, in a partially TLR2-dependent but TLR4/MD-2–independent manner. Either TLR or CD14 neutralization blocked activation to all bacterial strains tested with the exception of some Gram-positive strains in whole blood in which partial inhibition was noted. This study identifies dominant TLRs involved in responses to whole bacteria. It also validates the concept that host cell activation by bacterial pathogens can be therapeutically reduced by anti-TLR4, -TLR2, and -CD14 mAbs.

Comparison of two in vivo and two ex vivo tests to assess the antibacterial activity of several antiseptics

An ex vivo test was adapted to mimic the in vivo conditions of testing antiseptic activity on human forearms and in the European Standard Hygienic Handwash Test (BSEN 1499). The study was to validate the ex vivo protocols using 4.8% (w/v) para-chloro- meta-xylenol (PCMX, neat Dettol), 0.5% (w/v) triclosan in 70% (v/v) isopropanol, and 2% (v/v) povidone-iodine against a high bacterial inoculum (>10 8 cfu/mL) of Escherichia coli NCTC 10538. Two ex vivo tests using human skin samples, including one introducing a mechanical rubbing effect, were compared with two corresponding in vivo tests (the forearm test and the BSEN handwashing test). All antiseptics assessed in vivo (forearm and handwash tests) produced reductions in bacterial counts that were significantly greater than those for the non-medicated soft soap control. When assessed ex vivo without rubbing, only PCMX and povidone-iodine achieved reductions significantly greater than soft soap. When assessed ex vivo with mechanical rubbing, only PCMX and triclosan achieved reductions significantly greater than soft soap. Overall, the antiseptics at the concentrations tested were more active when tested in vivo than ex vivo. The addition of a mechanical effect, either in vivo by the volunteers washing their hands or ex vivo by a drill rubbing two skin samples against each other, produced a significantly greater reduction in bacterial concentrations. The ex vivo tests were easily adapted to mimic in vivo protocols. The value of such tests, particularly the one that includes a rubbing effect, may be significant as they avoid the need for human volunteers.

Splenic Norepinephrine and Serum Corticosterone Level Fluctuations Associated with Bacteria-Induced Stress

Corticosterone (CORT) and norepinephrine (NE), two effector molecules of the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-lymphoid (SL) axes, respectively, differentially influence murine host resistance to Listeria monocytogenes (LM). Serum CORT and splenic NE levels early (≤24 h) after infection correlated positively with host resistance, as long as the LM burden did not exceed approximately 10⁶ cfu LM per spleen. As previously reported, mice with right-circling preference (R-mice) have significantly greater host resistance to LM than those with left-circling preference (L-mice) and early after infection, R-mice had significantly higher serum CORT levels than L-mice. However, rapid pathogenesis with a high bacterial burden induced high activation of the HPA and SL axes, which prevented observable differences in the defense against LM, especially later in infection. With the high bacterial inoculum (10⁵ LM), the splenic NE levels significantly increased, but no differences among R- and L-mice were discernible. We suggest that endogenous asymmetry of neuroimmune circuits contributes to differential host resistance, but the level of stress (bacterial inoculum) is critical. With regard to the neuroendocrine factors assessed, CORT, but not NE, levels significantly correlated with the enhanced defenses of R-mice in comparison to L-mice. The differential host resistance based on brain laterality seems to be more a function of the HPA axis and possibly other CNS effects on peripheral immunity than neurotransmitter release by the sympathetic innervation of the spleen.

Efficacy of once- and thrice-daily dosing of aminoglycosides in in-vitro models of infection

The bactericidal efficacy of amikacin, isepamicin and netilmicin was studied against Pseudomonas aeruginosa and Serratia marcescens over a treatment period of 30 h using two one-compartment in-vitro models with differently designed culture compartments. High bacterial inocula were exposed to fluctuating drug concentrations, simulating human serum concentrations (t1/2 = 2 h) during clinical treatment. The same daily dose was administered as 1 h infusions given every 8 h or every 24 h, resulting in peak concentrations of 8 and 24 mg/l for netilmicin, and 24 and 72 mg/l for amikacin and isepamicin, respectively. Once-daily dosing was more bactericidal during initial treatment in the in-vitro models (P less than 0.01) and at least as effective as thrice-daily dosing in preventing bacterial regrowth, despite a prolonged period of subinhibitory drug concentration before administration of the second dose. Lower ratios of peak concentration to MIC were needed to achieve bactericidal activity (greater than 99.9% reduction of cfu) after 24 h treatment against S. marcescens compared with P. aeruginosa (P less than 0.01). All nine regimens providing peaks of at least four times the MIC were bactericidal against S. marcescens after 24 h exposure. In contrast, a bactericidal effect against P. aeruginosa occurred only during two of six experiments with peaks of four to nine times the MIC. Similar results were obtained in both in-vitro models of infection. These data suggest insufficient intrinsic activity of the aminoglycosides studied for single drug treatment of P. aeruginosa in the absence of host-defence mechanisms.

Comparison of single and multiple doses of prophylactic antibiotics in experimental streptococcal endocarditis.

Single-doses or short-term administration of beta-lactam antibiotics alone or combined with aminoglucoside antibiotics have failed to consistently prevent experimental streptococcal endocarditis induced by high inocula of bacteria poorly susceptible to killing by these antibiotics. The optimal duration of administration of antibiotics for successful prophylaxis under these circumstances has not been established. We therefore tested, in rats with catheter-induced sterile aortic vegetations, the duration of administration of antibiotic necessary to prevent endocarditis induced by bacterial inocula 100 to 10,000 times the 90% infective dose of two tolerant viridans-group streptococci and two Streptococcus faecalis strains. Multiple-dose regimens of amoxicillin alone or of amoxicillin combined with gentamicin were studied. Against the two viridans group streptococci, successful prophylaxis was achieved with multiple doses of amoxicillin alone given over 24 to 48 hr and by the combination of amoxicillin and gentamicin given for 6 to 24 hr. Against the two S. faecalis strains, multiple-dose regimens with amoxicillin alone failed, but the combination of amoxicillin and gentamicin was successful when administered for 48 to 72 hr. Thus, after challenge with high bacterial inocula, repeated doses of a beta-lactam antibiotic alone were sufficient to prevent viridans streptococcal endocarditis, but multiple doses of a bactericidal combination (beta-lactam plus aminoglucoside), as necessary for the treatment of established endocarditis, were a prerequisite for successful prophylaxis of S. faecalis endocarditis.

Influence of mixed culture conditions on yeast‐wall hydrolytic activity of Bacillus circulans WL‐12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma

Okagbue, R.N. &amp; Lewis, M.J. 1985. Influence of mixed culture conditions on yeast‐wall hydrolytic activity of Bacillus circulans WL‐12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma. Journal of Applied Bacteriology59, 243–255.In mixed culture Bacillus circulans WL‐12 hydrolysed cell walls of Phaffia rhodozyma and rendered astaxanthin extractable from the yeast. pH control was critical to survival and lytic activity of the bacillus; the optimum range was 6.2–6.8. The optimum range of temperature was 20–24d̀C. Glucose (1–2%) was efficient in minimizing catabolite repression of the lytic enzyme complex of the bacillus. Slow‐feeding of glucose improved ultimate yields of lytic enzyme but did not acclerate yeast cell wall modification. A relatively high inoculum level of B. circulans accelerated modification of P. rhodozyma in the mixed culture: when the bacterial inoculum was four times that of the yeast, over 80% of total astaxanthin was extractable in 48 h. High bacterial inoculum size also stimulated yeast autolysis and necessitated early harvest of the mixed culture. Results obtained in shake flasks were duplicated in 5‐litre fermentors and suggest that the mixed culture has potential industrial value for producing a biomass containing biologically‐available astaxanthin. Extractability of astaxanthin was also achieved when mixed culture filtrate was incubated with pure cultured Phaffia cells. When suitably fortified with nutrients, the filtrate also supported simultaneous yeast growth and modification of the yeast cell walls. A scheme incorporating mixed culture with B. circulans WL‐12 and re‐use of culture filtrate has been proposed for enzymatic processing of Phaffia rhodozyma for inclusion in animal diets.