LiF Reduces MICs of Antibiotics against Clinical Isolates of Gram-Positive and Gram-Negative Bacteria.

Antibiotic resistance is an ever-growing problem yet the development of new antibiotics has slowed to a trickle, giving rise to the use of combination therapy to eradicate infections. The purpose of this study was to evaluate the combined inhibitory effect of lithium fluoride (LiF) and commonly used antimicrobials on the growth of the following bacteria: Enterococcus faecalis, Staphyloccoccus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, and Streptococcus pneumoniae. The in vitro activities of ceftazidime, sulfamethoxazole-trimethoprim, streptomycin, erythromycin, amoxicillin, and ciprofloxacin, doxycycline, alone or combined with LiF were performed by microdilution method. MICs were determined visually following 18-20 h of incubation at 37°C. We observed reduced MICs of antibiotics associated with LiF ranging from two-fold to sixteen-fold. The strongest decreases of MICs observed were for streptomycin and erythromycin associated with LiF against Acinetobacter baumannii and Streptococcus pneumoniae. An eight-fold reduction was recorded for streptomycin against S. pneumoniae whereas an eight-fold and a sixteen-fold reduction were obtained for erythromycin against A. baumannii and S. pneumoniae. This suggests that LiF exhibits a synergistic effect with a wide range of antibiotics and is indicative of its potential as an adjuvant in antibiotic therapy.

Characterization of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients by different typing methods.

Pseudomonas (P.) aeruginosa strains isolated from the sputum of cystic fibrosis patients (CFP) are frequently difficult to type by conventional typing methods. The purpose of this study was to develop a random amplified polymorphic DNA (RAPD) analysis for the routine typing of these strains. Sixty P. aeruginosa non-repetitive strains recovered from CFP in a teaching hospital were typed. Thirty-five non-serotyped strains were studied by RAPD-PCR analysis with primers 272 and 208. RAPD data were performed to establish the relatedness between bioptype, antibiotic susceptibility and genotype. Fifty-five percent of strains are multiresistant, and no relation was found between antibiotype and biotype. A possible correlation between various phenotypes belonging to a single genotype was observed. RAPD typing characterized 30 distinct genotypes and two small clusters of strains were observed among isolates with each primer. Strains belonging to one cluster were present in two (6%) of the 35 strains. Strains belonging to the other cluster were present in three (8%) of the 35 strains. The occurrence of these clusters indicates that cross-infection may occur. The results indicate also that only the RAPD method can establish a clonal relation whereas the other methods may only reflect phenotypical differences, and thus are inadequate to type these strains.

Prevalence of and risk factors for sexually-transmitted infections in hidden female sex workers.

OBJECTIVES: We wanted to determine the age-specific prevalence of selected sexually transmitted infections while assessing the risk factors among hidden female sex workers (HFSW). METHODS: One hundred HFSW over 15 years of age were recruited in an impoverished area of Antananarivo, Madagascar. After oral informed consent, blood and endocervical swabs were tested for specific antigens, antibodies, and pathogens using molecular, serologic, and microscopic examinations. A risk factor analysis was conducted with odds ratios and 95% confidence intervals. RESULTS: Thirty-two percent, 27, 12, and 7% of HFSW were infected respectively with Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis. Specific antibodies against, syphilis were detected in 11%. None were HIV-positive. The main factors associated with STI were: young age, being married, lower education level, early age for first intercourse, and a history of genital infection.

[Various typing methods of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients]. / Différentes méthodes de typage des souches de Pseudomonas aeruginosa isolées des patients atteints de mucoviscidose.

Typing methods are essential to understand the epidemiology of bacterial infections. Strain typing is important for the detection of sources or routes of infections, identification between endemic and epidemic strains and prevention of transmission between patients. Some Pseudomonas aeruginosa cystic-fibrosis strains could not be typed with conventional typing methods. Due to the diverse phenotypic nature of P. aeruginosa, phenotyping methods are not discriminatory enough to identify strains belonging to the same genotype. Thus, molecular typing methods are required. These methods should be applied when data from phenotypic typing analysis becomes ambiguous, such as in cystic fibrosis. Molecular typing methods, developed over the past decade, are highly discriminatory in capacity and reproducibility. However, they are more likely applied in specialized laboratories since they are expensive and increase the workload. A reliable and low-cost typing system is required for better defining the epidemiology of this pathogen and designing more rational policies of infection control. Comparison between typing methods will pinpoint the limits and effectiveness of each method and will improve in turn the choice of a nonspecialized laboratory in terms of simplicity, time and cost.

Sustained interleukin-6 and interleukin-8 expression following infection with Chlamydia trachomatis serovar L2 in a HeLa/THP-1 cell co-culture model.

Chlamydia trachomatis, an intracellular obligate bacterium, remains responsible for a large spectrum of disorders that can progress to chronic diseases, resulting in severe sequelae, such as tubal infertility and blindness. These sequelae may be due to deleterious immune responses induced by repeated or persistent infections. By initiating and regulating inflammation as well as immune responses, pro-inflammatory cytokines secreted by local infected epithelial and immune cells, such as monocytes, may play an essential role in immunity and in the immunopathogenesis of chlamydial diseases. In this study, we mimicked the in vivo interaction between epithelial cells and monocytes by co-culturing epithelial-like HeLa cells with monocyte-like THP-1 cells. Pro-inflammatory cytokines [interleukin-beta (IL-1beta), IL-6, IL-8, IL-10, IL-12p70 and tumour necrosis factor-alpha (TNF-alpha)] were measured by multiplexed cytometric bead array assay over a period of 18 days. We observed that pro-inflammatory cytokine secretion was augmented after C. trachomatis infection in HeLa and THP-1 cells. However, this heightened secretion was subsequently reduced. When infected HeLa cells were co-cultured with THP-1 cells, IL-6 and IL-8 secretion was sustained, IL-1beta expression followed a bell-shaped curve and IL-10, IL-12p70 and TNF-alpha synthesis was down regulated. IL-6 and IL-8 may be involved in the immunopathogenesis of chronic chlamydial infections. We also observed that throughout C. trachomatis persistence induced by doxycycline (Dox) treatment, IL-1beta, IL-6, IL-8 and TNF-alpha expression was reduced, whereas the synthesis of IL-10 and IL-12p70 remained unchanged but not sustained. Thus, during chlamydial persistence infection evoked by treatment with Dox, none of the tested cytokines showed sustained expression.

Pharmacokinetics of cationic liposome-encapsulated doxycycline in mice challenged with genital infection by Chlamydia trachomatis.

Our previous studies demonstrated the efficacy of cationic liposome-encapsulated doxycycline (CaL-DOX) on the course of infection with Chlamydia trachomatis in vitro and in vivo. In this investigation, the pharmacokinetics of CaL-DOX are reported. Female mice inoculated intravaginally with 4.84 x 10(5) inclusion-forming units were treated intramuscularly, 3 days postinfection, as determined by a preliminary study, with 0.1 ml of unencapsulated doxycycline (DOX) or CaL-DOX, at 10 microg/ml body weight for 3 consecutive days. Sampling was performed at 12, 24, 48, 72 and 96 h after treatment. The microbiological test was used to measure the DOX concentration in sera, liver, kidneys and genital organs. The maximum concentration in kidneys, liver and genital organs was higher in mice treated with DOX than in those treated with CaL-DOX. However, in sera, the amount was similar to that in mice treated with DOX. The DOX concentration found in mice treated with CaL-DOX was much less than in those treated with unencapsulated DOX. This may have a direct impact on the secondary effects caused by the medication. Decreased secondary effects should have a positive outcome on patient physical and mental health. Even if this study is the only one of its kind so far, CaL-DOX could be an alternative solution for the treatment of chlamydial infections.

[In vitro inhibition of Chlamydia trachomatis growth by liposome-encapsulated cyclines]. / Inhibition in vitro de la croissance de Chlamydia trachomatis par des cyclines encapsulées dans les liposomes.

The antichlamydial activity of tetracycline (Tet) and doxycycline (Dox) encapsulated in cationic (CaL), anionic (AnL) and neutral (NtL) liposomes has been evaluated in vitro by adding serial dilutions of antibiotics (minimum inhibitory concentration, MIC: 0.12-0.007 microgram/ml; MBC: 4 to 0.25 micrograms/ml) to HeLa 229 cell monolayers inoculated with Chlamydia trachomatis L2/434/Bu (10(3) ufi/ml). Following 72 h incubation at 37 degrees C under a 5% CO2 atmosphere, the chlamydial inclusions were stained by the May-Giemsa method to determine the MICs. After a second and third passage, the MBC1 and MBC2 were determined in antibiotic-free medium. The chlamydial inclusions were then counted to assess the degree of growth inhibition at each antibiotic dilution tested for MBC1 and MBC2 determinations. The MIC, MBC1 and MBC2 of the various antichlamydial agents were as follows: Tet (0.12; 4; 4), AnL-Tet (0.01; 1; 1), NtL-Tet (0.03; 1; 2), Dox (0.06; 1; 2), CaL-Dox (0.03; 0.5; 2), AnL-Dox (0.01; 1; 2), and NtL-Dox (0.03; 0.5; 0.5). It was found that Tet and Dox liposome-encapsulated antibiotics were more active than their non-encapsulated counterparts, and the inclusion count showed a higher inhibitory activity of the former antibiotics on chlamydial growth. The inhibition of chlamydial growth by AnL-Tet may be of bactericidal nature. In conclusion, liposome-encapsulated drugs could be of value in the treatment of chlamydial infections.

Effects of cationic liposome-encapsulated doxycycline on experimental Chlamydia trachomatis genital infection in mice.

In a previous study, we demonstrated that cationic liposome-encapsulated doxycycline (CaL-Dox) was two-fold more effective than free doxycycline against Chlamydia trachomatis in vitro. Here, we evaluated the effects of two CaL-Dox regimens in comparison with unencapsulated doxycycline on the course of chlamydial genital infection in mice. Progesterone-treated, female CF-1 mice were challenged intravaginally with 1.2 x 10(5) inclusion-forming units (ifu) of C. trachomatis. Two days post-infection, the animals were divided into four treatment groups for im injection of doxycycline at 10 mg/kg body weight bd for 3 (3 Dox) or 7 days (7 Dox), or of CaL-Dox at the same dose level for 3 (3 CaL-Dox) or 7 days (7 CaL-Dox) consecutively. An infected fifth group served as a control and was given an empty CaL preparation. C. trachomatis were isolated after five blind passages from 82% of infected control mice, 61.4% of 3 Dox, 52.2% of 3 CaL-Dox, 29% of 7 Dox and 20% of 7 CaL-Dox animals. Histopathological reactions were found in various tissues of the genital tract in 79.5% of infected control mice, 80.9% of 3 Dox, 65.2% of 3 CaL-Dox, 47.1% of 7 Dox and 25.7% of 7 CaL-Dox animals. Total antichlamydial antibody titres were lower in 7 CaL-Dox mice than in all the other groups (P < 0.005). The results showed that progesterone-treated CF-1 mice are suitable for investigation of both lower and upper genital tract infection with a lymphogranuloma venereum biovar strain of C. trachomatis. Neither 7 CaL-Dox nor 3 CaL-Dox treatment was more effective than unencapsulated 7 Dox doses in the bacteriological cure of chlamydial genital infection in mice. However, 7 CaL-Dox prevented tissue damage in the genital tract significantly more than all the other regimens (P < 0.05). These results suggest that liposome-encapsulated doxycycline, particularly CaL-Dox, may have potential for the clinical treatment of chlamydial infections.

In-vitro anti-chlamydial activities of free and liposomal tetracycline and doxycycline.

The purpose of this study was to evaluate the anti-chlamydial activities in vitro of liposome-encapsulated doxycycline (Dox) and tetracycline (Tet) in comparison with free Dox and Tet. Dox and Tet encapsulated in cationic (CAL), anionic (ANL) and neutral (NTL) liposomes by sonication, were quantified by high-performance liquid chromatography. Anti-chlamydial activities were determined by addition of serial dilutions of antibiotics (MIC 0.12-0.007 mg/L; MBC 4-0.25 mg/L) to HeLa 229 cell monolayers inoculated with Chlamydia trachomatis L2/434/Bu (10(3) ifu/well). After incubation for 72 h at 37 degrees C, chlamydial inclusions were stained by the May-Grünwald Giemsa method to establish MICs. MBCs were determined in chlamydial agent-free medium after second passages. Dox-encapsulation efficiencies were 28.6 SEM 6.4% in cationic (CAL-Dox), 49.1 SEM 6.7% in anionic (ANL-Dox) and 21.0 SEM 0.8% in neutral (NTL-Dox) liposomes. Tet-encapsulation efficiencies were 3.5 SEM 0.3% in anionic (ANL-Tet) and 2.2 SEM 0.6% in neutral (NTL-Tet) liposomes; no Tet was detected in cationic (CAL-Tet) liposomes. MIC values were 0.06 mg/L for Dox, 0.12 mg/L for Tet, 0.03 mg/L for CAL-Dox, NTL-Dox and NTL-Tet, and 0.01 mg/L for ANL-Dox and ANL-Tet. MBCs were 4 mg/L for Tet, 0.5 mg/L for CAL-Dox and NTL-Dox, and 1 mg/L for Dox, ANL-Dox, ANL-Tet, NTL-Tet and NTL-Tet. For MICs, the relative increase in anti-chlamydial activity observed with liposomal formulations compared to the corresponding free antibiotic ranged from 2- to 6-fold with Dox and from 4- to 10-fold with Tet. For MBCs, the relative increases in anti-chlamydial activity were 2- and 4-fold with liposome-encapsulated Dox and Tet, respectively. Dox was better encapsulated than Tet in all liposomes. Liposome-encapsulated drugs showed greater anti-chlamydial activities than their free forms; thus, these drug formulations have potential in the treatment of chlamydial infections.

Beta-lactamases and outer membrane investigations in beta-lactam-resistant Comamonas acidovorans strains.

Imipenem-induced beta-lactamase (level of expression, specific activity and kinetic parameters (Vmax and Km) in response to nitrocefin) and outer membrane proteins (OMPs) (hydrophobicity, permeability and electrophoretic pattern) were characterized in, one beta-lactam sensitive (PAC-9), one resistant (PAC-1) and two resistant laboratory mutants (PAC-9M, PAC-9M2) of Comamonas acidovorans strains. Beta-lactamases from both mutant strains showed different Vmax values compared to the parental strains. Beta-lactam resistance was found to be associated in PAC-1 with inducible beta-lactamase production and OMP alteration by the appearance of a 102-KDa protein. Moreover, PAC-1 was less permeable to nitrocefin than PAC-9. These data indicate that C. acidovorans resistance to beta-lactam resulted from synergy between beta-lactamase and OMP alterations.

Incorporation rates, stabilities, cytotoxicities and release of liposomal tetracycline and doxycycline in human serum.

Tetracycline and doxycycline were encapsulated in cationic, anionic and neutral liposomes. The amounts of antibiotic encapsulated, the stability of each preparation at 4 degrees C for 4 weeks, and the kinetics of the release of entrapped drug into human sera were assessed by high-performance liquid chromatography. The toxicities of the liposome preparations on human erythrocytes and HeLa 229 cells were evaluated in vitro. The results showed that doxycycline was entrapped more efficiently than tetracycline, and that doxycycline-entrapped liposomes were more stable at 4 degrees C and in human sera, and less cytotoxic than tetracycline-entrapped liposomes.

Single-dose pharmacokinetics of ampicillin and tobramycin administered by hypodermoclysis in young and older healthy volunteers.

1. To test the feasibility of administering antibiotics by subcutaneous infusion to the elderly, we compared the pharmacokinetics of tobramycin (single dose of 80 mg) given by hypodermoclysis (HDC) with the kinetics of the antibiotic injected intravenously (i.v.) in 10 young (< 50 years old) and 10 elderly (> 65 years old) healthy volunteers. Similar studies were performed with ampicillin (single dose of 1 g) in 12 young and 10 older healthy volunteers. 2. Compared with the i.v. route, HDC delayed the time to reach the maximal plasma concentration (tmax) of tobramycin in young volunteers: 32 +/- 6 (s.d.) min vs 88 +/- 46, P < 0.005, and older volunteers: 27 +/- 4 min vs 89 +/- 15, P < 0.005. Administration of the antibiotics by HDC was well tolerated. The plasma concentration of tobramycin 30 min after the end of infusion (C60) was lower (P < 0.05) following HDC than after the i.v. route in both young, 2.2 +/- 0.7 vs 3.5 +/- 0.8 micrograms ml-1, and elderly subjects, 2.2 +/- 0.8 vs 3.8 +/- 0.9. micrograms ml-1. 3. The area under the curve (AUC) of tobramycin given by HDC was slightly smaller than when given i.v., i.e. in young subjects: 740 +/- 225 (s.d.) vs 893 +/- 223 micrograms ml-1 min, NS, and in the elderly: 980 +/- 228 vs 1056 +/- 315 micrograms ml-1 min, NS. 4. When ampicillin was administered by HDC, the tmax was also delayed in young volunteers: 45 +/- 18 vs 23 +/- 6 min, and in the elderly: 49 +/- 18 vs 27 +/- 4 min, P < 0.005, the AUC was greater by HDC than i.v. in the young volunteers: 4527 +/- 1658 micrograms ml-1 min vs 3810 +/- 1033 micrograms ml-1 min and in the elderly: 6795 +/- 2094 micrograms ml-1 min vs 4217 +/- 1518 micrograms ml-1 min, and the C60 was higher by HDC in the young: 27 +/- 7 vs 24 +/- 9 micrograms ml-1, and in the elderly: 32 +/- 9 vs 23 +/- 11 micrograms ml-1, P < 0.05. 5. In conclusion, HDC delays the entry of the antibiotic into the systemic circulation, but did not affect the amount available. HDC was well tolerated and could become an adequate method for antibiotic administration to the elderly.

Comparative in vitro activity of nine antistaphylococcal agents against 275 recent isolates of Gram-positive cocci.

The aim of this study was to compare the in vitro activities of 9 antistaphylococcal agents including teicoplanin (TEI) against 275 non-repetitive clinical strains representing 15 species of staphylococci and 27 strains of Enterococcus (E.) faecalis, isolated from various specimens between 1991-1992 at a Canadian teaching hospital. The NCCLS agar dilution method was used (10(4) colonyforming units/spot). In terms of MIC(90), TEI and vancomycin (VAN) appeared to be the most potent antibiotics against all staphylococci tested (TEI: 2.0-4.0 mug/ml; VAN: 1.0-2.0 mug/ml; ciprofloxacin (CPF): 0.25-32 mug/ml; cefazolin (CEF): 8.0-256 mug/ml; methicillin (MET): 2.0->256 mug/ml; imipenem (IMP): 1.0-32 mug/ml; erythromycin (ERT): 16->256 mug/ml; ampicillin (AMP): 16-128 mug/ml; fusidic acid (FSA): 0.5-16 mug/ml). Multiple resistant strains, including MET-resistant Staphylococcus (Staph.) aureus and Staph. epidermidis, were susceptible to TEI and VAN with respective MICs of 2-4 mug/ml and 1-2 mug/ml regardless of specimen type. Moreover, TEI was highly active against E. faecalis (MIC(90) for TEI and VAN: 0.5 and 4.0 mug/ml, respectively).

Preparation, properties and the effects of amikacin, netilmicin and tobramycin in free and liposomal formulations on Gram-negative and Gram-positive bacteria.

The most common problems limiting the medical use of aminoglycosides have been the nephro- and oto-toxicities and the increasing bacterial resistance. It has been shown that encapsulation of drugs into liposomes enhances their efficacy while reducing their toxicities. The present in vitro study was designed to evaluate the antimicrobial activities of free and liposomal amikacin, netilmicin and tobramycin. We, therefore, encapsulated these drugs into liposomes prepared by sonication. The drug contained in liposomes was measured by enzyme multiplied immunoassay technique (EMIT) after lysis of the vesicles by 0.2% Triton X-100. The comparative encapsulation efficiency of the three antibiotic preparations was assessed. Aminoglycosides kinetic release from liposomes in presence of normal human sera was also studied in vitro over a 48 h period at 37 degrees C under 5% CO(2). The MICs of these encapsulated drugs to Pseudomonas aeruginosa, Xanthomonas maltophilia, Escherichia coli, Streptococcus faecalis and Staphylococcus aureus were determined and compared to those of respective free drugs using an agar dilution method. The amikacin and tobramycin encapsulation efficiencies were significantly (P

Serotyping clinical isolates of Pseudomonas aeruginosa in relation to infection site antibiotic susceptibility and beta-lactamase production.

The present study investigates the association of O-serotype with isolation site, antibiotic resistance, and beta-lactamase production to determine the profiles of Pseudomonas aeruginosa clinical isolates circulating in a Canadian teaching hospital. The most frequently encountered serotypes of Pseudomonas aeruginosa strains isolated from inpatients at Hôtel-Dieu de Montréal hospital during the period 1992-1993 were O1, O3, O5, O6, O10, and O11 (9-13%) isolated from pus, urine, sputa and other sources. Serotype O1 was the most sensitive in contrast to serotype O3 which was resistant to virtually all antibiotics tested except to imipenem and quinolone. Of the strains of Pseudomonas aeruginosa, more than 50% with the most encountered serotype produced a high level of beta-lactamase.

Comparison of the bactericidal action of amikacin, netilmicin and tobramycin in free and liposomal formulation against Pseudomonas aeruginosa.

The rates at which free, cationic and anionic liposomal forms of amikacin, netilmicin and tobramycin kill Pseudomonas aeruginosa were studied in vitro. Control inocula with no antibiotic yielded 6.76, 9.53 and 9.74 log CFU/ml at 0, 6 and 24 h, respectively. Empty anionic or cationic liposomes had no effect on bacterial growth. The killing rates of free antibiotics against the bacterial strain were not enhanced by the addition of either empty anionic or cationic liposomes. After 6 and 24 h of exposure at 1, 2 and 4 times the minimum inhibitory concentrations, free amikacin, netilmicin and tobramycin demonstrated a more rapid bactericidal effect than encapsulated anionic or cationic liposomes. The killing rates of liposomal aminoglycosides were lower than those of free aminoglycosides at identical concentrations, suggesting that only fractions of the encapsulated drugs were released from liposomes.

Incorporation, release and in-vitro antibacterial activity of liposomal aminoglycosides against Pseudomonas aeruginosa.

Amikacin, netilmicin and tobramycin were incorporated into either anionic or cationic liposomes prepared by sonication. The influence of lipid constituents (charges) on encapsulation efficiency was determined after lysis of vesicles by 0.2% (v/v) Triton X-100. The in-vitro activities of the liposomal aminoglycosides were evaluated against Pseudomonas aeruginosa by agar dilution and compared with free antibiotics. Normal human pooled sera, incubated at 37 degrees C, were supplemented with anionic or cationic liposomes containing known fixed concentrations of amikacin, netilmicin or tobramycin. At various time intervals (0-48 h), samples were taken and antibiotic concentrations determined by the enzyme multiplied immunoassay technique (EMIT). The encapsulation efficiency of cationic liposomes (amikacin 17.1 +/- 1.55%, netilmicin: 5.63 +/- 1.13%, tobramycin 6.7 +/- 0.5%) was approximately 30% higher than that of anionic liposomes (amikacin 12.3 +/- 0.95%, netilmicin 4.0 +/- 0.06%, tobramycin 5.13 +/- 0.18%). Anionic and cationic liposomes in human serum still retained 79.13 +/- 4.04% and 82.71 +/- 2.6% of amikacin, 50.67 +/- 1.8% and 38.6 +/- 0.8% of netilmicin, and 89.09 +/- 1.0% and 88.93 +/- 0.4% of tobramycin, respectively, after 48 h of incubation at 37 degrees C under 5% CO2. The MICs of amikacin (2, 16 and 2 mg/L), netilmicin (2, 1 and 4 mg/L) and tobramycin (1, 2 and 4 mg/L) in free, anionic or cationic liposomal formulations, respectively, were relatively comparable except for anionic liposomal amikacin for which the MIC was increased eight-fold. Empty cationic or anionic liposomes had no effect on bacterial growth. Cationic liposomes containing aminoglycosides should be evaluated further for the treatment of pseudomonal infection.

Rapid method for isolating detergent-insoluble outer membrane proteins from Pseudomonas aeruginosa.

The present study compares two techniques for isolating outer membrane proteins (OMPs) of Pseudomonas aeruginosa, Method A - selective solubilization with sodium lauryl sarcosinate, and Method B - isopycnic sucrose gradient centrifugation, using three criteria: the amount of proteins obtained, polypeptide patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their practical aspects. Method A appears to be superior to Method B. It yields a higher outer membrane protein content and a similar polypeptide pattern as Method B, but is more rapid and less cumbersome.