Novel membrane proteins present in teicoplanin-resistant, vancomycin-sensitive, coagulase-negative Staphylococcus spp.

Clinical isolates of Staphylococcus epidermidis and Staphylococcus haemolyticus resistant to teicoplanin (MIC 64 mg/L) and sensitive to vancomycin (MIC 2 mg/L), were compared with vancomycin- and teicoplanin-sensitive isolates (MICs 1 mg/L) of the same species. No apparent differences between the sensitive and resistant strains of either pair were found with respect to binding of teicoplanin to the bacteria, or to the amino acid content or degree of cross-linkage of purified peptidoglycan. The resistant strains did not inactivate teicoplanin in the surrounding medium. Analysis of the membrane proteins of the resistant S. epidermidis strain grown in the presence or absence of sub-inhibitory levels of teicoplanin (4 mg/L), showed the presence of a 39 kDa protein which was either absent, or present in considerably reduced amounts, in the sensitive strain. Fractionation of cell components after lysis of protoplasts showed that the 39 kDa protein was present predominantly in the membrane fraction but also in small amounts in the wall fraction. Similar investigations with S. haemolyticus revealed the presence of a 35 kDa protein in membranes of the resistant strain: the amount was increased substantially by growth in sub-inhibitory levels of teicoplanin. Membranes prepared by mechanical disintegration of bacteria or by osmotic lysis of protoplasts showed large apparent differences in the amounts of the 39 kDa protein.

In-vitro studies with ramoplanin (MDL 62,198): a novel lipoglycopeptide antimicrobial.

Ramoplanin is a novel lipoglycopeptide antimicrobial complex, isolated from the fermentation products of a strain of Actinoplanes sp. (ATCC 33076), which comprises three closely related polypeptides, each containing chlorinated phenyl moieties and D-mannose. The in-vitro activity of ramoplanin was compared with those of vancomycin and teicoplanin. Ramoplanin was very active against Staphylococcus spp., irrespective of methicillin susceptibility, with all isolates inhibited by 1 mg/l or less. Concentrations of vancomycin and teicoplanin required to inhibit the same population of bacteria were 4 and 16 mg/l, respectively. Ramoplanin was also very active against Streptococcus spp. (alpha- and beta-haemolytic species, Str. pneumoniae and Enterococcus faecalis, Corynebacterium spp. (including Cory. jeikeium), Listeria monocytogenes, Gardnerella vaginalis, Propionibacterium acnes and Gram-positive anaerobic bacteria, with all isolates inhibited by 2 mg/l, or less. In general, the activity of ramoplanin against these species was either equal to or only slightly less than teicoplanin and equal to or somewhat greater than vancomycin. With the exception of Bacteroides melaninogenicus and Bact. bivius, ramoplanin was not active against Gram-negative bacteria.

In vitro activity of teicoplanin, vancomycin, A16686, clindamycin, erythromycin and fusidic acid against anaerobic bacteria.

The in vitro activity of teicoplanin and A16686, two new glycopeptide antibiotics was determined against 196 isolates of anaerobic bacteria. The activity of teicoplanin and A16686, in comparison with that of vancomycin, clindamycin, erythromycin and fusidic acid was 2 to 16 times higher against the gram positive anaerobes, namely, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Clostridium species, Peptococcus species and Peptostreptococcus species. However, Bacteroides fragilis was resistant to teicoplanin and A16686 while Bacteroides melaninogenicus and Bacteroides bivius were found to be sensitive.

Resistance studies with ofloxacin.

The selection of ofloxacin-resistant mutants from susceptible wild-type bacterial populations was investigated by three methods. Resistant mutants selected from populations of Escherichia coli (NCTC 10418) and Staphylococcus aureus (NCTC 6571) by single-step passage at either four or eight times the MIC occurred at a frequency of less than 1 x 10(-10). Ofloxacin-resistant mutants of Pseudomonas aeruginosa (NCTC 10662), selected at four times the MIC, occurred with a 100-fold greater frequency at 3.3 X 10(-8), however, mutants of this species selected at eight times the MIC, occurred at a frequency of less than 1 X 10(-10). Sequential selection of ofloxacin-resistant mutants during multiple passages at 1/2 MIC, resulted in a 16- to 32-fold increase in MICs for isolates of P. aeruginosa and Klebsiella aerogenes, a four- to eight-fold increase in MICs for some strains of E. coli and a two- to four-fold increase in MICs for Staph. aureus and Streptococcus spp. During prolonged exposure of populations of E. coli (NCTC 10418) and Staph. aureus (NCTC 6571) to concentrations of ofloxacin at 1, 10 and 100 times the MIC no resistant-mutants were selected after seven days incubation. In similar experiments with P. aeruginosa (NCTC 10662) ofloxacin-resistant mutants were selected from populations exposed to the MIC which required 64 mg/l ofloxacin for inhibition. No ofloxacin-resistant mutants were selected from populations of P. aeruginosa (NCTC 10662) exposed to either ten or 100 times the MIC.(ABSTRACT TRUNCATED AT 250 WORDS)

The comparative in-vitro activity of ofloxacin.

The antibacterial activity of ofloxacin, a new fluoroquinolone, was evaluated against a wide range of clinical bacterial isolates and compared with that of nalidixic acid, norfloxacin, enoxacin, pefloxacin and ciprofloxacin by determination of minimum inhibitory concentrations (MICs). Ofloxacin was very active against nalidixic acid-susceptible isolates of the Enterobacteriaceae (MIC less than or equal to 0.12 mg/l) and was also active against strains resistant to nalidixic acid (MIC less than or equal to 2 mg/l). The activity was similar to norfloxacin, enoxacin and pefloxacin but some four-fold less than that of ciprofloxacin. All of the fluoroquinolones were highly active against Vibrio cholerae (MIC less than or equal to 0.015 mg/l), V. parahaemolyticus (MIC less than or equal to 0.12 mg/l) Aeromonas hydrophila (MIC less than or equal to 0.03 mg/l), Plesiomonas shigelloides (MIC less than or equal to 0.015 mg/l), Campylobacter jejuni (MIC less than or equal to 0.5 mg/l), Neisseria spp., Haemophilus influenzae, H. ducreyi, Bordetella pertussis and Legionella pneumophila (MIC less than or equal to 0.06 mg/l for all species). Ofloxacin, ciprofloxacin and pefloxacin (MIC less than or equal to 1, 2 and 2 mg/l, respectively) showed similar activity against Staphylococcus spp. and were somewhat more active than enoxacin (MIC less than or equal to 4 mg/l) and norfloxacin (MIC less than or equal to 8 mg/l). Ofloxacin was moderately active against beta-haemolytic Streptococcus spp. (MIC less than or equal to 2 mg/l), Corynebacterium diphtheriae (MIC less than or equal to 1 mg/l) and Cory. jeikeium (MIC less than or equal to 2 mg/l) and somewhat less active against alpha- and non-haemolytic Streptococcus spp., Str. pneumoniae and Listeria monocytogenes (MIC less than or equal to 4 mg/l for all species) and Str. faecalis (MIC less than or equal to 8 mg/l). The activity of ofloxacin, against these species, was similar to ciprofloxacin and four to eight times greater than norfloxacin, enoxacin and pefloxacin. Ofloxacin, and all of the fluoroquinolones, were less active against anaerobic than aerobic bacteria. Clostridium perfringens (MIC less than or equal to 1 mg/l) was more susceptible to ofloxacin than were other anaerobic species and Cl. difficile (MIC less than or equal to 16 mg/l) was more resistant. Ofloxacin was the most active compound tested against Chlamydia trachomatis SA2f (MIC less than or equal to 0.5 mg/l) with only ciprofloxacin (MIC less than or equal to 1 mg/l) approaching similar activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The in vitro activity of ramoplanin (A-16686/MDL 62,198), vancomycin and teicoplanin against methicillin-susceptible and methicillin-resistant Staphylococcus spp.

Ramoplanin (A-16686/MDL 62,198) is a novel lipoglycopeptide antimicrobial, comprised of three closely related polypeptides containing chlorinated phenyl moieties and D-mannose, isolated from the fermentation products of Actinoplanes sp. ATCC 33076. The antimicrobial activity of ramoplanin is limited to Gram-positive bacteria and its reportedly unacceptable administration side-effects suggest that any potential clinical role will be limited to the topical therapy of superficial skin infections and the eradication of bacteria, representing a possible nosocomial cross-infection source, from carriage sites. In this study the MICs of ramoplanin have been determined for methicillin-susceptible and methicillin-resistant isolates of Staphylococcus aureus, S. epidermidis and S. haemolyticus and compared with those of two glycopeptide antimicrobials, vancomycin and teicoplanin. MICs were determined using an agar incorporation technique in Mueller-Hinton medium with an inoculum of 10(5) cfu. Ramoplanin was 2-8 times more active than either vancomycin or teicoplanin against methicillin-susceptible and methicillin-resistant isolates of S. aureus and methicillin-susceptible isolates of S. epidermidis. Isolates of methicillin-resistant S. epidermidis and both methicillin-susceptible and -resistant isolates of S. haemolyticus were generally less susceptible to teicoplanin than to vancomycin. Ramoplanin was significantly more active than either vancomycin or teicoplanin against these isolates. These results suggest that the clinical evaluation of ramoplanin as a topical antibacterial agent for the control of superficial infections caused by Staphylococcus spp. and for the eradication of methicillin-resistant S. aureus from carriage sites, is justified.

In vitro activity of aztreonam, cefuroxime and ceftazidime against gram-negative rods isolated from hospital patients with urinary tract infection.

The in vitro activity of aztreonam, cefuroxime and ceftazidime was determined against 2,372 Gram-negative rods (including Pseudomonas spp.) isolated from hospital patients with urinary tract infections during 1985. Minimum inhibitory concentrations (MICs) were determined using an agar incorporation technique in Mueller-Hinton agar. The inoculum used was approximately 10(5) colony forming units (cfu) contained in 10 microliter Mueller-Hinton broth, which was applied to the surface of the agar plates using a multipoint inoculator. Following inoculation plates were incubated aerobically at 37 degrees C for 18 h. The MIC of each antimicrobial for each organism examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum. The minimum concentration required to inhibit the growth of 90% (MIC90) of the bacterial isolates in each genus or species examined was also determined. In general the antibacterial spectrum of aztreonam was comparable to that of ceftazidime and superior to that of cefuroxime. Against Escherichia coli, which accounted for 72% of the isolates examined, aztreonam (MIC90 less than or equal to 0.25 microgram/ml) was slightly more active than ceftazidime (MIC90 0.5 microgram/ml) and considerably more active than cefuroxime (MIC90 8 micrograms/ml). Aztreonam was active against Pseudomonas spp. (MIC90 16 micrograms/ml), although somewhat less so than ceftazidime (MIC90 4 micrograms/ml). Cefuroxime showed low activity against this genus (MIC90 greater than 128 micrograms/ml).

The comparative in vitro activity of twelve 4-quinolone antimicrobials against enteric pathogens.

The minimal inhibitory concentrations (MICs) of twelve 4-quinolone antimicrobials were determined for Salmonella typhi (25), Salmonella spp. (50), Shigella spp. (50), Campylobacter jejuni (100), Vibrio cholerae (10), Vibrio parahaemolyticus (10), Yersinia enterocolitica (25), Aeromonas hydrophila (25) and Plesiomonas shigelloides (10). MICs were determined using an agar dilution technique in Mueller-Hinton agar (Oxoid, England) supplemented with 10% lysed horse blood. Antibiotic containing plates were inoculated with approximately 10(4) colony forming units of each organism, contained in 10 microliters of Mueller-Hinton broth (Oxoid, England), using a multipoint inoculator. Following inoculation plates were incubated aerobically for 18 hours at 37 degrees C, except for plates inoculated with Campylobacter jejuni which were incubated microaerophilically for 48 hours at 37 degrees C. The MICs of each antimicrobial for each isolate examined, together with the minimum concentrations of each antimicrobial required to inhibit 50% (MIC50) and 90% (MIC90) of the isolates examined, were also determined. The more recently synthesized 4-quinolones showed very good activity against all of the enteric pathogens examined with ciprofloxacin being the most active (MIC90: Salmonella typhi 0.015 microgram/ml, Salmonella spp. 0.015 microgram/ml, Shigella spp. 0.015 microgram/ml, Campylobacter jejuni 0.12 microgram/ml, Vibrio cholerae 0.008 microgram/ml, Vibrio parahaemolyticus 0.06 microgram/ml, Yersinia enterocolitica 0.015 microgram/ml, Aeromonas hydrophila 0.015 microgram/ml and Plesiomonas shigelloides 0.015 microgram/ml. Where considered clinically appropriate these compounds may have a useful role in the treatment and prevention of diarrhoeal disease caused by these enteric pathogens.

The comparative activity of twelve 4-quinolone antimicrobials against Haemophilus influenzae and Streptococcus pneumoniae.

The minimal inhibitory concentrations (MICs) of twelve 4-quinolone antimicrobials were determined for 100 isolates of Haemophilus influenzae (including 30 beta-lactamase producing strains) and 100 isolates of Streptococcus pneumoniae. MICs were determined using an agar dilution technique in Mueller-Hinton agar supplemented with 10% lysed horse blood. The inoculum used was approximately 10(4) colony-forming units, contained in 10 microliters of Mueller-Hinton broth, which was applied to the agar plates using a multipoint inoculator. Following inoculation, plates were incubated at 37 degrees C for 18 h in an atmosphere enriched to 10% carbon dioxide. The MIC of each antimicrobial for each isolate examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum. The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the organisms examined were also determined. The more recently synthesised 4-quinolones showed considerably greater activity than nalidixic acid and pipemidic acid against clinical isolates of Haemophilus influenzae and Streptococcus pneumoniae. There was no apparent difference between the MICs observed for beta-lactamase producing and non-beta-lactamase producing strains of Haemophilus influenzae. Ciprofloxacin was the most active 4-quinolone examined (MIC90 for Haemophilus influenzae 0.008 microgram/ml; Streptococcus pneumoniae 2 micrograms/ml). Clinical studies on a possible role for some of the more recently synthesised 4-quinolones in the management of patients with respiratory infection are indicated.

Comparative in vitro studies with 4-quinolone antimicrobials.

The minimal inhibitory concentrations (MICs) of nalidixic acid, pipemidic acid, cinoxacin, oxolinic acid, flumequine, pefloxacin, acrosoxacin, amifloxacin, norfloxacin, enoxacin, ofloxacin and ciprofloxacin were determined for a range of clinical isolates. MICs were determined using an agar dilution technique in Mueller-Hinton agar supplemented with 10% lysed horse blood. The inoculum used was approximately 10(4) colony forming units, contained in 10 microliters Mueller-Hinton broth, which was applied to the agar plates using a multipoint inoculator. Following inoculation, plates were incubated in conditions appropriate for the organisms under investigation. The MIC of each antimicrobial for each isolate examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum. The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the organisms examined were also determined. All of the more recently synthesised 4-quinolones showed considerably greater activity than the parent compounds, nalidixic acid, pipemidic acid and cinoxacin, against the range of organisms used in this study. Ciprofloxacin and ofloxacin were the two most active of the 4-quinolones examined.

The comparative activity of twelve 4-quinolone antimicrobials against gram-positive and gram-negative anaerobes.

The minimal inhibitory concentrations (MICs) of twelve 4-quinolone antimicrobials were determined for the Bacteroides fragilis group (50), Bacteroides melaninogenicus (20), Bacteroides bivius (10), Fusobacterium spp. (10), anaerobic Gram-positive cocci (50) and Clostridium spp. (20). MICs were determined using an agar dilution technique in Mueller-Hinton agar supplemented with 10% lysed horse blood. The inoculum used was approximately 10(4) colony-forming units, contained in 10 microliter of Mueller-Hinton broth, which was applied to the agar plates using a multipoint inoculator. Following inoculation, plates were incubated at 37 degrees C for 48 h in an anaerobic atmosphere. The MIC of each antimicrobial for each isolate examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum. The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the organism examined were also determined. All of the more recently synthesised 4-quinolones showed increased activity against the anaerobic bacteria used in this study. Ciprofloxacin and ofloxacin were the most active compounds examined (Bacteroides fragilis group MIC90 ciprofloxacin 4 micrograms/ml; ofloxacin 4 microgram/ml; Bacteroides melaninogenicus MIC90 ciprofloxacin 2 micrograms/ml, ofloxacin 2 micrograms/ml; Bacteroides bivius MIC90 ciprofloxacin 16 micrograms/ml, ofloxacin 32 micrograms/ml; Fusobacterium spp. MIC90 ciprofloxacin 2 micrograms/ml, ofloxacin 4 micrograms/ml; Clostridium spp. MIC90 ciprofloxacin 1 microgram/ml, ofloxacin 1 microgram/ml and anaerobic Gram-positive cocci MIC90 ciprofloxacin 4 micrograms/ml, ofloxacin 4 micrograms/ml).