Aminoglycoside-inactivating enzymes in clinical isolates of Streptococcus faecalis. An explanation for resistance to antibiotic synergism.

Clinical isolates of enterococci (Streptococcus faecalis) with high-level resistance to both streptomycin and kanamycin (minimal inhibitory concentration >2,000 mug/ml), and resistant to synergism with penicillin and streptomycin or kanamycin were examined for aminoglycoside-inactivating enzymes. All of the 10 strains studied had streptomycin adenylyltransferase and neomycin phosphotransferase activities; the latter enzyme phosphorylated amikacin as well as its normal substrates, such as kanamycin. Substrate profiles of the neomycin phosphotransferase activity suggested that phosphorylation occurred at the 3'-hydroxyl position, i.e., aminoglycoside 3'-phosphotransferase. A transconjugant strain, which acquired high-level aminoglycoside resistance and resistance to antibiotic synergism after mating with a resistant clinical isolate, also acquired both enzyme activities. Quantitative phosphorylation of amikacin in vitro by a sonicate of the transconjugant strain inactivated the antibiotic, as measured by bioassay, and the phosphorylated drug failed to produce synergism when combined with penicillin against a strain sensitive to penicillin-amikacin synergism.No differences were found in the sensitivity of ribosomes from a sensitive and resistant strain when examined in vitro using polyuridylic acid directed [(14)C]-phenylalanine incorporation in the presence of streptomycin, kanamycin, or amikacin. Therefore, we conclude that aminoglycoside-inactivating enzymes are responsible for the aminoglycoside resistance, and resistance to antibiotic synergism observed in these strains.

Plasmid-mediated resistance to antibiotic synergism in enterococci.

Mating experiments have shown that high-level resistance (minimal inhibitory concentration greater than 2,000 microgram/ml) to streptomycin and kanamycin, and resistance to penicillin-streptomycin and penicillin-kanamycin synergism are transferable by conjugation from resistant clinical isolates of enterococci to a sensitive recipient strain. Cesium chloride-ethidium bromide ultracentrifugation revealed a satellite (plasmid) band in resistant clinical isolates and the transconjugant strains but not in the sensitive recipient. Examination of these satellite bands by agarose gel electrophoresis and electron microscopy demonstrated a common plasmid with a weight of 45 megadaltons. Novobiocin treatment of a resistant clinical isolate produced simultaneous loss of high-level resistance to streptomycin and kanamycin, and of resistance to penicillin-aminoglycoside synergism. These results suggest that (a) high-level resistance to streptomycin and kanamycin among some clinical isolates of enterococci is associated with a 45 megadalton plasmid, and (b) the same plasmid is also responsible for the resistance to penicillin-aminoglycoside synergism observed in these strains.

Resistance to gentamicin, tobramycin and amikacin among clinical isolates of bacteria.

Susceptibility to the administration of gentamicin, tobramycin and amikacin was determined for all isolates of aerobic and facultative gram-negative bacilli submitted for testing to the clinical bacteriology laboratory of the Massachusetts General Hospital between July 1, 1974, and June 30, 1976. In this 24-month period more than 46,000 isolates of bacteria were tested by the single-disc diffusion (Bauer-Kirby) method. Resistance to one or more of the aforementioned aminoglycosidic aminocyclitol antibiotics was found among 4,114 stains. Correlation with quantitative susceptibility test methods revealed that disc-diffusion methods using 10 microng discs accurately predicted resistance to gentamicin and tobramycin, but overestimated the prevalence of resistance to amikacin by 20 to 60%. Most of the gentamicin-resistant Enterobacteriaceae in this study were also cross-resistant to tobramycin but were susceptible to amikacin. Many gentamicin-resistant strains of Ps. aeruginosa were susceptible to both tobramycin and amikacin. Resistance to amikacin tended to be of relatively low magnitude (most had minimal inhibitory concentrations (MIC's) between 31 and 125 microng/ml), but organisms which were resistant to the administration of amikacin were usually resistant to the other two aminoglycosidic antibiotics as well.

In vitro activity of trimethoprim alone compared with trimethoprim-sulfamethoxazole and other antimicrobials against bacterial species associated with upper respiratory tract infections.

Trimethoprim-sulfamethoxazole has been used to treat various respiratory tract infections. Nevertheless, for many patients, intolerance of the sulfonamide component precludes use of this combination. This study examined the activity of trimethoprim alone in comparison to that of trimethoprim-sulfamethoxazole and other antimicrobials against bacterial species implicated in respiratory tract infections. For Haemophilus influenzae, minimal inhibitory concentrations of trimethoprim were equal to or one dilution greater than those of trimethoprim-sulfamethoxazole, with 56 of 58 strains inhibited by the former at < or = 0.25 microgram/ml. All oxacillin-susceptible Staphylococcus aureus and 96.7% of Streptococcus pyogenes were inhibited by trimethoprim < or = 2 micrograms/ml. In contrast, only 50% of Streptococcus pneumoniae were inhibited by this concentration of trimethoprim, whereas 93.3% were susceptible to the combination at < or = 2/38 micrograms/ml. All oxacillin-resistant S. aureus and all Moraxella catarrhalis were resistant to trimethoprim, although many of the former and all of the latter were susceptible to trimethoprim-sulfamethoxazole.

Comparative in vitro activity of levofloxacin and ofloxacin against gram-positive bacteria.

The in vitro activity of levofloxacin against 506 Gram-positive bacteria was compared with those of D(-)-ofloxacin, ofloxacin, ciprofloxacin, and sparfloxacin. Levofloxacin was generally twice as active as ofloxacin against these organisms (range, 0-3 twofold dilutions). Sparfloxacin appeared to have the greatest activity overall, but for several groups of organisms minimum inhibitory concentrations (MIC90s) of this compound were within one twofold dilution of those of levofloxacin. Resistance to levofloxacin (MIC > or = 8 micrograms/ml) was not encountered among streptococcal species, was rare in methicillin-susceptible staphylococci (1.7%), and was infrequent in vancomycin-susceptible Enterococcus faecalis and Enterococcus faecium (8.7%). Resistance was more common among vancomycin-resistant enterococci and methicillin-resistant staphylococci.

In vitro activity of BAY 12-8039, a new fluoroquinolone, against species representative of respiratory tract pathogens.

The in vitro antibacterial activity of BAY 12-8039, a novel 8-methoxy-quinolone, was compared with those of other quinolones, amoxicillin/clavulanate, cefuroxime and erythromycin against species commonly implicated in respiratory tract infections as well as viridans group streptococci. The new compound was highly active against methicillin-susceptible staphylococci (MIC90 0.125 microgram/ml), penicillin-susceptible and penicillin-resistant pneumococci (MIC90 0.5 and MIC50 0.25 microgram/ml, respectively), penicillin-susceptible and penicillin-resistant viridans group streptococci (MIC90 0.5 and 0.25 microgram/ml, respectively), group A streptococci (MIC90 0.25 microgram/ml), M. catarrhalis (MIC90 0.125 microgram/ml) and H. influenzae (MIC90 0.063 microgram/ml), irrespective of beta-lactamase production. It was, however, less active against methicillin-resistant staphylococci (MIC50 and MIC90, 2 and 4 micrograms/ml, respectively). The new compound demonstrated bactericidal activity at concentrations 2, 4, 8 times the MIC against representative isolates of the above collection. At a concentration of eight times the MIC, the frequency of spontaneous resistance ranged from 2.5 x 10(-7) to < 4 x 10(-8). These results suggested that BAY 12-8039 would be a promising agent for the eradication of respiratory tract pathogens and that clinical trials assessing its efficacy for the management of infections caused by these organisms are warranted.

In vitro activity of WY-49605, a penem antimicrobial.

The in vitro activity of the penem antimicrobial WY-49605 was compared with those of other agents available for oral administration. Based on concentrations inhibiting 90% of isolates (MIC(90)s), the penem inhibited methicillin-susceptible staphylococci (MIC(90) = 0.25 microg/ml), penicillin-susceptible streptococci (MIC(90) < or = 0.12 microg/ml) and several other Gram-positive genera at concentrations comparable or superior to the most active comparison agents. WY-49605 and cefpodoxime were the most active agents against members of the family Enterobacteriaceae. Most strains of Enterococcus faecalis and Bacteroides fragilis were susceptible to the new agent at concentrations < or =4microg/ml, while Pseudomonas aeruginosa, Enterococcus faecium, and methicillin-resistant Staphylococcus aureus were resistant to all agents tested.

In vitro activity of an oral streptogramin antimicrobial, XRP2868, against gram-positive bacteria.

The comparative in vitro potency of XRP2868, a new oral semisynthetic streptogramin antibiotic, was evaluated against gram-positive bacteria. XRP2868 inhibited all staphylococci at < or = 1 microg/ml and all non-pneumococcal streptococci at < or = 0.25 microg/ml and was fourfold more potent than quinupristin-dalfopristin against Staphylococcus aureus and Enterococcus faecium.

Antimicrobial activity of quinupristin-dalfopristin combined with other antibiotics against vancomycin-resistant enterococci.

Interactions between quinupristin-dalfopristin and six other antimicrobials were examined by checkerboard arrays against 50 clinical isolates of vancomycin-resistant Enterococcus faecium selected to represent a range of susceptibilities to individual agents. Unequivocal synergistic or antagonistic interactions at clinically relevant concentrations were infrequently encountered when the streptogramin was combined with chloramphenicol, ampicillin, imipenem, vancomycin, or teicoplanin. Combinations with doxycycline resulted in synergistic inhibition in 36% of checkerboards. Against 10 strains of Enterococcus faecalis, synergistic interactions were found when quinupristin-dalfopristin was combined with doxycycline (four strains), either glycopeptide (three strains), or ampicillin (two strains). Combination with quinupristin-dalfopristin increased the ampicillin MIC from 1 to 4 microg/ml for one strain. For 10 strains of E. faecium, interactions were also assessed by time-kill methods using concentrations of the agents attainable in human serum. Most of these antimicrobials augmented killing by quinupristin-dalfopristin to a minor degree. Against 2 of the 12 strains in this collection that were not highly resistant to gentamicin, the combination of quinupristin-dalfopristin (2 microg/ml) plus gentamicin (5 microg/ml) resulted in killing approaching 3 log(10) CFU/ml. With the exception of doxycycline, inhibitory interactions between quinupristin-dalfopristin and other agents tested against vancomycin-resistant strains of E. faecium were uncommon at clinically relevant concentrations.

Therapeutic potential of rifampin in enterococcal infections.

Rifampin is active against enterococci in vitro; virtually all clinical isolates are inhibited by concentrations of less than or equal to 16 micrograms/ml. However, rifampin is bacteriostatic, not bactericidal, and resistance emerges rapidly when the drug is employed alone against enterococci in vitro. The combination of rifampin with beta-lactam drugs, aminoglycosides, or vancomycin generally gives indifferent results. Limited data from studies done in vivo are contradictory and do not document unequivocally that rifampin is strikingly active, even in combination with other agents, against enterococcal infections. Whether rifampin will ever play a role in therapy for enterococcal infections in humans remains to be determined.

Emergence of gentamicin-resistant bacteria: experience with tobramycin therapy of infections due to gentamicin-resistant organisms.

A computerized system for testing and surveillance of bacterial susceptibility to antibiotics was used in monitoring the emergence of gentamicin-resistant strains of aerobic and facultative gram-negative bacilli at Massachusetts General Hospital since the release of gentamicin for clinical use in 1971. During the period studied, there was a significant increase in the prevalence of gentamicin-resistant bacteria, particularly among Pseudomonas, Acinetobacter (Herellea), and Proteus and, more recently, among Enterobacter and Klebsiella. Most gentamicin-resistant strains of Pseudomonas aeruginosa and Acinetobacter calcoaceticus var. anitratum (Herellea varginicola) retained susceptibility to tobramycin. Of the other gentamicin-resistant organisms, most were also resistant to tobramycin. Twelve patients with infections caused by gentamicin-resistant organisms were treated with tobramycin. All 12 patients were seriously ill, and all but one had failed to respond to previous therapy with gentamicin. Nine patients responded favorably to tobramycin, and six were cured. P. aeruginosa and A. calcoaceticus var. anitratum were most frequently the infecting organisms in these patients. Patients received tobramycin for three to 42 days; no significant drug-related toxicity was noted. These results emphasize the increasing clinical importance of gentamicin-resistant bacteria and suggest that tobramycin may be effective for treatment of some, but not all, infections caused by gentamicin-resistant bacteria.

Comparative synergistic activity of nafcillin, oxacillin, and methicillin in combination with gentamicin against.

The effectiveness of three semisynthetic, penicillinase-resistant penicillins alone and in combination with gentamicin was tested against 29 clinical isolates of enterococci. The minimal inhibitory concentrations of nafcillin were considerably lower than those of oxacillin and methicillin but were slightly higher than those of penicillin. At clinically achievable concentrations, the combination of nafcillin plus gentamicin produced enhanced killing against 13 of 14 strains of enterococci and was synergistic (by very rigid criteria) against 10 of 14 strains. In contrast, combinations of oxacillin plus gentamicin were synergistic against only 3 of 14 strains, and methicillin plus gentamicin produced synergistic killing against only 1 of 14 strains.

In vitro activity of the new oxazolidinone AZD2563 against Enterococci.

The activity of a new oxazolidinone antimicrobial, AZD2563, was assessed against >500 clinical isolates of enterococci representing six species. All isolates, including those resistant to other antibiotic classes, were inhibited by AZD2563 at concentrations

In vitro activities of the glycylcycline GAR-936 against gram-positive bacteria.

The in vitro activities of GAR-936, the 9-t-butylglycylamido derivative of minocycline, were compared with those of doxycycline, minocycline, and tetracycline against 527 gram-positive clinical isolates. GAR-936 inhibited all strains, including those resistant to other tetracyclines, at concentrations of

Detection of enterococcal high-level aminoglycoside resistance with MicroScan freeze-dried panels containing newly modified medium and Vitek Gram-Positive Susceptibility cards.

Both conventional and modified MicroScan Type 5 panels and Vitek Gram-Positive Susceptibility cards were compared with agar dilution screen plates for their abilities to detect high-level resistance to gentamicin and streptomycin in 235 enterococcal isolates, including 167 Enterococcus faecalis and 63 E. faecium isolates. The modified Type 5 panels contained dextrose-phosphate broth instead of Mueller-Hinton broth in their high-level-resistance screen wells. The sensitivities for detection of gentamicin and streptomycin high-level resistance were 100 and 100% (E. faecalis) and 100 and 94% (E. faecium) for the modified MicroScan panels, 100 and 89% (E. faecalis) and 100 and 98% (E. faecium) for the conventional MicroScan panels, and 81 and 86% (E. faecalis) and 85 and 94% (E. faecium) for the Vitek cards. All specificities were 100% except for the Vitek cards with streptomycin, where it was 96%. Isolates that showed resistance on the streptomycin agar screen plates were rescreened on plates containing 32,000 micrograms/ml to detect ribosomally mediated resistance. For all three systems, every failure to detect streptomycin high-level resistance occurred in isolates with enzymatic, not ribosomal, resistance. The modified MicroScan Type 5 panels are a suitable method for detecting enterococcal high-level resistance to gentamicin and streptomycin. The Vitek cards are too insensitive for this purpose.

Penicillin-tobramycin synergism against enterococci: a comparison with penicillin and gentamicin.

Combinations of penicillin plus tobramycin have been compared with penicillin plus gentamicin against 27 strains of enterococci isolated from blood cultures. Penicillin plus gentamicin was synergistic against all strains. The combination of penicillin plus tobramycin was equally effective against strains of Streptococcus facalis, but was ineffective against all four strains of S. faecium tested.

Antimicrobial susceptibility of flavobacteria.

Antimicrobial susceptibility patterns of 28 clinical isolates of Flavobacterium sp. were determined by standard disk diffusion technique and by antimicrobial dilution in agar. Rifampin, clindamycin, trimethoprim-sulfamethoxazole, cefoxitin, and vancomycin are among the antimicrobial agents which may be clinically useful to treat infections caused by flavobacteria. All 28 isolates were resistant to erythromycin with minimal inhibitory concentrations of 32 mug/ml or more. Currently recommended interpretive zones of inhibition by disk diffusion did not reliably predict antimicrobial susceptibility of the 28 flavobacteria isolates when compared with the agar dilution technique, and, therefore, a more direct measurement of minimal inhibitory or bactericidal concentration is recommended.

Resistance to beta-lactam antibiotics in Streptococcus faecium.

Clinical isolates of Streptococcus faecium are characteristically resistant to beta-lactam antibiotics. Two strains, selected for hypersusceptibility to penicillin, were derived from normally resistant isolates treated with novobiocin. These strains were also found to be hypersusceptible to other beta-lactams. Differences in beta-lactam susceptibility between the original isolates and the hypersusceptible strains could not be attributed to alterations in penicillin-binding protein affinities, and no evidence of a relative permeability barrier was found in the resistant strains. Isolated cell membranes prepared from resistant strains were found to possess two protein bands which were absent or greatly diminished in the membranes of susceptible strains. Hypersusceptibility to beta-lactam antibiotics in these strains may be due to the absence or alteration of one or more cell membrane proteins distinct from the penicillin-binding proteins of these organisms.

Resistance to antibiotic synergism in Streptococcus faecalis: further studies with amikacin and with a new amikacin derivative, 4'-deoxy, 6'-N-methylamikacin.

Streptococcus faecalis strains may resist penicillin-aminoglycoside synergy by the production of plasmid-mediated aminoglycoside-modifying enzymes. One of these enzymes, aminoglycoside 3'-phosphotransferase, has been shown to have a broad range of substrate specificity, including amikacin. We have studied a derivative of amikacin, 4'-deoxy, 6'-N-methylamikacin (BB-K311), against 11 clinical blood isolates of S. faecalis. Minimal inhibitory concentrations of BB-K311 were quite similar to those of amikacin, ranging from 125 to 1,000 micrograms/ml. In assays for antibiotic synergy, penicillin and amikacin produced enhanced killing compared with the penicillin alone only against those three strains which lacked the phosphotransferase enzyme. The other eight enzyme-positive strains actually demonstrated significant antagonism between penicillin against all 11 strains, regardless of enzyme production. Analysis of substrate profiles with crude preparations of the aminoglycoside 3'-phosphotransferase enzyme confirmed that BB-K311 was a very poor substrate for modification, as expected from the synergy studies. Use of other aminoglycoside analogs confirmed the 3'-OH site of modification. These findings suggest that removing the 4'-OH group in amikacin effectively blocks 3'-phosphorylation by S. faecalis enzyme.

Ribosomal resistance of clinical enterococcal to streptomycin isolates.

The mechanism of high-level resistance to streptomycin was studied in 12 clinical isolates of Streptococcus faecalis. Six strains produced streptomycin-modifying enzymes. Each of three enzyme-negative strains tested demonstrated ribosomal resistance to streptomycin. Lack of ribosomal susceptibility is a significant cause of high-level streptomycin resistance among clinical enterococcal isolates.