Antimicrobial Susceptibility and Selection of Resistance Among Staphylococcus epidermidis Isolates Recovered from Patients with Infections of Indwelling Foreign Devices

Twenty-seven isolates of Staphylococcus epidermidis from patients with prosthetic valve endocarditis or infected cerebrospinal fluid shunts were examined for susceptibility to antimicrobial agents. Subpopulations resistant to 20 and 100 μg of methicillin per ml were present in 63% of the isolates (methicillin-resistant isolates). Subpopulations resistant to 20 μg of nafcillin and cephalothin per ml were found in every methicillin-resistant isolate but with frequencies (10^{−5.0 ± 0.5} and 10^{−6.4 ± 0.9}, respectively) which were not always detectable by susceptibility testing. Resistance to ≥1.6 μg of penicillin per ml was found in 80% of isolates. Cephalothin, cefazolin, and cefamandole were more active than cefoxitin or cephradine, and gentamicin was more active than tobramycin or amikacin; rifampin was the single most active agent against all isolates. There was no difference in susceptibility between prosthetic valve endocarditis and cerebrospinal fluid shunt infection isolates. Among methicillin-resistant isolates, the phenotypic expression of resistance to methicillin or nafcillin but not to cephalothin could be enhanced by 48 h of incubation with each drug. Isolates containing no methicillin-resistant subpopulations were killed by incubation with methicillin, nafcillin, or cephalothin. High-level resistance to rifampin emerged in both methicillin-resistant and methicillin-sensitive isolates after 8 to 24 h of incubation with this drug. The presence or absence of antibiotic-resistant subpopulations among S. epidermidis isolates and their selection during treatment should be considered when therapy is devised.     

Activity of ten cephalosporins on biomass of methicillin-susceptible and -resistant Staphylococcus aureus

The growth curves automatically recorded and printed during the action of 10 cephalosporins on methicillin-susceptible and methicillin-resistant Staphylococcus aureus showed the following. (i) The biomass of methicillin-susceptible S. aureus exposed to the cephalosporins increased before lysis occurred (inoculum, 10⁶ colony-forming units per ml). Lysis was more rapid with cephalothin and cephaloridine, whose minimal inhibitory concentrations were lowest. (ii) The same biomass increase followed by lysis occurred with methicillin-resistant S. aureus, and the speed of lysis was not different from those of cephalothin (without any regrowth), cefoxitin (with regrowth of a few strains), and cephaloridine (regrowth of all strains), with methicillin-susceptible strains. A 2-log increase of inoculum (10⁸ colony-forming units per ml) did not modify significantly the speed of lysis with cephalothin, cephaloridine, and cefoxitin, but regrowth sometimes occurred. The early transitory lysis caused by cephaloridine, cephalothin, cefamandole, and cefoxitin was not suppressed by preincubation with 32 μg of methicillin per ml, but regrowth occurred more frequently. No lysis could be observed with cefazolin, cefotaxime, cephalexin, cephradine, cefuroxime, and cefaclor unless high concentrations were achieved. (iii) From a practical point of view, the early response of the growth curve (4 h) could not determine in every case whether a strain of S. aureus was resistant or susceptible to cephalosporin. A further study of the growth curve (18 of 24 h) was necessary for this purpose. Results obtained after a few hours with automated systems should be interpreted with great caution.     

Comparative Susceptibility of Haemophilus Species to Cefaclor, Cefamandole, and Five Other Cephalosporins and Ampicillin, Chloramphenicol, and Tetracycline

The minimal inhibitory concentration of cefaclor, cephalexin, cephradine, cefamandole, cephalothin, cephapirin, cefazolin, ampicillin, chloramphenicol, and tetracycline for inhibition of 198 freshly isolated clinical strains of Haemophilus species (23 H. influenzae type b, 157 H. influenzae non-type b, 14 H. parainfluenzae, and 4 H. aphrophilus) was determined simultaneously by a slightly modified WHO-ICS agar dilution method. Nine strains were resistant to ampicillin. There was no correlation between ampicillin resistance and minimal inhibitory concentration of other antibiotics. All strains were susceptible to chloramphenicol, and all except five were susceptible to tetracycline. Cefaclor was the most active oral cephalosporin, and cefamandole was the most active parenteral cephalosporin. Among the seven cephalosporins tested, cefamandole was the most effective compound. All but two strains were inhibited by cefamandole at 2 mug or less per ml.     

Antistaphylococcal activity of amoxicillin and ticarcillin when combined with clavulanic acid. Evaluation of oxacillin-resistant and oxacillin-susceptible isolates

The beta-lactamase inhibitor, clavulanic acid, was combined with amoxicillin and with ticarcillin for in vitro studies with 586 staphylococci: 97 stock cultures of oxacillin-resistant strains recovered before 1982, and 489 blood or wound isolates collected from 40 separate medical centers during 1987-1988 (300 were oxacillin resistant). Over 92% of the staphylococci produced beta-lactamase enzymes and were thus resistant to both penicillins. However, with the addition of clavulanic acid, oxacillin-susceptible strains were rendered susceptible to low concentrations of amoxicillin and ticarcillin. Staphylococcus aureus strains with borderline or partial borderline resistance to penicillinase-resistant penicillins occurred infrequently (72 of 325 S. aureus isolates). Those strains were susceptible to both clavulanic acid combinations, because their methicillin resistance is thought to be due to an excess beta-lactamase production. Strains with chromosomally mediated intrinsic heteroresistance were relatively resistant to both drug combinations. Minimal inhibitory concentration (MIC) breakpoints that best separated those heteroresistant strains from oxacillin-susceptible isolates were as follows: amoxicillin/clavulanic acid, less than or equal to 2.0/1.0 micrograms/ml for susceptible; and ticarcillin/clavulanic acid, less than or equal to 4.0/2.0 micrograms/ml for susceptible. When the broth was supplemented by 2% NaCl, MICs for both drug combinations were increased by less than one doubling dilution. Although oxacillin and methicillin broth microdilution tests were more reliable when 2% NaCl was added, tests with the two drug combinations were only minimally improved by adding 2% NaCl to the broth medium.     

Mechanism of action of cephalosporins and resistance caused by decreased affinity for penicillin-binding proteins in Bacteroides fragilis.

The susceptibilities of 52 clinical isolates of Bacteroides fragilis to five monoanionic cephalosporins were examined. Cefoperazone showed the highest antibacterial activity, followed by ceftezole, cefazolin, cefamandole, and cephalothin. There were two groups of resistant strains: one group (ca. 15%), of which B. fragilis G-232 was a typical sample, was resistant to ceftezole (MIC, 100 micrograms/ml), cefazolin (MIC, 100 micrograms/ml), and cephalothin (MIC, 200 micrograms/ml) but not cefoperazone (MIC, 6.25 micrograms/ml) or cefamandole (MIC, 25 micrograms/ml). On the basis of studies of stability to beta-lactamase, outer membrane permeation, and affinity for penicillin-binding proteins (PBPs), we conclude that decreased affinity for PBP 3 may play an important role in the resistance to ceftezole, cefazolin, and cephalothin in B. fragilis G-232. Another group (also ca. 15%), of which B. fragilis G-242 was a representative, was resistant to all five cephalosporins (MIC, 100 to 400 micrograms/ml) and produced a high amount of beta-lactamase. Similar broad-spectrum resistance was seen in a mutant of strain G-232 that had a greater-than-30-fold increase in beta-lactamase production.     

Susceptibility testing of methicillin-resistant Staphylococcus aureus with three commercial microdilution systems

The antimicrobial susceptibilities of 100 methicillin-resistant Staphylococcus aureus isolates were determined concurrently by API Uniscept KB, Micro-Media, MicroScan, standardized disk diffusion, and reference broth microdilution to evaluate whether these commercial microdilution systems would reliably defect methicillin-resistant S. aureus. The methicillin minimal inhibitory concentration for all isolates was greater than or equal to 16 micrograms/ml as determined by the reference minimal inhibitory concentration panels containing 2% NaCl supplemented Mueller-Hinton broth. Using the breakpoints established by the National Committee for Clinical Laboratory Standards for reporting susceptible and resistant methicillin results, there was 100% agreement between the reference methods and API Uniscept KB at 24 hr. The Micro-Media and MicroScan systems had 47% and 8% very major discrepancies at 24 hr, respectively. At 48 hr, these two systems exhibited 15% and 0% very major discrepancies. Micro-Media and MicroScan were in agreement with the reference microdilution method (+/- 1 log2 dilution) for 62% and 68% of the strains at 24 hr, respectively and 88% and 85% of the isolates at 48 hr, respectively. The results of this study indicate that API Uniscept KB would provide a practical and reliable method for the detection of methicillin-resistant S. aureus.     

Comparative activity of CGP 31608, nafcillin, cefamandole, imipenem, and vancomycin against methicillin-susceptible and methicillin-resistant staphylococci.

The activity of CGP 31608 for 53 strains of Staphylococcus aureus and 48 strains of S. epidermidis, both methicillin susceptible and resistant, was compared with that of vancomycin, nafcillin, cefamandole, and imipenem. Microdilution MICs were determined in Mueller-Hinton broth with or without 2.5% NaCl at an inoculum of 3 x 10(5) CFU/ml with a 20-h, 37 degrees C incubation. The MICs of imipenem and CGP 31608 for methicillin-resistant strains were lower than the MICs of nafcillin or cefamandole for these strains; these differences diminished in the presence of 2.5% NaCl. Subpopulations were detected in strains of methicillin-resistant S. aureus and S. epidermidis that were resistant to all the beta-lactam antibiotics tested at 5 micrograms/ml. This resistant subpopulation produced progeny that were uniformly resistant to high concentrations of each of the beta-lactams.     

Bactericidal action of nafcillin, vancomycin, and three cephalosporins against nafcillin-susceptible and nafcillin-resistant coagulase-negative staphylococci

Coagulase-negative staphylococci (S. epidermidis, 43 strains; S. warneri, 16 strains; S. haemolyticus, five strains; and others, four strains) were tested by the agar dilution method for nafcillin susceptibility: 53 were susceptible with a minimal inhibitory concentration (MIC) of less than or equal to 2 micrograms/ml; four were of indeterminate susceptibility, MIC = 4-16 micrograms/ml; and 11 were resistant, MIC greater than or equal to 32 micrograms/ml. The bactericidal activities from 0 to 24 hr for nafcillin, vancomycin, cephalothin, cefazolin, and cefamandole, each at 16 micrograms/ml in broth, were determined for all the isolates. The data indicate that a nafcillin agar dilution susceptibility test result of resistance does not consistently predict lack of killing activity by the cephalosporins. It is likely that each cephalosporin would have to be tested against individual coagulase-negative staphylococci in order to determine a suitable therapeutic or prophylactic cephalosporin, if a cephalosporin were to be used. Vancomycin was bactericidal for all the nafcillin-resistant coagulase-negative organisms tested.     

In vitro susceptibility of Streptococcus bovis to six antibiotics

A recent report of two isolates of Streptococcus bovis resistant to killing by low levels of penicillin G prompted this study of the in vitro susceptibility of 100 clinical isolates of S bovis to six antibiotics. Using a microdilution method, we found the minimum bactericidal concentrations for 99% of the isolates to be as follows: penicillin G, 0.5 microgram/ml; methicillin, 16 micrograms/ml; ampicillin, 0.5 microgram/ml; cephalothin, 2 micrograms/ml; clindamycin, 2 micrograms/ml; and vancomycin, 2 micrograms/ml. The high-level resistance of S bovis to penicillin G previously described was not seen. This study suggests that penicillin G can still prove effective in treating most infections caused by S bovis, including infective endocarditis, and that cephalothin and vancomycin are useful alternative agents.     

Clinical isolates of Streptococcus pneumoniae with different susceptibilities to ceftriaxone and cefotaxime

Ceftriaxone and cefotaxime are extended-spectrum cephalosporins previously demonstrated to possess very similar in vitro activities against Streptococcus pneumoniae. Anecdotal reports of isolates with divergent in vitro susceptibilities to ceftriaxone and cefotaxime have been published. To determine the prevalence of pneumococcal isolates with divergent ceftriaxone and cefotaxime susceptibilities, we tested 1,000 clinical isolates collected by U.S. laboratories in 2001-2002 by broth microdilution and E-test. The percentages of isolates susceptible to ceftriaxone and cefotaxime were significantly different by both broth microdilution (98.6 and 96.6%, respectively; P < 0.05) and E-test (98.3 and 95.8%; P < 0.001). The differences observed were due solely to the activities of the two agents against penicillin-resistant isolates. Twenty-six of 188 penicillin-resistant isolates (13.8%) demonstrated different ceftriaxone and cefotaxime MIC interpretative phenotypes when tested by broth microdilution; 18 isolates were concurrently ceftriaxone susceptible and cefotaxime intermediate, 6 were ceftriaxone intermediate and cefotaxime resistant, and 2 were ceftriaxone susceptible and cefotaxime resistant (1.1% of penicillin-resistant isolates; 0.2% of all isolates tested). Sixteen of the 26 isolates (65%) were from southern U.S. states. The 26 isolates had serogroups and serotypes (6, 9, 14, 19, and 23) commonly associated with penicillin-resistant isolates; SmaI pulsed-field gel electrophoresis identified 18 isolates (69%) dispersed among five subtype groups and 8 isolates that were unrelated to any of the other isolates. We conclude that certain isolates of penicillin-resistant pneumococci are less susceptible to cefotaxime than to ceftriaxone and that these isolates are not the result of the spread of a single clone. Whether such isolates have increased in prevalence over time remains unknown.     

Antibiotic-resistant Staphylococcus epidermidis in patients undergoing cardiac surgery.

Staphylococcus epidermidis isolates containing subpopulations resistant to 100 microgram of methicillin per ml were found on the chests of only 3 of 80 (4%) patients before cardiac surgery, whereas these highly resistant staphylococci were isolated from the chest wounds of 43 of 80 (54%) patients 5 days postoperatively. The percentage of patients colonized with methicillin-resistant S. epidermidis increased with time postoperatively. Methicillin-resistant postoperative isolates also contained organisms resistant to other antibiotics frequently used during these patients' hospitalizations. The percentages of patients with organisms resistant to various antibiotics were: nafcillin (100%), penicillin (100%), cephalothin (93%), cefamandole (80%), streptomycin (67%), and gentamicin (20%). Preoperative methicillin-susceptible isolates were generally susceptible to other antibiotics. Two patients with S. epidermidis prosthetic valve endocariditis caused by multiple antibiotic-resistant isolates were among the study patients. Antibiotic susceptibility patterns of each isolate from these two patients were identical to those of postoperative chest isolates from the same patient.     

Susceptibility and Synergy Studies of Methicillin-Resistant Staphylococcus epidermidis

Methicillin-resistant Staphylococcus epidermidis is an important cause of cerebrospinal fluid shunt infections and prosthetic valve endocarditis. Agar dilution minimum inhibitory concentrations were determined for 100 strains of methicillin-resistant S. epidermidis which were isolated from clinical specimens. Vancomycin inhibited all 100 strains at /=3.12 mug/ml. Of the five cephalosporins and one cephamycin tested, cefamandole was the most active in vitro, inhibiting 97 strains at /=10(2) kill by the combination over the most effective single antibiotic at 24 h) was demonstrated with vancomycin (1.56 mug/ml) plus cefamandole (6.25 mug/ml) in 14 of 14 strains, vancomycin plus cephalothin (6.25 mug/ml) in 14 of 14 strains, vancomycin plus rifampin (0.008 to 0.012 mug/ml) in 6 of 12 strains, rifampin plus cefamandole in 9 of 12 strains, and rifampin plus cephalothin in 10 of 12 strains. The emergence of populations of bacteria resistant to 0.2 mug of rifampin per ml developed in three of five methicillin-resistant S. epidermidis strains tested. The addition of either vancomycin, cephalothin, or cefamandole to the rifampin prevented the emergence of resistance in these three strains. Clinical trials of synergistic antibiotic combination therapy for serious methicillin-resistant S. epidermidis infections are indicated.     

Ciprofloxacin-resistant methicillin-resistant Staphylococcus aureus in an acute-care hospital.

Use of ciprofloxacin as an alternative to vancomycin for treatment of methicillin-resistant Staphylococcus aureus infection has been paralleled by the emergence of resistant strains. This phenomenon has also been noticed in our hospital. To confirm our observation, methicillin and ciprofloxacin susceptibilities were tested by disk diffusion and broth microdilution techniques. We studied 83 methicillin-resistant Staphylococcus aureus isolates obtained from various sources over a 4-month period. Ciprofloxacin resistance (MIC, greater than 2 micrograms/ml) was detected in 69 isolates (83%). Prior use of ciprofloxacin was reported for 24 of 69 patients with ciprofloxacin-resistant strains and 0 of 14 patients with ciprofloxacin-susceptible strains. The day of detection during the hospital stay and the location of the source patient were not significantly different between resistant and susceptible strains. Bacteriophage typing showed a higher occurrence of nontypeable strains among ciprofloxacin-resistant strains (54%). Review of our microbiology register showed a progressive increase in the rate of resistance to ciprofloxacin during the first year of use, with initial rates being about 10% and recent rates being higher than 80%. On the other hand, methicillin-susceptible S. aureus remained uniformly susceptible to ciprofloxacin (98.4%). We conclude that prior use of ciprofloxacin is an important factor for the selection of ciprofloxacin-resistant strains and that ciprofloxacin has limited usefulness against methicillin-resistant S. aureus.     

Species dependent variability in the susceptibility of coagulase-negative staphylococci to various antimicrobial agents

The in-vitro activity of 20 beta-lactam antibiotics and vancomycin was determined against three different species of coagulase-negative-staphylococci. All isolates were susceptible to vancomycin (MIC90 3.12-6.25 mg/l) although occasional tolerance was seen. Most isolates (85%) of Staphylococcus hominis were susceptible to methicillin and other beta-lactams including cephalothin, cefapirin, cefamandole, imipenem and BMY28142, while 90% of Staph. haemolyticus isolates were resistant to these agents. Staph. epidermidis was intermediate in susceptibility with 50% of isolates being methicillin resistant (MR). These MR isolates exhibited cross-resistance or tolerance to the other beta-lactam agents except cefamandole.     

Species differences in susceptibilities of Proteeae spp. to six cephalosporins and three aminoglycosides

Six cephalosporins and three aminoglycosides were examined for activity against 1,693 isolates belonging to six species of Proteeae. The most notable species-specific differences included the marked susceptibility of Providencia alcalifaciens and Proteus mirabilis to cephalothin, the resistance of Proteus vulgaris to cefamandole, and the resistance of Providencia stuartii to gentamicin and tobramycin. The third-generation cephalosporins cefotaxime and moxalactam were substantially more inhibitory than were cefoperazone, cefamandole, and cefoxitin. P. stuartii, generally the most resistant species, was, however, markedly susceptible to moxalactam and cefotaxime.     

Antibiotic Therapy of Experimental Staphylococcus epidermidis Endocarditis

Endocarditis was produced in rabbits with a methicillin-resistant Staphylococcus epidermidis isolate. Subpopulations resistant to other semisynthetic penicillins and cephalosporins were detected in the isolate. Their presence was probably responsible for the increase in minimum bactericidal concentrations and minimum inhibitory concentrations when tests with high inocula, rather than low inocula were pursued. Rabbits were treated for either 2 or 7 days with nafcillin, cephalothin, cefamandole, vancomycin, rifampin, or gentamicin. Spontaneous death was uncommon in either controls (84% survival) or treated animals (80 to 94% survival). There was no significant difference in the number of bacteria in vegetations of rabbits treated for 7 days with cephalothin, cefamandole, nafcillin, or no antibiotic (control). There was a significant reduction in total bacteria in vegetations of rabbits given vancomycin, gentamicin, or rifampin for 7 days as compared with cephalothin, cefamandole, nafcillin or control. Gentamicin or rifampin sterilized significantly more vegetations after 7 days than cephalothin, cefamandole, nafcillin, or control; rifampin was more effective in sterilizing vegetations than either gentamycin or vancomycin after 2 days. Mutants resistant to 10 μg of rifampin per ml comprised the total bacterial population cultured from vegetations of 2 of 17 rabbits treated with this antibiotic for 7 days; there was no change in the susceptibility of vegetation isolates to other antibiotics. Rifampin, vancomycin, or gentamicin may prove to be more effective in humans than cephalosporins or semisynthetic penicillins in the treatment of methicillin-resistant S. epidermidis endocarditis.     

Susceptibility of multiply resistant Haemophilus influenzae to newer antimicrobial agents

One hundred and six isolates of Haemophilus influenzae from a national antimicrobial surveillance study demonstrated resistance to two or more of 10 primary antimicrobial agents by mechanisms other than or in addition to beta-lactamase. Of particular note were strains multiply resistant to ampicillin (by beta-lactamase production), chloramphenicol, trimethoprim/sulfamethoxazole, and tetracycline in various combinations. All of the aforementioned strains were shown to be highly susceptible to amoxicillin/clavulanate, the second generation cephalosporins cefuroxime and cefonicid, and the third generation cephalosporins cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, moxalactam, and cefixime. However, 68 strains that demonstrated resistance or marginal susceptibility (MIC greater than or equal to 2 micrograms/ml) to ampicillin by mechanisms other than beta-lactamase, also demonstrated reduced susceptibility to amoxicillin/clavulanate (MICs up to 8 micrograms/ml) and the second generation cephalosporins (MICs up to 32 micrograms/ml). While the latter strains were susceptible to the third generation cephalosporins, MICs were often 10-fold higher than MICs of ampicillin susceptible isolates or of beta-lactamase producing isolates. All of the multiply antimicrobial-resistant strains were highly susceptible (MIC less than or equal to 0.25 micrograms/ml) to the two quinolones ciprofloxacin and pefloxacin.     

Susceptibility of Pseudomonas paucimobilis to 24 antimicrobial agents.

Pseudomonas paucimobilis (group IIK, biotype 1) clinical isolates showed in vitro resistance to ampicillin, carbenicillin, cephalothin, cefoxitin, cefamandole, moxalactam, cefotaxime, cefoperazone, mezlocillin, azlocillin, piperacillin, and ticarcillin. Those agents to which the microbes were shown to be susceptible were tetracycline, chloramphenicol, gentamicin, tobramycin, kanamycin, amikacin, netilmicin, sisomicin, trimethoprim-sulfamethoxazole, ceftazidime, ceftriaxone, and ceftizoxime.     

Methicillin-Resistant Staphylococcus aureus Susceptibility Testing by an Automated System, Autobac I

The Autobac I system was used to evaluate the antibiotic susceptibility pattern of methicillin-resistant Staphylococcus aureus isolates. The results of the Autobac I were compared with the results of the disk diffusion method. The disk diffusion susceptibility pattern showed resistance to methicillin/oxacillin, penicillin, erythromycin, clindamycin, and kanamycin. All isolates were susceptible to cephalothin, chloramphenicol, tetracycline, and gentamicin. There was at least 96% agreement using the Autobac I system with all antibiotics except methicillin and clindamycin. Seventy percent of 57 isolates were recorded as susceptible to methicillin, whereas 9% had an intermediate susceptibility. With clindamycin, 14% were recorded as susceptible and 7% were recorded as intermediate. Upon prolonged incubation of the Autobac I cuvette, the agreement between the two methods was 44% for methicillin and 93% for clindamycin. Changes in the environmental conditions, such as use of 5% sodium chloride broth and a 32 degrees C incubation temperature, did not increase the detection of methicillin-resistant isolates by the Autobac I system.     

The in vitro spectrum of the cephalosporins.

The cephalosporins may currently be classified according to their relative susceptibility to beta-lactamases. Cefoxitin, cefamandole, cefatrizine, and cephanone are relatively resistant to the gram-negative beta-lactamases, whereas cephalothin, cefamandole, and cefoxitin are resistant to staphylococcal beta-lactamase. Although the inhibitory activity of cephalothin is representative of that of cephaloridine, cephalexin, cefazolin, cephapirin, cephacetrile, and cephradine, there are significant differences between its activity and that of cefoxitin, cefamandole, and cefatrizine, especially against Enterobacter, Serratia, indole-positive Proteeae, Bacteroides fragilis, and ampicillin-resistant Haemophilus influenzae.