Marked Inoculum Effect Research Articles

Efficacy of Ceftaroline against Methicillin-Susceptible Staphylococcus aureus Exhibiting the Cefazolin High-Inoculum Effect in a Rat Model of Endocarditis.

Certain Staphylococcus aureus strains exhibit an inoculum effect (InE) with cefazolin (CFZ) that has been associated with therapeutic failures in high-inoculum infections. We assessed the in vitro activities of ceftaroline (CPT), CFZ, and nafcillin (NAF) against 17 type A β-lactamase (βla)-producing, methicillin-susceptible S. aureus (MSSA) strains, including the previously reported TX0117, which exhibits the CFZ InE, and its βla-cured derivative, TX0117c. Additionally, we determined the pharmacokinetics of CPT in rats after single intramuscular doses of 20 and 40 mg/kg of body weight and evaluated the activities of CPT (40 mg/kg every 8 h [q8h]), CFZ, and NAF against TX0117 and TX0117c in a rat model of infective endocarditis. No InE was observed for CPT or NAF, whereas a marked InE was detected for CFZ (MIC, 8 to ≥128 μg/ml). CPT and NAF treatment against TX0117 resulted in mean bacterial counts of 2.3 and 2.1 log10 CFU/g in vegetations, respectively, compared to a mean of 5.9 log10 CFU/g in the CFZ-treated group (CPT and NAF versus CFZ, P = 0.001; CPT versus NAF, P = 0.9830). Both CFZ and CPT were efficacious against the βla-cured derivative, TX0117c, compared to time zero (t0) (P = <0.0001 and 0.0015, respectively). Our data reiterate the in vivo consequences of the CFZ InE and show that CPT is not affected by this phenomenon. CPT might be considered for high-inoculum infections caused by MSSA exhibiting the CFZ InE.

Effect of Resistance Mechanisms on the Inoculum Effect of Carbapenem in Klebsiella pneumoniae Isolates with Borderline Carbapenem Resistance.

We aimed to examine the effects of resistance mechanisms on several resistance phenotypes among carbapenem-resistant Klebsiella pneumoniae isolates with borderline carbapenem MICs. We compared carbapenemase-negative K. pneumoniae with carbapenemase-producing K. pneumoniae (CPKP) isolates with similar MICs. CPKP isolates exhibited a marked inoculum effect and were more resistant to the bactericidal effect of meropenem. This suggests that MIC measurements alone may not be sufficient in predicting the therapeutic efficacy of carbapenems against CPKP.

Treatment options for extended-spectrum beta-lactamase-producers.

A review of antibiotic options for the treatment of infections caused by extended-spectrum beta-lactamase-producing isolates is presented. The use of the third-generation cephalosporin, cefotaxime, for infections caused by isolates producing ceftazidimase-type extended-spectrum beta-lactamases is controversial, despite in vitro susceptibility to the antibiotic in many instances. The fourth-generation cephalosporin, cefipime, although active against most extended-spectrum beta-lactamases, is reported to show a marked inoculum effect. The cephamycins, such as cefoxitin. are generally effective against Enterobacteriaceae producing TEM- and SHV-derived extended-spectrum beta-lactamases, but Klebsella pneumoniae strains are prone to cephamycin resistance as a result of porin loss. The use of beta-lactamase inhibitor combinations is variable. Sulbactam is less effective than clavulanate for the inhibition of SHV-derived extended-spectrum beta-lactamases and a marked inoculum effect has been noted, while the efficacy of tazobactam against SHV-derived extended-spectrum beta-lactamase producers is controversial. Furthermore, extended-spectrum beta-lactamases are often encoded by multi-resistant plasmids carrying genes conferring resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim and other antimicrobials, severely limiting even alternative therapies. Extensive susceptibility testing before the institution of antibiotic therapy is thus vital.

Treatment options for extended-spectrum β-lactamase-producers

A review of antibiotic options for the treatment of infections caused by extended-spectrum β-lactamase-producing isolates is presented. The use of the third-generation cephalosporin, cefotaxime, for infections caused by isolates producing ceftazidimase-type extended-spectrum β-lactamases is controversial, despite in vitro susceptibility to the antibiotic in many instances. The fourth-generation cephalosporin, cefipime, although active against most extended-spectrum β-lactamases, is reported to show a marked inoculum effect. The cephamycins, such as cefoxitin, are generally effective against Enterobacteriaceae producing TEM- and SHV-derived extended-spectrum β-lactamases, but Klebsiella pneumoniae strains are prone to cephamycin resistance as a result of porin loss. The use of β-lactamase inhibitor combinations is variable. Sulbactam is less effective than clavulanate for the inhibition of SHV-derived extended-spectrum β-lactamases and a marked inoculum effect has been noted, while the efficacy of tazobactam against SHV-derived extended-spectrum β-lactamase producers is controversial. Furthermore, extended-spectrum β-lactamases are often encoded by multi-resistant plasmids carrying genes conferring resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim and other antimicrobials, severely limiting even alternative therapies. Extensive susceptibility testing before the institution of antibiotic therapy is thus vital.

Comparison of the in vitro activity of levofloxacin and other antimicrobial agents against vancomycin-susceptible and vancomycin resistant Enterococcus species

The in vitro activities of levofloxacin and other antibiotics against 133 clinical isolates of vancomycin-resistant and vancomycin-susceptible enterococci were evaluated. Only 14 (39%) of the vancomycin-susceptible isolates and 11 (11%) of the vancomycin-resistant isolates were susceptible to levofloxacin. Levofloxacin exhibited a marked inoculum effect for all enterococci tested. These results suggest that levofloxacin may be of limited use in the treatment of serious enterococcal infections.

Optimal times above MICs of ceftibuten and cefaclor in experimental intra-abdominal infections.

The duration of time that serum drug levels remain above the MIC (time above the MIC) for the pathogen has been shown to be the most significant parameter determining the efficacies of beta-lactam antibiotics. In the described study, we investigated the optimal time above the MIC of ceftibuten and cefaclor using a nonneutropenic mouse model of intra-abdominal infections caused by Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Streptococcus pneumoniae. The abilities of the drugs to protect mice against the organisms were determined in mouse protection tests, and the doses were fractionated to produce various dosing regimens with different times above the MIC. All drug-organism combinations showed a significant correlation (r > 0.9) between drug efficacy and the time above the MIC. Also, with ceftibuten treatment, the different dosing regimens that produced equal times above the MIC resulted in the same efficacy, whereas with cefaclor, an apparent dose-dependent effect was observed. These results showed that for a 100% recovery from K. pneumoniae and E. coli infections, the optimal times above the MIC with ceftibuten treatment were 2.2 and 1.6 h, respectively. Relatively high doses of both antibiotics were required to ensure recovery from S. pneumoniae infections. In vitro time-kill studies demonstrated that cefaclor exhibits a marked inoculum effect against the pathogens, and there was a concentration-dependent killing at a large inoculum size. On the other hand, ceftibuten showed no inoculum effect. It is suggested that optimization of both dose and time above the MIC appears to be necessary for the treatment of S. aureus infections with cefaclor, and this may apply to other beta-lactams tht exhibit marked inoculum effects.

Susceptibility survey of piperacillin alone and in the presence of tazobactam

Among 325 fresh isolates of bacteria from 153 hospital patients with serious infections, 142 were susceptible to piperacillin, 129 were resistant and a further 54 exhibited a marked inoculum effect. Tazobactam restored the susceptibility of resistant isolates of Staphylococcus aureus and Staphylococcus epidermidis (except methicillin-resistant isolates), Haemophilus influenzae, Moraxella catarrhalis, Acinetobacter spp. and Bacteroides fragilis. Among 87 Enterobacteriaceae that were resistant to piperacillin, or showed an inoculum effect, 78 (89%) were restored to susceptibility by tazobactam. Pseudomonas spp. resistant to piperacillin were mostly resistant to the combination.

Effect of inoculum size on bacteriolytic activity of cefminox and four other beta-lactam antibiotics against Escherichia coli.

MICs and turbidimetric experiments revealed a negligible inoculum effect with two Escherichia coli strains exposed to cefminox and cefoxitin, whereas a marked inoculum effect was revealed after exposure to cefotaxime, ceftizoxime, and imipenem. The activities of the cephamycins were associated with spheroplast formation and bacteriolysis at concentrations close to the MIC, whereas the other agents induced the formation of filaments or, in the case of imipenem, rounded cells.

Ceftriaxone-sulbactam combination in rabbit endocarditis caused by a strain of Klebsiella pneumoniae producing extended-broad-spectrum TEM-3 beta-lactamase

We studied the activity of the combination of sulbactam and ceftriaxone against a Klebsiella pneumoniae strain producing TEM-3, a new extended-broad-spectrum beta-lactamase, in an endocarditis model. In vitro, ceftriaxone was strongly inactivated in the presence of TEM-3 (MBC, 128 micrograms/ml with an inoculum of 5 x 10(5) CFU/ml). A marked inoculum effect was demonstrated with sulbactam: effective concentrations of inhibitor needed to reduce the MIC and MBC of ceftriaxone to similar levels increased from 1 microgram/ml in the presence of an inoculum of 5 x 10(5) CFU/ml to 20 micrograms/ml in the presence of an inoculum of 1 x 10(7) CFU/ml. In vivo, sulbactam given at 200 mg/kg of body weight every 12 h, a dosage higher than that previously reported to be effective against rabbit endocarditis caused by other microorganisms, was not sufficient to restore the complete activity of ceftriaxone given at 30 mg/kg once daily for 4 days. This insufficient activity may be correlated with the presence of a high level of beta-lactamase inside the vegetations, as indicated by a quantitative in vitro assay of beta-lactamase activity in the cardiac vegetation, suggesting an insufficient inactivation of the extended-broad-spectrum beta-lactamase in vivo.

In-vitro susceptibility of the Bacteroides fragilis group and the inoculum effect of newer beta-lactam antibiotics on this group of organisms.

The susceptibility and inoculum effect of 190 selected Bacteroides fragilis group isolates were determined with six beta-lactam antibiotics by two different methods: agar dilution and broth microdilution. The results were analysed by species within this group of pathogens. The evaluation showed that imipenem had superior activity and was very stable to increased inoculum concentration. Cefoxitin and piperacillin exhibited very good activity and very small inoculum effect; latamoxef showed stability to changes in the inoculum concentration but only intermediate activity; while ceftizoxime and cefoperazone exhibited a marked inoculum effect and relatively poor activity. Resistance among the species was as follows: B. distasonis greater than B. thetaiotaomicron greater than B. ovatus greater than B. fragilis. The inoculum effect was higher when tests were performed in broth rather than in agar. In general, the effect was more pronounced among B. ovatus and less among B. distasonis isolates.

An in vitro evaluation of HRE 664, a novel penem antibiotic

HRE 664 is a novel parenteral penem with a broad antimicrobial spectrum. In this study, HRE 664 inhibited 90% of Enterobacteriaceae at ≤2.0 μg/ml, Haemophilus influenzae and Neisseria gonorrhoeae at ≤0.5 μg/ml, oxacillin-susceptible staphylococci at ≤0.13 μg/ml, oxacillin-resistant staphylococci at ≤8.0 μg/ml, enterococci at ≤8.0 μg/ml, and streptococci at ≤0.13 μg/ml. All strains of Pseudomonas aeruginosa were resistant (MICs were >32 μg/ml). HRE 664 exhibited a minimal inoculum effect and good bactericidal activity with all organisms tested except an oxacillin-resistant Staphylococcus aureus . With the latter, there was a marked inoculum effect and no on-scale bactericidal endpoints. No measurable hydrolysis of HRE 664 occurred with any of the five Gram-negative bacterial β-lactamases tested. When stored at −20°C, HRE 664 showed no significant loss of activity for up to 6 wk, but detectable deterioration occurred thereafter. At −60°C, no loss in HRE 664 potency was observed for up to 12 wk. Proposed HRE 664 MIC quality control parameters are: 0.03–0.13 μg/ml for S. aureus ATCC 29213, 0.25–1.0 μg/ml for Escherichia coli 25922, and 2.0–8.0 μg/ml for Enterococcus faecalis ATCC 29212.

Effect of inoculum size on in vitro activity of norfloxacin against fecal anaerobic bacteria: Rationale for selective decontamination of the digestive tract

Previous studies have shown that anaerobic bacterial are susceptible to norfloxacin at the levels attained in the feces. Conversely, studies in laboratory animals and neutropenic humans using norfloxacin for selective decontamination of the digestive tract have shown that norfloxacin markedly reduces the aerobic enteric flora without reducing fecal anaerobic flora. In an effort to resolve this paradox, the effect of a 10 9 colony-forming units (cfu)/ml inoculum, which is more reflective of actual fecal counts than the standard 10 5 cfu/ml inoculum, on the activity of norfloxacin against two fecal Escherichia coll isolates and 16 fecal anaerobic isolates was studied. The results showed a marked inoculum effect at 10 9 cfu/ml for most anaerobic isolates but not for the E. coli strains tested. At 256 μg/ml, all E. coli were killed while the anaerobic bacteria maintained colony counts greater than or equal to 10 9 cfu/ml. Hence, the lack of anaerobic activity (minimal inhibitory concentration greater than or equal to 512 μg/ml) at higher fecal inocula might explain the utility of norfloxacin in selective decontamination of the bowel.

In vitro activity and beta-lactamase stability of two oral cephalosporins, ceftetrame (Ro 19-5247) and cefetamet (Ro 15-8074).

Ceftetrame (Ro 19-5247) and cefetamet (Ro 15-8074), two new orally administered aminothiazolyl imimomethoxy cephalosporins, inhibited hemolytic streptococci and Streptococcus pneumoniae at less than or equal to 0.5 micrograms/ml but were less active against staphylococci than were cephalexin and cefaclor. They did not inhibit S. faecalis, S. faecium, Listeria monocytogenes, Corynebacterium JK species, or Pseudomonas aeruginosa. Haemophilus influenzae, Branhamella catarrhalis, and Neisseria gonorrhoeae, including ampicillin-resistant isolates, were inhibited at less than 0.25 micrograms/ml. Both agents inhibited Escherichia coli, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis, Salmonella species, Shigella species, Citrobacter diversus, and Aeromonas hydrophila resistant to ampicillin, cephalexin, and cefaclor at less than or equal to 2 micrograms/ml, although many isolates of Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens resistant to cefotaxime were not inhibited by these agents. A marked inoculum effect was noted for Enterobacteriaceae carrying the Richmond-Sykes type 1A chromosomally mediated beta-lactamases, but plasmid-mediated beta-lactamases did not hydrolyze the compounds. Both drugs inhibited the chromosomally mediated beta-lactamase of E. cloacae, P99.

Beta-lactamase-positive strains of Haemophilus influenzae: susceptibility to and inactivation of beta-lactam antibiotics.

Susceptibility and time-kill studies were done with low and high inocula of both beta-lactamase-positive and -negative strains of Haemophilus influenzae with cefamandole, ampicillin, cefoperazone, mezlocillin, moxalactam, and ceftriaxone. Bioassay was done to test for antibiotic inactivation by beta-lactamase-positive strains. All six antibiotics were highly active against the low inoculum (10(4) to 10(5) colony-forming units/ml) of beta-lactamase-negative strains; ceftriaxone, moxalactam, and cefoperazone were equally active against the same inoculum concentration of beta-lactamase-positive strains. In contrast, cefamandole, mezlocillin, and ampicillin were less active against the low inoculum of beta-lactamase-positive H influenzae. A marked inoculum effect occurred with the high inoculum (10(7) to 10(8) CFU/ml) with all six antibiotics, regardless of beta-lactamase production. In time-kill studies, marked differences in bacterial killing resulted after low and high inocula. Ampicillin, cefamandole, cefoperazone, and mezlocillin were rapidly inactivated by the high inoculum of beta-lactamase-positive H influenzae.

Activities of tobramycin and azlocillin alone and in combination against experimental osteomyelitis caused by Pseudomonas aeruginosa.

Azlocillin and tobramycin were used alone and in combination in the treatment of chronic osteomyelitis due to Pseudomonas aeruginosa in rabbits. This combination showed in vitro synergy measured by both the checkerboard technique and time-kill curves. A marked inoculum effect was demonstrated in vitro with azlocillin and the infecting strain of P. aeruginosa. The minimal inhibitory concentration of azlocillin, with an inoculum of 10(5) organisms, was 12.5 micrograms/ml; when the inoculum size was increased to 10(7) organisms, the minimal inhibitory concentration rose to more than 500 micrograms/ml. In therapeutic trials, the combination of azlocillin and tobramycin, given for 28 days, was significantly better than either no therapy or azlocillin alone, but was not significantly better than tobramycin alone. Even after 4 weeks of combined therapy with azlocillin and tobramycin, P. aeruginosa was recovered from the bones of 60% of the treated rabbits.

Comparative in-vitro activity of ceftazidime (GR 20263) and other -lactamase stable cephalosporins against pseudomonas: Effect of inoculum size and divalent cation supplementation

The anti-pseudomonal activity of ceftazidime was determined by an agar-dilution technique against 92 strains of Pseudomonas aeruginosa and 22 non-aeruginosa isolates and compared with that of cefoperazone, moxalactam and ceftizoxime. Against Ps. aeruginosa, ceftazidime and cefoperazone were comparable in activity and both were significantly more active than moxalactam and ceftizoxime (P < 0001). At 16 mg/l, 95% of strains were inhibited by ceftazidime, compared to 94% by cefoperazone, 80% by moxalactam and 25% by ceftizoxime. A three- to four-fold increase in geometric mean MIC for ceftazidime, moxalactam and ceftizoxime, and a 23-fold increase for cefoperazone was observed with a larger inoculum of 10(6) cfu, demonstrating a marked inoculum effect. Additionally, divalent cation (calcium and magnesium) concentrations of the test medium also influenced the in-vitro results for ceftazidime and cefoperazone, but not for moxalactam or ceftizoxime. Against non-aeruginosa strains, cefoperazone was most active while ceftizoxime was less effective.

Susceptibility of Enterobacter to Cefamandole: Evidence for a High Mutation Rate to Resistance

Cefamandole minimum inhibitory concentrations (MICs) of 10 strains of Enterobacter were determined by the ICS agar dilution and broth dilution procedures. Agar dilution MICs ranged from 1 to 8 mug/ml, with an inoculum of 10(4) organisms/spot. Broth dilution MICs were consistently higher, with an inoculum of approximately 7 x 10(5) organisms/ml. Seven strains showed MICs of >/=64 mug/ml. There was a marked inoculum effect in broth, and skipped tubes were often observed. Variants resistant to 32 mug/ml or more were isolated by direct selection and were shown to occur at a frequency of approximately 10(-6) to 10(-7). A mutant showing a 16-fold increase in agar dilution MIC was also isolated by indirect selection. These variants and others isolated from broth in the presence of cefamandole were tested for ability to inactivate the antibiotic, using both a biological and a chemical procedure. Two distinct classes of variants were seen. Twelve of 28 were shown by both methods to inactivate the antibiotic, whereas the others, including the indirectly selected mutant, did not. The wild types were also negative by both tests. The higher cefamandole MICs of Enterobacter in broth, thus, appeared to reflect a high frequency of resistant variants that were not detected with the inoculum and end point criteria usually used in agar dilution methods. The ability of some variants to inactivate cefamandole may have resulted from a mutation that extended the activity of Enterobacter cephalosporinase to include this antibiotic.

Susceptibility of recent isolates of Pseudomonas aeruginosa to gentamicin, polymyxin, and five penicillins, with observations on the pyocin and immunotypes of the strains.

The susceptibilities of recently isolated strains of Pseudomonas aeruginosa to gentamicin, polymyxin B, carbenicillin, ampicillin, penicillin G, and two newer penicillins were tested with the inocula-replicating technique by using undiluted and 10(-3) dilutions of the cultures. With either inoculum, polymyxin B was the most active agent, and a comparison with previous data from this laboratory showed that the susceptibility of P. aeruginosa to this antibiotic had not changed over the past 20 years. Gentamicin was nearly as active as polymyxin, all but 2 of the 141 strains tested with the diluted inoculum being inhibited by 6.25 mug/ml or less. AB-2288, an agent resembling carbenicillin, was four times more active than carbenicillin or BLP-1654; the last two were equally active against the 10(-3) inoculum. A more marked inoculum effect was noted with the penicillin analogues tested, the increase in minimum inhibiting concentration with the undiluted culture being eight-fold for carbenicillin and at least 16-fold for AB-2288 and BLP-1654. Pyocin typing and serotyping failed to demonstrate any clearly predominating types.

Minimum inhibitory concentrations of carbenicillin against Pseudomonas aeruginosa. Investigations on the inoculum effect.

Carbenicillin, a recently introduced semi-synthetic penicillin, has a relatively high activity against Pseudomonas aeruginosa . A marked inoculum effect on the minimum inhibitory con

Susceptibility of Recent Isolates of Pseudomonas aeruginosa to Gentamicin, Polymyxin, and Five Penicillins, with Observations on the Pyocin and Immunotypes of the Strains 1

The susceptibilities of recently isolated strains of Pseudomonas aeruginosa to gentamicin, polymyxin B, carbenicillin, ampicillin, penicillin G, and two newer penicillins were tested with the inocula-replicating technique by using undiluted and 10(-3) dilutions of the cultures. With either inoculum, polymyxin B was the most active agent, and a comparison with previous data from this laboratory showed that the susceptibility of P. aeruginosa to this antibiotic had not changed over the past 20 years. Gentamicin was nearly as active as polymyxin, all but 2 of the 141 strains tested with the diluted inoculum being inhibited by 6.25 mug/ml or less. AB-2288, an agent resembling carbenicillin, was four times more active than carbenicillin or BLP-1654; the last two were equally active against the 10(-3) inoculum. A more marked inoculum effect was noted with the penicillin analogues tested, the increase in minimum inhibiting concentration with the undiluted culture being eight-fold for carbenicillin and at least 16-fold for AB-2288 and BLP-1654. Pyocin typing and serotyping failed to demonstrate any clearly predominating types.