Tigecycline (Tygacil): the first in the glycylcycline class of antibiotics

Nickie D. Greer, PharmD, BCPS1
1From the Department of Pharmacy Services, Baylor University Medical Center, Dallas, Texas.

Corresponding author.

Corresponding author: Nickie D. Greer, PharmD, BCPS, Department of Pharmacy Services, Baylor University Medical Center, 3500 Gaston Avenue, Dallas, Texas 75246 (e-mail: NickiG@BaylorHealth.edu).

Tigecycline is the first drug in the glycylcycline class of antibiotics. Although it is structurally related to minocycline, alterations to the molecule resulted in its expanded spectrum of activity and decreased susceptibility to the development of resistance when compared with other tetracycline antibiotics. Tigecycline has a broad spectrum of activity, including activity against drug-resistant gram-positive organisms. Randomized trials have shown tigecycline to be efficacious for the treatment of complicated intraabdominal infections and complicated skin and skin structure infections. The dose of tigecycline is 50 mg intravenously every 12 hours after a 100-mg loading dose. Nausea, vomiting, and diarrhea were the most common adverse events reported with tigecycline therapy and may result in discontinuation of therapy.

Resistant organisms remain a concern in hospitalized patients and are becoming an increased concern in community-acquired infections. Gram-positive organisms continue to increase in resistance, and very few agents are available to treat these infections. Available options include vancomycin, linezolid, daptomycin, and quinupristin/ dalfopristin. These antimicrobials have improved treatment of resistant gram-positive organisms but may have adverse events that require discontinuation. For this reason, new antimicrobials are needed to treat resistant organisms without sacrificing safety.

Tetracyclines are broad-spectrum antibiotics that have been available since the mid-1900s, but use in recent years has been limited by widespread resistance to these agents. Resistance generally occurs by alterations in tetracycline efflux or ribosomal protection. The glycylcyclines were developed to help overcome these resistance mechanisms. Tigecycline, approved by the Food and Drug Administration (FDA) on June 15, 2005, is the first drug in this class of antimicrobials. Alterations to the tetracycline structure allow tigecycline to maintain activity against tetracycline-resistant organisms, including resistant gram-positive organisms such as penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), and vancomycin-resistant Enterococcus (VRE) species. Tigecycline is approved for the treatment of complicated skin and skin structure infections (cSSSI) and complicated intraabdominal infections caused by susceptible organisms (1).

Tigecycline is a glycylcycline antimicrobial structurally related to minocycline. Like minocycline, tigecycline binds to the bacterial 30S ribosome, blocking the entry of transfer RNA. This ultimately prevents protein synthesis by halting the incorporation of amino acids into peptide chains and thus limits bacterial growth (1–3). However, the addition of an N,N,-dimethylglycylamido group at the 9 position of the minocycline molecule increases the affinity of tigecycline for the ribosomal target up to 5 times when compared with minocycline or tetracycline (4). This allows for an expanded spectrum of activity and decreased susceptibility to the development of resistance (1, 2, 5).

Pharmmacokinetics and Pharmacodynamics

Tigecycline pharmacokinetics were evaluated after single and multiple doses were given over 30 to 60 minutes and are summarized in Table 1 (1–3, 6).

Because it is administered intravenously, tigecycline is 100% bioavailable (1). Tigecycline is highly protein bound and has a large volume of distribution (7–9 L/kg) when compared with the other available tetracyclines. Concentrations of tigecycline in the gallbladder, lung, and colon are higher than serum concentrations (1), while concentrations of tigecycline in the bone and synovial fluid are lower than serum concentrations (7). In vitro studies have shown that tigecycline is not extensively metabolized (1). A slight amount of metabolism may occur via glucuronidation. Tigecycline has an elimination half-life of approximately 36 hours (1, 3). It is mainly eliminated as unchanged drug and metabolites in the bile and feces (59%). Another 22% of the drug is excreted as unchanged drug in the urine.

Based on pharmacokinetic studies, no dosage adjustment is required based on age, sex, or race (1–3, 8).
A study evaluated the pharmacokinetics of tigecycline in patients with mild, moderate, and severe hepatic impairment (Child-Pugh class A, B, and C, respectively) (1). Compared with healthy control subjects, patients with mild hepatic impairment did not have alterations in the pharmacokinetic profile. Patients with moderate hepatic impairment had an increase in the half-life of tigecycline by 23% and a decrease in systemic clearance by 25%. Tigecycline clearance was reduced by 55% and the half-life was lengthened by 43% in patients with severe hepatic impairment. Based on this information, dosage adjustments are not recommended in patients with mild or moderate hepatic impairment. Patients with severe hepatic impairment should receive a 100-mg loading dose of tigecycline followed by a maintenance dose of 25 mg every 12 hours.

A single-dose study compared pharmacokinetic parameters of tigecycline in 6 healthy subjects, 6 patients with a creatinine clearance <30>1, 2). Half of the patients with ESRD received tigecycline prior to hemodialysis and half received it after hemodialysis. Patients with renal impairment (creatinine clearance <30>

Multiple studies have been conducted to evaluate the pharmacodynamic parameters of tigecycline (2–4). One study evaluated the pharmacodynamic activity of tigecycline against Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae using the neutropenic murine thigh model. All isolates were susceptible to tigecycline, with activity dependent on the time above the minimum inhibitory concentration (MIC). The study found that optimal activity was achieved when tigecycline levels were maintained above the MIC for >50% of the dosing interval. A postantibiotic effect of 9 hours and 5 hours was noted against Streptococcus pneumoniae and Escherichia coli, respectively, following a 3-mg/kg dose. This postantibiotic effect was shown to be superior to that of minocycline for all tested species.

Tigecycline has been shown to be a bacteriostatic agent against Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, and Staphylococcus aureus (4). Bacteriostatic activity and bacteriocidal activity have been reported against Streptococcus pneumoniae.

Spectrum of Activity

As shown in Table 2, tigecycline has a broad spectrum of activity against many gram-positive, gram-negative, and anaerobic organisms (1, 3, 4, 9). Coverage includes many multidrug-resistant strains of gram-positive organisms, such as MRSA and MRSE, penicillin-resistant Streptococcus pneumoniae, and VRE species. Tigecycline has also shown activity against organisms that have developed resistance to tetracycline via various mechanisms.

Minocycline remains more active than tigecycline against methicillin-susceptible Staphylococcus aureus and Staphylococcus epidermidis, while tigecycline is more active against the methicillin-resistant strains of these organisms. Tigecycline has demonstrated improved activity against Streptococcus species, including penicillin-susceptible, penicillin-intermediate, and penicillin-resistant strains of Streptococcus pneumoniae. Tigecycline also has improved activity against Enterococcus species when compared with the other tetracycline agents. Tigecycline, minocycline, and doxycycline have better activity against Listeria monocytogenes when compared with tetracycline.

Equal activity is seen against many gram-negative organisms among the tetracyclines (1, 3, 4). Tigecycline does display improved activity in vitro when compared with the other tetracyclines against the following organisms: Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella species, Salmonella species, Serratia marcescens, and Shigella species. Tigecycline and minocycline cover Acinetobacter species (10). Tigecycline is less active than minocycline against Stenotrophomonas maltophilia and Burkholderia cepacia. None of the tetracyclines have coverage against Pseudomonas aeruginosa. Tigecycline also does not have activity against certain Proteus strains, including Proteus mirabilis.

Tigecycline may be more effective than other tetracyclines against anaerobic organisms such as Bacteroides fragilis, Peptostreptococcus species, and Propionibacterium acnes. Tigecycline is also active against some atypical organisms.

LaPlante and Rybak conducted a study to evaluate the activity of arbekacin, tigecycline, daptomycin, and vancomycin against glycopeptide-intermediate staphylococci (GISS) and heterogeneous glycopeptide-intermediate staphylococci (hGISS) (11). An hGISS is a staphylococci isolate that is susceptible to vancomycin but contains subpopulations that are intermediately susceptible to vancomycin. A total of 47 strains of hGISS and GISS were obtained from various locations worldwide. The majority of isolates were Staphylococcus aureus (n = 35). MICs were determined according to guidelines from the National Committee for Clinical Laboratory Standards. A total of 25 isolates (53.2%) were categorized as GISS (MIC, 8 μg/mL to vancomycin), and 22 isolates (46.8%) were categorized as hGISS (MIC, 4 μg/mL to vancomycin). The minimum inhibitory concentration required to inhibit the growth of 90% of organisms (MIC90) for arbekacin, daptomycin, tigecycline, and vancomycin was 2, 1, 25 mg or 50 mg of tigecycline intravenously every 12 hours for 7 to 14 days. Patients in the 25-mg group received a loading dose of 50 mg, and patients in the 50-mg group received a loading dose of 100 mg. Patients could be discharged home after 3 days and continue receiving infusions as an outpatient. The primary endpoint was clinically observed cure rate at the “test-of-cure” visit (approximately 3 weeks after initiation of therapy) among evaluable patients. Cure was defined as the resolution of all signs and symptoms of the infection to the extent that no further antibiotic therapy was needed. The secondary endpoints were clinical cure rates at the end of treatment and microbial eradication rates.

At the end of therapy, 78% of patients in the 25-mg group and 85% of patients in the 50-mg group were considered cured. At the test-of-cure visit, 67% of patients in the 25-mg group and 74% of patients in the 50-mg group were deemed clinically cured. Six patients per group experienced cure at the end of treatment and then were deemed a “failure” at the test-of-cure visit. At the end of therapy, bacterial eradication occurred in 62% of patients in the 25-mg group and 74% of patients in the 50-mg group. At the test-of-cure visit, bacterial eradication occurred in 56% of patients in the 25-mg group vs 69% of patients in the 50-mg group. Adverse events were similar in the 25-mg and 50-mg groups, with nausea being the most common adverse event noted. Nausea occurred in 28% of patients, but only two patients discontinued therapy due to this adverse event. Laboratory abnormalities probably related to tigecycline included elevated serum levels of transaminases, alkaline phosphatase, and blood urea nitrogen as well as anemia. All of these were rare occurrences. The authors 0.5, and 8 μg/mL, respectively, against GISS isolates. Time-kill results showed that daptomycin demonstrated bactericidal activity throughout the 24-hour period. Arbekacin and tigecycline displayed bacteriostatic activity at 24 hours.

Clinical Efficacy

Dose-ranging study Postier and colleagues conducted a multicenter, randomized, open-label, dose-ranging study to evaluate the efficacy and safety of two doses of tigecycline for the treatment of skin and skin structure infections in 160 patients (12). Patients received either concluded that tigecycline is safe and efficacious for the treatment of cSSSI. This dose-ranging study showed that 50 mg twice daily of intravenous tigecycline had higher rates of cure, so this dose was chosen for further clinical study.

Complicated intraabdominal infections The Infectious Diseases Society of America published updated guidelines for the treatment of complicated intra-abdominal infections in 2003 (13). The guidelines recommend treating mild to moderate community-acquired intraabdominal infections with ampicillin/sulbactam, cefazolin plus metronidazole, or ertapenem. If patients can take oral therapy, use of a fluoroquinolone, such as levofloxacin, plus metronidazole or amoxicillin/clavulanate alone is acceptable treatment. High-risk patients, defined as patients with other medical conditions or immunosuppression, are recommended to receive treatment with carbapenems (e.g., imipenem/cilastatin, ertapenem, or meropenem), piperacillin/tazobactam, a fluoroquinolone plus metronidazole, or a third- or fourth-generation cephalosporin (e.g., ceftriaxone, cefepime) plus metronidazole. Hospital-acquired intraabdominal infections are more likely to be caused by resistant organisms due to length of hospital stay and prior antimicrobial therapy. In this case, combination therapy is usually indicated and should include one of the above regimens plus vancomycin if resistant gram-positive organisms are suspected. Empiric enterococcal coverage is not routinely recommended in patients with community-acquired infections and is recommended in hospital-acquired infections only when Enterococcus isolates are recovered.

Babinchak and colleagues reported on a pooled analysis of two phase 3 double-blind trials conducted to evaluate the safety and efficacy of tigecycline vs imipenem/cilastatin for the treatment of complicated intraabdominal infections in 1642 patients (14). Patients were entered into the study if they were at least 18 years of age and required surgery to treat a complicated intraabdominal infection (Table 3).

Exclusion criteria for the study are listed in Table 4. Patients with neutropenia, active malignancy, AIDS, and abdominal malignancy within the past 6 months were excluded from the study, which makes it difficult to extrapolate the results to these populations. Additionally, patients with hepatic and renal dysfunction were excluded from the study, as were patients receiving concomitant ganciclovir.

Patients received tigecycline (100-mg loading dose followed by 5 mg every 12 hours, both given intravenously) or imipenem/cilastatin 500 mg intravenously every 6 hours. Imipenem/cilastatin doses were adjusted for body weight and creatinine clearance results. The primary endpoint was clinical response at the test-of-cure visit (12–42 days after therapy) for the microbiologically modified-intention-to-treat (m-mITT) and microbiologically evaluable populations (n = 1262 and n = 1025, respectively). Safety was assessed on the modified-intention-to-treat (m-ITT) population (n = 1642). Baseline characteristics were similar between the groups. Cure rates for the m-mITT population were 80.2% for the patients treated with tigecycline vs 81.5% for the patients in the imipenem/ cilastatin group (P <>

Adverse events on the whole were similar between the groups. However, patients treated with tigecycline experienced significantly more nausea and vomiting than patients treated with imipenem/cilastatin(P = 0.01 and P = 0.008, respectively). Patients in the tigecycline group also experienced higher rates of infection and leukocytosis than patients in the imipenem/ cilastatin group (P < p =" 0.03," p =" 0.038).">

This study did show the noninferiority of tigecycline. However, infection and postoperative wound infection rates were significantly higher in the tigecycline group. The rate or type of infection was not addressed by the authors, so it is unclear if these represented recurrent infections or new resistant infections. The authors stated that although the wound infection rates were higher in the tigecycline group, the rates were similar to those published in the literature. No comment was made on the higher rate of leukocytosis in these patients, but that may be an adverse effect to watch for as use of tigecycline increases.

Complicated skin and skin structure infectionsThe Infectious Diseases Society of America published practice guidelines for the treatment of skin and soft tissue infections in 2005 (15). The guidelines recommend nafcillin or cefazolin for patients with cSSSI caused by suspected methicillin-sensitive Staphylococcus aureus. Clindamycin or vancomycin may be used for those patients who are allergic to penicillin. Vancomycin or linezolid may be used for the treatment of cSSSI caused by suspected MRSA. A penicillin plus a beta-lactamase inhibitor (e.g., ampicillin/sulbactam or piperacillin/tazobactam), carbapenems, or a fluoroquinolone plus clindamycin may be used for cSSSI caused by multiple organisms.

Ellis-Grosse and colleagues reported on pooled data from two randomized, double-blind trials evaluating the safety and efficacy of tigecycline vs vancomycin/aztreonam for the treatment of cSSSI in 1116 patients (16). Inclusion and exclusion criteria are shown in Table 5

Patients received a tigecycline 100-mg intravenous loading dose followed by 50 mg intravenously every 12 hours. Vancomycin was given at a dose of 1 g intravenously every 12 hours and could be adjusted based on renal function. Aztreonam was given at a dose of 2 g intravenously every 12 hours and could be discontinued after 48 hours at the investigators' discretion. Treatment was continued for up to 14 days. The primary endpoint was clinical response within the clinically evaluable and the clinical m-ITT populations at the test-of-cure visit (days 12–92 after the last dose of study drug). The clinical m-ITT group included 1057 patients, and the clinically evaluable group included 833 patients. The cure rates in the clinical m-ITT groups were 79.7% in the tigecycline group and 81.9% in the vancomycin/aztreonam group (P <>

Significantly more patients in the tigecycline group had gastrointestinal adverse events (diarrhea, anorexia, dyspepsia, nausea, and vomiting) than patients in the vancomycin/aztreonam group (46% vs 21%, P <>

Resistance

Because of widespread use of the various tetracycline agents in the past, resistant organisms are commonly seen. Tetracycline resistance typically results from one of the following: chemical modification, efflux of the antibiotic out of the cell, or decreased binding of the antibiotic to the target receptor due to ribosomal protection (2, 3, 5, 17, 18). Numerous tetracycline-resistant genes have been identified. These genes code for efflux or ribosomal protection. Gram-negative organism resistance to tetracycline, minocycline, and doxycycline is mainly due to efflux genes. Gram-positive organism resistance to tetracycline is due to efflux genes, but efflux is not as important for resistance to the tetracycline derivatives.

Tigecycline has shown activity against organisms containing either efflux or ribosomal protection genes (2, 3). This activity is thought to be due to tigecycline's increased binding affinity for the ribosomes. The tetracycline-resistance gene cannot disrupt the tigecycline-ribosome bond, rendering the gene unable to interact with the ribosome and protect it. It is also speculated that the large side chain added to the tigecycline molecule keeps the efflux pump from pumping it out of the cell.

The development of resistance to tigecycline by organisms normally sensitive to tigecycline has not been noted to date, either in nature or in the laboratory (2, 3). Two veterinary Salmonella strains resistant to earlier investigational glycylcyclines have been isolated, but no resistance to tigecycline was noted.

Adverse Effects

Adverse effect information is compiled from data collected on 1415 patients in the phase 3 clinical trials (1, 3, 4). Adverse effects were mild in most patients and similar to placebo (Table 6). In 5% of patients in the tigecycline groups vs 4.7% in the comparator groups, therapy was discontinued due to adverse events. The most common adverse events reported with tigecycline therapy were similar to adverse effects seen with the tetracycline class and included nausea (29.5%), vomiting (19.7%), and diarrhea (12.7%). These events are thought to be caused by an irritant effect of tigecycline on the gastric mucosa.

Hepatic toxicity is a rare occurrence with tetracyclines and occurs mainly in patients who receive high doses (3). Tigecycline was shown to increase hepatic enzymes and bilirubin in clinical trials. Hyperbilirubinemia occurred more often in the tigecycline group than in the comparator group.

Infection-related adverse events were more common with tigecycline therapy (1). The incidence of sepsis and septic shock was significantly higher in tigecycline patients (1.5%) than in comparators (0.5%). Clinical studies reported the higher rate of infection but did not clarify what factors may have contributed to this finding. It may be due to baseline differences in patients' severity of illness prior to initiation of tigecycline.

Tetracyclines can inhibit bone growth in children and cause dental staining due to deposition of drug in the teeth and bone (1, 3). The latter may occur at any time from the last months of gestation to age 8. Tigecycline, like other tetracyclines, is not recommended for pregnant women or children younger than 8 years.

One case of possible tigecycline-related Clostridium difficile infection has been reported to date (1, 4). Additionally, post-marketing reports have associated tigecycline with the development of acute pancreatitis (1).

Dose/Dosage Forms

The recommended dose of tigecycline is 50 mg every 12 hours after a 100-mg loading dose (1). If a patient has severe hepatic impairment (Child-Pugh class C), a dose of 25 mg every 12 hours should be given after a loading dose of 100 mg. Doses are given intravenously over 30 to 60 minutes.
Pharmacoeconomics

Table 7 lists the cost of tigecycline and other antimicrobials for a 7- and 10-day course of therapy (Baylor University Medical Center acquisition costs). For MRSA and other resistant organisms, vancomycin remains the least expensive and daptomycin the most expensive agent. Tigecycline is less expensive for a course of therapy than both linezolid and daptomycin. Tigecycline is more expensive than imipenem/cilastatin and approximately equivalent in cost to aztreonam plus vancomycin. Tigecycline is also approximately equivalent in cost to imipenem/cilastatin plus vancomycin. For a 10-day course of therapy, tigecycline monotherapy costs about $250 more than piperacillin/tazobactam plus vancomycin; $250 more than 4. Nathwani D. Tigecycline: clinical evidence and formulary positioning. ampicillin/sulbactam plus vancomycin; and $600 more than levofloxacin plus metronidazole.

Recommendation

Tigecycline should be used when coverage is needed for resistant gram-positive organisms as well as gram-negative and anaerobic organisms. It should not be used for patients who are not at risk of resistant infections or in patients who have infections sensitive to vancomycin, unless the patient is not responding to vancomycin therapy. Empiric use of tigecycline may be appropriate in select patients in the intensive care unit, although care must be taken that Pseudomonas aeruginosa coverage is added to those patients at risk of infections caused by that organism. To avoid the development of resistance, tigecycline should not be used for infections caused by gram-positive organisms only, unless other agents have failed to work.

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Postier RG, Green SL, Klein SR, Ellis-Grosse EJ, Loh E, Tigecycline 200 Study Group. Results of a multicenter, randomized, open-label efficacy and safety study of two doses of tigecycline for complicated skin and skin-structure infections in hospitalized patients. Clin Ther. 2004;26(5):704–714. [PubMed] [Full Text]

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Proc (Bayl Univ Med Cent). 2006 April; 19(2): 155–161.

Successful Clearance of Catheter-Related Bloodstream Infection by Antibiotic Lock Therapy Using Ampicillin

Published Online, 31 January 2006, DOI 10.1345/aph.1G446.The Annals of Pharmacotherapy: Vol. 40, No. 2, pp. 347-350. DOI 10.1345/aph.1G446© 2006 Harvey Whitney Books Company.

Robert L Elwood, MD
Fellow, Pediatric Infectious Disease, Department of Pediatric Infectious Disease, Walter Reed Army Hospital Center, Washington, DC
Steven E Spencer, MD
Chief, Pediatric Infectious Disease, Department of Pediatric Infectious Disease, Walter Reed Army Hospital Center

Reprints: Dr. Elwood, Department of Pediatric Infectious Disease, Walter Reed Army Hospital Center, 6900 Georgia Ave. NW, Washington, DC 20307-5001, fax 202/782-4699, Robert.Elwood@NA.AMEDD.ARMY.MIL

OBJECTIVE:

To report a case in which ampicillin was used successfully as lock therapy for a central venous intravascular catheter and to discuss the implications of ampicillin used in this modality.

CASE SUMMARY:

A 14-month-old girl with a long-term central venous catheter acquired a polymicrobial (Escherichia coli and Enterococcus durans) bloodstream infection. The central venous catheter was suspected as the source for the bacteremia based on the timing and number of positive blood cultures in relation to therapy with antibiotics. Antibiotic sensitivity testing revealed ampicillin monotherapy to be an ideal choice to treat both organisms. A combination of systemic therapy via a temporary catheter and antibiotic lock therapy of the central venous catheter was then instituted using ampicillin without anticoagulants. The patient tolerated this therapy without complications, and follow-up cultures demonstrated effective clearance of the bacteria.

DISCUSSION:

Antibiotic lock therapy has been shown to be useful in the treatment of catheter-related bloodstream infections. However, many antibiotics have yet to be tested with this modality. Ampicillin, which is frequently used in the treatment of Enterococcus and E. coli infections, has not previously been reported as a single agent for lock therapy.

CONCLUSIONS:

Ampicillin may be a useful agent with the relatively new modality of lock therapy for central venous catheters. Further studies are needed to demonstrate possible compatibility of this agent with anticoagulants, such as heparin, as well as its efficacy in treating catheter-related bloodstream infections.
Key Words: ampicillin, antibiotic lock therapy.

Abstract

Evaluation of high-dose daptomycin for therapy of experimental Staphylococcus aureus foreign body infection

Research article

Heinz J. Schaad , Manuela Bento , Daniel P. Lew and Pierre Vaudaux BMC Infectious Diseases 2006, 6:74 doi:10.1186/1471-2334-6-74

Published 11 April 2006

Abstract (provisional)

Background

Daptomycin is a novel cyclic lipopeptide whose bactericidal activity is not affected by current antibiotic resistance mechanisms displayed by S. aureus clinical isolates. This study reports the therapeutic activity of high-dose daptomycin compared to standard regimens of oxacillin and vancomycin in a difficult-to-treat, rat tissue cage model of experimental therapy of chronic S. aureus foreign body infection.

Methods

The methicillin-susceptible S. aureus (MSSA) strain I20 is a clinical isolate from catheter-related sepsis. MICs, MBCs, and time-kill curves of each antibiotic were evaluated as recommended by CLSI, including supplementation with physiological levels (50 mg/L) of Ca2+ for daptomycin. Two weeks after local infection of subcutaneously implanted tissue cages with MSSA I20, each animal received (i.p.) twice-daily doses of daptomycin, oxacillin, or vancomycin for 7 days, or was left untreated. The reductions of CFU counts in each treatment group were analysed by ANOVA and Newman-Keuls multiple comparisons procedures.

Results

The MICs and MBCs of daptomycin, oxacillin, or vancomycin for MSSA strain I20 were 0.5 and 1, 0.5 and 1, or 1 and 2 mg/L, respectively. In vitro elimination of strain I20 was more rapid with 8 mg/L of daptomycin compared to oxacillin or vancomycin. Twice-daily administered daptomycin (30 mg/kg), oxacillin (200 mg/kg), or vancomycin (50 mg/kg vancomycin) yielded bactericidal antibiotic levels in infected cage fluids throughout therapy. Before therapy, mean (+/- SEM) viable counts of strain I20 were 6.68 +/- 0.10 log10 CFU/mL of cage fluid (n = 74). After 7 days of therapy, the mean (+/- SEM) reduction in viable counts of MSSA I20 was 2.62 (+/- 0.30) log10 CFU/mL in cages (n = 18) of daptomycin-treated rats, exceeding by >2-fold (P<0.01) n =" 19)" n =" 18)" n =" 19)">

Conclusions

The improved efficacy of the twice-daily regimen of daptomycin (30 mg/kg) compared to oxacillin (200 mg/kg) or vancomycin (50 mg/kg) may result from optimisation of its pharmacokinetic and bactericidal properties in infected cage fluids.

Full Length Article - BMC Infectious Diseases

Doxycycline plus streptomycin versus ciprofloxacin plus rifampicin in spinal brucellosis

April 11, 2006

Alp E, Koc RK, Durak AC, Yildiz O, Aygen B, Sumerkan B, Doganay M.

ABSTRACT: BACKGROUND:

The optimal treatment regimen and duration of the therapy is still controversial in spinal brucellosis. The aim of this study is to compare the efficacy, adverse drug reactions, complications and cost of ciprofloxacin plus rifampicin versus doxycycline plus streptomycin in the treatment of spinal brucellosis. METHODS: The patients diagnosed as spinal brucellosis between January 2002 to December 2004 were enrolled into the study. Patients were enrolled into the two antimicrobial therapy groups (doxycycline plus streptomycin vs. ciprofloxacin plus rifampicin) consecutively. Only the cost of antibiotic therapy was analysed for each patient.

RESULTS:

During the study period, 31 patients with spinal brucellosis were enrolled into the two antimicrobial therapy groups. Fifteen patients were included in doxycycline plus streptomycin group and 16 patients were included in ciprofloxacin plus rifampicin group. Forty-two levels of spinal column were involved in 31 patients. The most common affected site was lumbar vertebra (n=32, 76%) and involvement level was not different in two groups. Despite the disadvantages (older age, more prevalent operation and abscess formation before the therapy) of the patients in the ciprofloxacin plus rifampicin group, the duration of the therapy (median 12 weeks in both groups) and clinical response were not different from the DS. The cost of ciprofloxacin plus rifampicin therapy was 1.2 fold higher than the cost of doxycycline plus streptomycin therapy.

CONCLUSION:

Classical regimen (doxycycline plus streptomycin), with the appropriate duration (at least 12 weeks), is still the first line antibiotics and alternative therapies should be considered when adverse drug reactions were observed.

PMID: 16606473

[PubMed - as supplied by publisher]

Evolution of a Vancomycin-Intermediate Staphylococcus aureus Strain In Vivo:

Evolution of a Vancomycin-Intermediate Staphylococcus aureus Strain In Vivo: Multiple Changes in the Antibiotic Resistance Phenotypes of a Single Lineage of Methicillin-Resistant S. aureus under the Impact of Antibiotics Administered for Chemotherapy

K. Sieradzki,1 T. Leski,1 J. Dick,2 L. Borio,2 and A. Tomasz1*
The Rockefeller University, New York, New York 10021,1 John Hopkins Medical Institutions, Baltimore, Maryland 212872

*Corresponding author. Mailing address: The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8277. Fax: (212) 327-8688.

E-mail: tomasz@mail.rockefeller.edu.

Received October 2, 2002; Revised November 29, 2002; Accepted January 7, 2003.

Abstract

A number of methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered over a period of several weeks from blood samples and from the heart valve of a patient who underwent extensive vancomycin chemotherapy for persistent S. aureus bacteremia. Consecutive isolates showed gradually decreasing growth rates during in vitro cultivation and increasing vancomycin MICs, from an MIC of 1 μg/ml for the initial isolate to an MIC of 8 μg/ml for the final MRSA isolates, which also became tolerant to vancomycin. Major changes were observed in the oxacillin resistance phenotype of several of the isolates—apparently related to in vivo exposure to imipenem, which was also used during a period of chemotherapy. Both the gradually increasing vancomycin MICs and the changes in oxacillin resistance could be reproduced by appropriate exposure of the initial MRSA isolate to antibiotics in vitro. All isolates had the same pulsed-field gel electrophoresis pattern, spaA type, and multilocus sequence type (MLST), which was identified as a single-locus variant of ST5, the MLST characteristic of previously characterized MRSA isolates with reduced susceptibility to vancomycin in the United States and Japan.

Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin—so-called VISA isolates—have been identified in several countries, but identification of the mechanism of resistance has been made difficult by conflicting observations concerning the properties of individual VISA isolates (3, 9, 11, 15) and by the lack of isogenic isolates with different levels of vancomycin susceptibility. Here we describe the physiological properties of consecutive MRSA isolates recovered from a single patient during extensive vancomycin therapy (D. Flayhart, A. Hanlon, T. Wakefield, T. Ross, L. Borio, and J. Dick, Abstr. 101st Gen. Meet. Am. Soc. Microbiol., abstr. A-39, 2001), which was accompanied by gradually increasing vancomycin MICs for the isolates. Our observations strongly suggest that these VISA isolates represent the progeny of a single MRSA strain that emerged in vivo under the selective pressure of vancomycin therapy. The purpose of the studies described here was to compare the properties of the VISA isolates as they emerged during various stages of the patient's chemotherapy in order to better understand the mechanism of evolution of drug resistance in vivo.

Material and Methods

Strains and growth conditions. The S. aureus isolates recovered from clinical material in Baltimore, Md., are listed in Table 1. Bacteria were grown in tryptic soy broth (TSB) (Difco, Detroit, Mich.) at 37°C with aeration. Growth was monitored by measuring the optical density at 620 nm with an LKB spectrophotometer (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden). Viable titers were also determined routinely at sampling times by plating diluted cultures on tryptic soy agar (TSA) (Difco).

Antibiotic Susceptibility

Susceptibility testing was performed by the agar dilution method according to NCCLS recommendations (10). Oxacillin and vancomycin susceptibility profiles of bacterial cultures were also tested by population analysis (18). Selection of highly resistant subpopulations was achieved by picking single colonies from the population analysis plates containing elevated concentrations of antibiotics and using these as inocula for overnight cultures grown in antibiotic-free TSB.

Time-kill Curves

Kill rates were measured for exponentially growing cultures that at “zero” time (corresponding to an optical density at 620 nm of 0.1, or about 107 CFU per ml) received concentrations of vancomycin ranging from 2 to 100 times the corresponding MIC. Control cultures received no antibiotic. Portions of the cultures (200 μl) were removed at various intervals, serially diluted, and plated on TSA. Colonies were counted after 48 h of incubation at 37°C.

DNA manipulations

Chromosomal DNA for pulsed-field gel electrophoresis (PFGE) was prepared as described before (4). Chromosomal DNA for PCR was prepared as described before (2), except that the proteinase K digestion step was preceded by treatment of the bacterial cells with lysostaphin.

MLST and SPAA Typing

Multilocus sequence typing (MLST) based on the sequences of seven housekeeping genes (6, 8) and typing based on the polymorphic region of protein A (spaA typing) (6, 13) were performed by published procedures.

Detection of MECA

The presence of mecA was determined by PCR with 20 pmol of primers PF-MECP (5′-GATTGGGATCATAGCGTCATT-3′) and PR-MECP (5′-GTTTTTCGAGTCCCTTTTTACC-3′), which were used to amplify a 481-bp internal fragment of the gene. PCR was carried out for 30 cycles of 30 s at 95°C, 52°C for 30 s, and 72°C for 1 min and one final extension step at 72°C for 4 min.

Results and Discussion

Characterization of JH VISA isolates. All JH isolates were resistant to erythromycin, clindamycin, levofloxacin, and rifampin and susceptible to tetracycline, linezolid, and quinupristin-dalfopristin, and all isolates carried the mecA gene (data not shown). The dates of recovery of the MRSA isolates and antibiotic treatments are shown schematically in Fig. 1.

All JH strains had an indistinguishable PFGE pattern which was similar but not identical to the PFGE patterns of several previously described VISA isolates from the United States and Japan (Fig. 2).

Table 1 summarizes the date and clinical source of isolation, oxacillin and vancomycin MICs, and the results of three additional molecular typing techniques used for the characterization of the bacteria. Also included for comparison are the relevant properties of several other VISA isolates from the United States and strain Mu50 from Japan. All JH isolates—including the earliest isolate, JH1, subsequent isolates JH2 through JH14, and isolate JH15 (recovered from the nares of a family contact)—shared a common 1-4-1-4-12-1-28 multilocus sequence type and a common, invariant spaA type. The MLST of the JH isolates appears to be a hitherto-undescribed single-locus variant of ST5, which is the characteristic MLST of the New York clone of MRSA (6) and of several previously described MRSA isolates from the United States and Japan—including VISA isolates PC3, MI, NJ (1, 12, 15), and Mu50 (9).

Changes in Oxacillin and Vancomycin Susceptibilities as a function of the chronological date of isolation of JH strains

Cultures of the earliest isolate, JH1, and surveillance culture JH15 showed heterogeneous oxacillin resistance phenotypes when tested for population analysis profiles (PAPs). For the great majority of bacteria (>99.9%), oxacillin MICs were 0.75 μg/ml. The cultures also contained subpopulations of highly resistant cells for which oxacillin MICs were >400 μg/ml, but these subpopulations were present at a low (10−6) frequency in the cultures. A major increase in the oxacillin MICs for the majority of cells and a less heterogeneous PAP were detected in cultures of the isolates obtained next chronologically, JH2 and JH3; these isolates were recovered from the patient after a therapeutic regimen of imipenem treatment for noscomial pneumonia. Cultures of isolates recovered after this time (JH6 through JH14) showed heterogeneous oxacillin phenotypes; the MICs for the majority of cells were reduced to 1 to 3 μg/ml (Fig. 3).

Similar but much less extensive changes were also apparent with respect to the vancomycin susceptibility of the JH isolates. While the vancomycin MICs for the early isolates (JH1, JH2, and JH3) were within the range of susceptibility breakpoints, those for the late isolates (JH6, JH9, and JH14), recovered from the patient after an extended course of vancomycin therapy, gradually increased from 1 μg/ml to 4, 6, and eventually 8 μg/ml for isolates JH9 and JH14 (Fig. 3). Moreover, all of these isolates were capable, at low frequencies, of forming colonies on agar containing 8.0 or sometimes even 16 μg of vancomycin/ml.

If the changing oxacillin and vancomycin phenotypes were the products of selective antibiotic pressure in vivo, then it might be possible to reconstruct these phenotypic changes by applying similar antibiotic selection to the earliest isolate, JH1, in vitro. Figures 4 and 5 demonstrated this to be the case. A culture of strain JH1 was plated on agar containing increasing concentrations of imipenem (the β-lactam antibiotic used in the chemotherapy of the patient), and a rare colony capable of growing on plates containing 50 μg of imipenem/ml was used as an inoculum for an overnight TSB culture (strain JH1IMP50 in Fig. 4). Plating of the overnight culture on agar containing either imipenem or oxacillin at increasing concentrations demonstrated the selection of a more highly and homogeneously β-lactam-resistant culture from strain JH1 that mimicked the properties of strains JH2 and JH3 (Fig. 4).

With a similar procedure, it was also possible to generate from cultures of strain JH1 plated on agar containing increasing concentrations of vancomycin bacterial cultures that showed increased resistance to vancomycin, as indicated by a shift in the shape of the PAPs toward higher vancomycin MICs (see the PAPs of JH1 and JH1P4 in Fig. 5). Subculturing of such bacteria in liquid medium containing one-half the MIC of vancomycin allowed the selection of bacteria (JH1P4T4 in Fig. 5) for which vancomycin MICs were even higher, approaching those of in vivo-selected strains JH9 and JH14.

A careful comparison of the shapes of the PAP curves illustrating the oxacillin and vancomycin susceptibility profiles for the JH strains (Fig. 3) suggests that increasing vancomycin MICs were accompanied by a reverse change in the β-lactam antibiotic susceptibility profiles. For instance, for strains JH2 and JH3, for which oxacillin MICs were relatively high, vancomycin MICs were lower; in contrast, for strains JH9 and JH14, vancomycin MICs were the highest but oxacillin MICs were relatively low for the majority of the bacteria in cultures.

This inverse relationship between β-lactam and vancomycin susceptibility profiles could be reproduced in vitro. A colony of strain JH3 capable of growing on agar containing 8 μg of vancomycin/ml was used as an inoculum to select a homogeneous culture of these bacteria by overnight growth in TSB. In such cultures (JH3V8), the vancomycin MIC for the majority of the bacteria increased from 2 to 16 μg/ml. The phenotype of JH3V8 cultures was stable during growth in nonselective medium. Plating of cultures of JH3 and JH3V8 on oxacillin-containing agar plates produced PAPs with phenotypes that were the inverse of the vancomycin susceptibility profiles of these strains: the oxacillin MIC decreased from about 100 μg/ml for the majority of cells of JH3 to as low as 0.75 μg/ml for cells of JH3V8 (Fig. 6).

Changes in growth rate and susceptibility to the bactericidal effect of vancomycin as a function of the chronological dates of isolation of JH strains

The growth rates of the JH strains were determined by monitoring the rate of increase in the optical density during aerobic incubation at 37oC for TSB cultures in vitro. The doubling times of the cultures increased from 30 min for JH1 to 45 min for JH2 and JH3 and approximately 60 min for JH6, JH9, and JH14.

Cultures of JH1 and JH14 were compared for their susceptibility to the bactericidal effect of vancomycin during exposure of TSB cultures to various concentrations of the antibiotic administered at different MIC equivalents. Figure 7 shows that cultures of JH1 exposed to vancomycin at concentrations corresponding to 2, 5, and 10 times the MIC (2, 5, and 10 μg of vancomycin/ml, respectively) underwent an extensive loss of the viable titer (from about 108 CFU per ml to 8 × 106 CFU per ml) during a 4-h incubation period. There was no detectable decrease in the viable titer of cultures of JH14 during exposure to a similar range of vancomycin MIC equivalents (corresponding to 16, 40, and 80 μg of vancomycin/ml) (Fig. 7). After exposure of the cultures to 10 times the respective vancomycin MICs for 24 h, the viable titers of JH1 and JH14 were reduced to 6 × 103 and 5 × 104 CFU per ml, respectively.

Vancomycin susceptibility of MRSA strain PA237

Interestingly, the JH strains share a common and hitherto-undescribed MLST, 1-4-1-4-12-1-28, which is a single-locus variant of ST5, the genetic background of previously characterized VISA isolates from the United States and Japan (6). The JH series is closely related but not identical to the previously characterized VISA isolates. The close genetic relatedness of the JH series to other vancomycin-resistant strains (MI, NJ, PC3, and Mu50) and, in turn, of those strains to an MRSA clone that is widespread in hospitals in the New York metropolitan area is of obvious concern. Sieradzki et al. previously showed that several isolates belonging to this MRSA clone, although still susceptible to vancomycin, contained subpopulations of bacteria for which vancomycin MICs approached and even surpassed those for the majority of cells of various VISA isolates (15).

Strain PA237 was such an isolate recovered from a hospitalized patient in Pennsylvania. This strain belonged to the New York MRSA clone (ST5) and was analyzed for vancomycin susceptibility by population analysis (Fig. 8). Strain PA237 showed a heterogeneous vancomycin phenotype, since it contained subpopulations (at a frequency of approximately 10−4) of bacteria which could grow on agar containing 4.0 μg of vancomycin/ml. A colony picked from an agar plate containing 4.0 μg of vancomycin/ml (Fig. 8) and used as an inoculum for drug-free TSB was grown overnight and replated for population analysis. Now the vancomycin MIC for the majority of the bacteria was 4.0 μg/ml, and the population profile was similar to that of other VISA isolates. The similar genetic background of all VISA isolates described so far suggests that some determinant carried in the New York MRSA clone may predispose the bacteria to the development of vancomycin resistance.

All of the JH isolates characterized in this study were recovered from a single patient at various times after the onset of a prolonged course of chemotherapy with vancomycin (for the MRSA infection) and a shorter course of treatment with imipenem (for noscomial pneumonia) (Flayhart et al., Abstr. 101st Gen. Meet. Am. Soc. Microbiol.). All of the JH isolates had identical PFGE patterns as well as identical MLST and spaA types, suggesting that the JH isolates represented the progeny of the earliest MRSA isolate, JH1, and that the changing phenotypes of the subsequent isolates mirrored the selective pressure of chemotherapeutic agents molding the antibiotic susceptibility profiles of the bacteria.

The extremely low and heterogeneous oxacillin-resistant phenotype of JH1 was shown to change to a more homogeneous and more highly resistant phenotype in isolates JH2 and JH3—apparently as a consequence of the brief imipenem therapy given to the patient. Subsequently, in later isolates, such as JH6, JH9, and JH14 (isolated after the therapy with imipenem had been discontinued), the oxacillin PAP again changed to low and heterogeneous resistance, similar to the phenotype of the original isolate, JH1. This change might have been the result of continued vancomycin chemotherapy selecting for gradually increasing vancomycin MICs which—in turn—might have brought about the decline in oxacillin resistance. Such a seesaw-like effect between vancomycin resistance and β-lactam resistance was originally observed for in vitro-selected vancomycin-resistant S. aureus mutants (14) and was subsequently also noted for some clinical VISA isolates (15). The experiments illustrated in Fig. 6 actually reconstructed this phenomenon for isolate JH3 and its more highly vancomycin-resistant subpopulation. It was also possible to reconstruct the increased and more homogeneous oxacillin-resistant phenotype in vitro by applying selective imipenem pressure to isolate JH1 in the laboratory (Fig. 5).

An interesting property of the VISA isolates was the gradually decreasing growth rate of the bacteria, which seemed to parallel the increase in the vancomycin MIC. A similar phenomenon was noted for vancomycin-resistant mutants isolated in the laboratory (14). The slow growth rate for JH14 may be one of the factors responsible for the vancomycin tolerance of this isolate, as demonstrated in Fig. 8. The dependence of the bactericidal activity of antimicrobial agents on the rate of growth of target bacteria has been demonstrated repeatedly in the past (5, 7, 19).

Addendum

Since the submission of the manuscript, we also determined the structural type of SCCmec carried by isolates JH1, JH4, JH9, and JH14 by using a multiplex PCR method (10a). All isolates contained the same type II SCCmec, further confirming that these bacteria are close relatives of the New York-Japan pandemic MRSA clone (10b).

Acknowledgments

Partial support for this work was provided by a grant from the National Institutes of Health (RO1-AI45738).
The help of Marilyn Chung with multiplex PCR of the staphylococcal chromosomal cassette mec (SCCmec) is gratefully acknowledged.

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Journal of Clinical Microbiology