Pseudomonas aeruginosa Resistance to beta-lactam antibiotics

Pseudomonas aeruginosa Resistance to beta-lactam antibiotics

98 Infectious Diseases Newsletter 10(12) December 1991 and in foreign-device-associated infections as well. Many CF patients harbor large amounts of t...

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98 Infectious Diseases Newsletter 10(12) December 1991 and in foreign-device-associated infections as well. Many CF patients harbor large amounts of this organism, 106 to 10 t° cfu/ml sputum, and receive multiple antipseudomonal course. Thus, this group of patients gives the opportunity to study the development and mechanisms of resistance of P. aeruginosa. The results from such studies may also be important for physicians treating other groups of patients, since it may define a resistance problem that is occurring more frequently in patients other than those with cystic fibrosis.

Mechanisms of Resistance

P aeruginosa has intrinsic resistance to a large number of antibiotics, including many beta-lactam antibiotics. The outer membrane in effect constitutes a considerable penetration barrier, which is to a large extent responsible for the high intrinsic resistance of P. aeruginosa. Despite the development of new, more active beta-lactams, P. aeruginosa have continued to express resistance mechanisms against even the most potent agents. The resistance mechanisms are related to the drug's penetration of the cell wall, its inactivation by betalactamases (chromosomal or plasmid mediated) located in the periplasmic space, its affinity for the penicillin binding proteins, or a combination of these factors. In addition, the ability of the bacteria to form biofilms offers a very effective protection that will be discussed later in more detail. A major cause of beta-lactam resistance is the inactivation of the compound by beta-lactamase catalyzed hydrolysis. Compounds with increased stability to many beta-lactamases, such as aztreonam (monobactams) and third generation

cephalosporins, eg, ceftazidime, cefsulodin, and ureidopenicillins (eg, piperacillin) have been marketed but there are increasing numbers of reports of beta-lactamase-mediated resistance to these antibiotics. All P. aeruginosa strains possess a chromosomal beta- lactamase. This enzyme is usually expressed at very low basal level in the absence of antibiotics, but it can be induced to much higher levels in their presence. Induction is therefore a transient response to the presence of a beta-lactam antibiotic. On the other hand, strains exhibiting stable derepression permanently produce elevated amounts of the enzyme. Derepression may be partial, such that the organism produces an unusually high uninduced level of the enzyme but retains inducibility, or total, such that beta-lactamase expression is constitutive. Imipenem is known to be a very strong inducer, whereas piperacillin is a weak inducer in vitro. Ceftazidime and cefsulodine induce betalactamase production to rather high levels in vitro. Aztreonam is a weak inducer and acts as a beta-lactamase inhibitor as well. Many in vitro studies have shown the importance of the beta-lactamases as resistance mechanisms together with the penetration barrier posed by the cell wall. It has become clear that the interaction between the penetration rate of the cell wall and the rate of removal of the beta-lactam antibiotic by hydrolysis and/or trapping plays an important role in the activity of beta-lactam agents against P. aeruginosa, and modification of penicillinbinding proteins have been reported. But what happens in vivo during the antipseudomonal course? We studied the in vivo development of resistance of P. aeruginosa in CF patients as well as the activity of beta-

lactamase in CF sputum during the course. We found as expected that during a two-week course of antipseudomonal beta-lactam antibiotic therapy, the proportion of resistant P. aeruginosa cells increase significantly. By using a very large inoculum we were able to demonstrate the presence of several subpopulations exhibiting various beta-lactam antibiotic sensitivities in each sputum sample, including preexisting resistant subpopulations. The resistant bacteria were present at a significant proportiorr--10-20% of total P. aeruginosa - - in sputum before treatment. This should be distinguished from the more usual condition, where one resistant cell may exist per 106 to 107 cells. At the end of treatment this pattern was changed and most P. aeruginosa present in the sputum showed decreased sensitivity to beta-lactam antibiotics. The heterogeneity of the population before treatment changed to a greater homogeneity with less variation in levels of resistance (Fig. 1). The majority of the resistant P. aeruginosa isolates selected in vivo were stable, partially derepressed producers of chromosomal Class I type beta-lactamase, and outer membrane analysis did not show any significant differences in the number and intensity of outer membrane protein bands. Inhibition of the beta-lactamase therefore should restore the activity of the beta-lactam antibiotic. In vitro sensitivity studies showed that addition of the beta-lactamase inhibitor tazobactam restored the beta-lactam activity in the in vivo selected resistant cells. The MIC values decreased for all 126 isolates from 185.2 ~tg/mi to 21.9 ~tg/mi (geometric mean) (Fig. 2). The MIC of one highly resistant strain decreased from 200 to 6.2 ~g/ml. These results are in accordance with

NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operatiou of any methods, products, instructions or ideas contained in the material herein. No suggested test or procedure should be carried out unless, in the reader's judgment, its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug dosages should be made. Discussions, views, and recommendations to medical procedures, choice of drugs and drag dosages are the responsibility of the authors. Infectious Diseases Newsletter (ISSN 0278-2316) is issued monthly in one indexed volume per year by Elsevier Scienca Publishing Co., Inc.. 655 Avenue of the Americas, New York, N e w York 10010. Printed in USA at Hanover, PA 17331. Subscription price per year: institutions, $158.00; individuals, $92.00. For postage outside the U.S., add $40.00 (Canada and Mexico require on additional postage). Second-class postage paid at New York, NY. and at additional mailing offices. Postmaster: Send address changes to Infectious Diseases Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas. New York. New York 100 I0.

© 1991 Elsevier Science Publishing Co., Inc. 0278-2316/91/$0.00 + 2.20

99 Infectious Diseases Newsletter 10(12)

December 1991

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the hypothesis that production of chromosomal Class I type beta- iactamase is a major resistance mechanism in the in vivo selected subpopulation. However, the interaction of resistance mechanisms, such as outer membrane permeability, penicillin binding protein affinity, and degradation by betalactamase, need further investigation. Imipenem seems to be different from the other beta-lactams used in antipseudomonal treatment in being betalactamase-stable in vitro, and changes in outer membrane proteins as resistance mechanisms have been reported. Even when an apparently susceptible e. aeruginosa is cultured from the patients prior to and during the treatment the beta-lactam antibiotics often seem to be ineffective. One among several possibilities are that the betalactam antibiotics are inactivated in the CF lungs by beta-lactamases. We investigated the in vivo activity of P. aeruginosa beta-lactamase in sputum from CF patients during antipseudomonal treatment and showed a significant increase to high levels in sputum from patients treated with piperacillin, imipenem, ceftazidime, and eefsuIodine in combination with tobramycin. Aztreonam was found to be masking/inhibiting the beta-lactamase activity in sputum. The presence of beta-lactamase in sputum at such high levels could lead to inactivation of beta-lactam antibiotics by hydrolysis or trapping, thus rendering them ineffective. Another point is that a change of antibiotic from a strong/middlestrong inducer such as imipenem and ceftazidime to another beta-lactam antibiotic could enhance the in vivo hydrolysis/inactivation because of a high local beta-lactamase activity, inlracellularly as well as extracellularly.


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Biofilm Mode of Growth

Another very important resistance factor is the inherent resistance of bacterial microcolonies and biofilms. It has been shown that P. aeruginosa establish biofilms on the mucosal surfaces


Infectious Diseases Newsletter 10(12) December 1991

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830) might prove to be a useful compound for the treatment of infections caused by P. aeruginosa. Tazobactam is an irreversible ("suicide") inhibitor of plasmid mediated as well as chromosomally mediated Class I beta-lactamases. This inhibitor restored the in vitro activity of piperacillin in P. aeruginosa strains isolated from 126 CF patients entering the Danish CF Centre (n = 126, MIC (geometric median) decreased from 185.2 I,tg/ml) to 21.9 lag/ml, and we suggest that clinical trials should be carried out, Combinations of the beta-lactamase inhibitor/beta-lactam antibiotic aztreonam with other beta-lactam antibiotics and tobramycin might also prove to be a useful compound for the treatment of infections caused by P. aerugi-





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Fig. 2. Sensitivity of 126 P. aeruginosa strains from 120 CF patients to piperacillin. IC50 is defined as the antibiotic concentration at which 50% of the inoculum is inhibited. of the pulmonary tracs and on intravenous, intra~erial, and urinary catheters as well as microcolonies. The bacteria adhere to these surfaces by using exopoly-saccharide glycocalyx polymers and initiate the development of adherent bacterial biofilms by cell division and glycocalyx formation. This heralds the onset of infections which readily become chronic, despite stringent antibiotic treatment regimens. Within the biofilm glycocalyx, the sessile bacteria are shielded from the action of surfactants, antibodies. antibiotics, and leucocytes. P. aeruginosa biofilms are highly resistant to beta-lactam antibiotics as well as aminoglycosides. In a previous study we have shown induction of beta-lactamase production in P. aeruginosa biofilms. Piperacillin and imipenem penetrated the biofilms and triggered the production of beta-lac-

tamase enzyme. The presence of such levels of beta-lactam-degrading enzymes in a biofilm would be expected to afford a large measure of protection from beta-lactam antibiotics, and the interplay of beta-lactarnase production with the other protective properties of the biofilm mode of growth, including decreased growth rate, could be a major reason for the persistence of these bacteria in chronic P. aeruginosa infections.

Prospective for Future Research The use of an effective beta-lactamase inhibitor in combination with a betalactam antibiotic such as tobramycin could revise the position of beta-lactam antibiotics in the treatment of P. aeruginosa infections. Such a betalactamase inhibitor is not available at the moment, but a novel betalactamase inhibitor tazobactam (YTR©1991 Elsevier Science Publishing Co., Inc. 0278-2316/91/$0.00 + 2.20

nosa. Bibliography Brown MRW, Allison DG, Gilbert P: Resistance of bacterial biofilms to antibiotics: A growth-rate related effect? J Antimicrob Chemother 22:777-783, 1988. Bryan LE, Kwan S, Godfrey AJ: Resistance of Pseudomonas aeruginosa mutants with altered control of chromosomal beta-lactamase to piperacillin, ceftazidhne, and cefsulodine. Antimicrob Agents Chemother 25:382-384, 1984. Bush K, Sykes RB: Beta-lactamase inhibitots in perspective. J Antimicrob Chemother 11:97-107, 1983. Curtis NAC, Orr D, Boulton MG, Ross GW: Penicillin-binding proteins of Pseudomonas aeruginosa. Comparison of two strains differing in their resistance to beta-lactam antibiotics. J Antimicrob Chemother 7:127-136, 1981. Costerton JW, Cheng KJ, Geesy GG, Ladd TI, Curtis N, Dasgupta M, Marrie TJ: Bacterial biofilms in nature and disease. Ann Rev Microbiol 41:435---464, 1987. Hoyle BD, Jas J, Costerton JW: The biofilm glycocalyx as a resistance factor. J Antimicrob Chemother 26:1---6, 1990. Giwercman B, Lambert PA, Rosdahl VT, Shand GH, H0iby N: Rapid emergence of resistance in Pseudomonas aeruginosa in cystic fibrosis patients due to in vivo selection of stable partially derepressed beta-lactamase producing strains. J Antimicrob Chemother

101 Infectious Diseases Newsletter 10(12) December 1991 26:247-259, 1990. Giwereman B, Jensen El', H¢iby N, Kharazmi A, Costerton JW: Induction of beta-lactamase production in Pseudomonas aeruginosa biofilm. Antimicrob Agents Chemother 35:1008--1010, 1991. Hancock REW: Bacterial outer membranes: Evolving concepts. Features 57:175-182, 1991. Hoiby N, Kock C: Pseudomonas aeruginosa infection in cystic fibrosis and its management. Thorax 45:881-884, 1990. Jensen ET, Kharazmi A, Lam K, Costerton JW, Hoiby N: Human polymorphonu-

clear leucocyte response to Pseudomonas aeruginosa grown in biof'dms. Infection and Immunity 58:2383-2385, 1990. Livermore DM: Penicillin-binding proreins, porins and outer-membrane permeability of carbenicillin-resistant and -susceptible strains of Pseudomonas aeruginosa, l Med Microb 18:261-270, 1984. Moosdeen F, Williams JD, Yamabe S: Antibacterial characteristics of YTR-830, a sulfone beta-lactamase inhibitor, compared with those of clavulanic acid and sulbactam. Antimicrob Agents Chemo-

ther 32:925-927, 1988. Sanders CC, Sanders WE: Clinical importance of inducible beaa-lactamases in gram-negative bacteria. Europ J Clin Microb 6:435--437, 1987. Stratton CW, Tansk F: Synergistic resistance mechanisms in Pseudomonas aeruginosa. J Antimicrob Chemother 19:413--416, 1987. Address correspondence to Birgit Giwercman, MD, Research Fellow, Department of Clinical Microbiology, 8223, Rigshospitalet, Julinne Mariesvej 28, 2100 X, Copenhagen, Denmark.


Lactobacillus acidophilus Liver Abscess Natalie Klein, MD, Paul E. Schoch, PhD, and Burke A. Cunha, MD Infectious Disease Division and Department of Pathology, WinthropUniversityHospital, Mineola, New York, and SUNY School of Medicine, Stony Brook, New York

Introduction Lactobacillus sp. are part of the nor-

mal flora of the mouth, female genital tract, and gastrointestinal tract. Although Lactobacilli have been implicated as a cause of dental caries, they are an unusual cause of other human infections. Nine cases of lactobacillemia were reported by Bayer et al. Three cases related to pregnancy, three were cases of endocarditis, two were cases associated with gastrointestinal disease, and one was dental abscess. Bourne et al described four cases of bacteremia with Lactobacil/us sp.--two had gastrointestinal disease, one had endocarditis, and one was associated with a postoperative fever. Lactobacillemia secondary to pyelonephrolithiasis and to urinary tract infection with stasis has also been reported. This is the first reported case implicating L. acidophitus as a cause of pyogenic liver abscess. Anaerobic bacteria are commonly isolated from liver abscess. In one series, anaerobes were isolated from 45% of liver abscesses. The most frequently encountered an-

aerobic bacteria were microaerophilic streptococci, Bacteroides sp., Fusobacterium sp., Clostridia, and Actinomyces.

Case Report An 86-year-old woman with a history of hypertension, noninsulin-dependent diabetes mellitus, an old left-sided cerebrovascular accident with residual expressive aphasia, and chronic cholecystitis was admitted after a fall at home. Two years prior to admission she had a normal barium enema and hepatobiliary scan. She denied any recent fever, chills, nausea, vomiting, or abdominal pain. Her family reported that she was allergic to penicillin. Examination revealed a mildly confused elderly female with a blood pressure of 130/70 mm Hg, a temperature of 100.8°F, and a pulse of 95/minute. A grade 2/6 systolic murmur was heard best at the apex; the lungs were clear. Abdominal examination revealed marked right upper quadrant tenderness but bowel sounds were present and there was no guarding or rebound. The white blood cell count © 1991 Elsevier Science Publishing Co., Inc. 0278-2316/91/$0.00 + 2.20

was 15A00/mm 3 with 92% neutrophils, 6% bands, 2% lymphocytes. The hemoglobin was 12.8 g/dl, the BUN was 20 mg/dl, the creatinine 1.4 mg/dl. The SGOT was 451 IU/L, LDH 802 IU/L, and CPK > 3000 IU/L with negative MB bands. A C T scan of the brain revealed an old left middie cerebral artery infarction. The patient was begun on aztreonam and clindamycin for presumed acute cholecystitis. A hepatobiliary scan showed nonvisualization of the gallbladder. An ultrasound o f the abdomen revealed a contracted gallbladder with multiple gallstones, and a hererogenous area in the medial segment of the left lobe of the liver suggestive of a mass. A C T scan confh'med the presence of a hypodense mass of the liver consistent with a hepatic abscess and the patient underwent a CTguided aspiration/drainage of the liver abscess. L. acidophilus grew from three sets of blood cultures drawn on the day of admission and was also isolated as the only organism from the liver abscess. The patient became afebrile on anti-