22 Correspondence - Oxford Journals

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Journal of Antimicrobial Chemotherapy (1998) 42, 547–566

Correspondence patterns. Furthermore, specimens are not obtained at all from many patients in the community. It may be that there are also international differences in requesting patterns and selection of patients for testing, although this possibility has not yet been explored. All of these differences are likely to bias the results and, ultimately, any conclusions arising from them. In this respect, typing of isolates and establishing mechanisms of resistance are of prime importance in that clonal spread of a single isolate has implications which are markedly different from the simultaneous emergence of several distinct clones. Finally, there is a need to develop more sophisticated methods of analysing changing patterns of susceptibility and relating them to other factors, such as the population studied, antimicrobial usage and systems of healthcare delivery, in order to determine the economic impact of resistance. To date, this has proved difficult to quantify.3 In conclusion, we must look beyond performing even larger multicentre national or international susceptibility testing programmes and try to improve the quality and relevance of the data such programmes generate.

Decrease in antibiotic susceptibility or increase in resistance?—defects of present antimicrobial susceptibility surveillance J Antimicrob Chemother 1998; 42: 547 Alasdair MacGowana* and Peter Bennettb a

Bristol Centre for Antimicrobial Research and Evaluation, Southmead Health Services NHS Trust and University of Bristol, Department of Medical Microbiology, Southmead Hospital, Bristol; b Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, UK *Correspondence address: Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK. Tel: 44-(0)117-959-5652; Fax: 44-(0)117-959-3154. Sir, In their recent leading article, Walker & Thornsberry1 highlighted the need for an adequate monitoring system for antibiotic susceptibility testing, both nationally and internationally. They point out that external quality assurance, a standardized methodology—preferably MICbased—and standardized definitions of susceptibility are required. This last point is not completely accurate because the publication of full data on susceptibility testing or access via the Internet can facilitate the reinterpretation of data with different breakpoints; recently published data from the Alexander Project point the way in this respect.2 In addition, Walker & Thornsberry have ignored several major sources of unquantified bias in the type of surveillance they describe. Firstly, such surveys report resistance as a function of isolates, while a parameter of greater interest would be resistance defined by episode of infection. Secondly, missing from their proposal are important linked epidemiological data relating to the patients from whom specimens are obtained, i.e., age, sex, type of infection, previous antimicrobial therapy and whether the infection was community- or hospital-acquired. In addition, all of the isolates tested are collected simply on the basis that they are recovered from specimens referred for diagnostic purposes. Our own local data indicate that there are marked differences amongst general practitioners and practice groups in terms of their requesting

References 1. Walker, R. D. & Thornsberry, C. (1998). Decrease in antibiotic susceptibility or increase in resistance? Journal of Antimicrobial Chemotherapy 41, 1–4. 2. Felmingham, D., Grüneberg, R. N. & The Alexander Project Group. (1996). A multicentre collaborative study of the antimicrobial susceptibility of community-acquired, lower respiratory tract pathogens 1992–1993: The Alexander Project. Journal of Antimicrobial Chemotherapy 38, Suppl. A, 1–57. 3. Coast, J., Smith, R. D. & Millar, M. R. (1996). Superbugs: should antimicrobial resistance be included as a cost in economic evaluation. Health Economics 5, 217–26.

547 © 1998 The British Society for Antimicrobial Chemotherapy

Correspondence

Reply

Antibiotic susceptibilities of verocytotoxinproducing Escherichia coli O157 and non-O157 strains isolated from patients and healthy subjects in Germany during 1996

J Antimicrob Chemother 1998; 42: 548 R. D. Walkera* and C. Thornsberryb a

Department of Microbiology, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824; bMRL Pharmaceutical Services, 7003 Chadwick Drive, Suite 235, Brentwood, TN 37027, USA

J Antimicrob Chemother 1998; 42: 548–550 Herbert Schmidt, Jeannette von Maldeghem, Matthias Frosch and Helge Karch Institut für Hygiene und Mikrobiologie der Universität Würzburg, Bau 17, Josef-SchneiderStrasse 2, D-97080 Würzburg, Germany

*Tel: 1-517-353-2296; Fax: 1-517-353-4426; E-mail: [email protected] Sir, We thank MacGowan & Bennett for their thoughtful comments concerning our recent leading article on the need for uniform methods to detect changes in bacterial susceptibility to antimicrobial agents. We agree with many of the issues they have raised. However, we also feel that they missed the major point of the article, that bacterial resistance is essentially identified by in-vitro susceptibility tests. In order for this to occur, the tests must be performed according to standardized methodologies. We acknowledge that susceptibility data can be viewed on the Internet. However, unless there is uniformity in testing and interpretation, data appearing on the Internet may be misleading. There are two ways of detecting resistance based on antimicrobial susceptibility tests. The first includes those tests performed in diagnostic laboratories to guide the clinical use of antimicrobials. However, the data produced by these tests tend to be less useful for surveillance purposes, especially if results are reported only in terms of the susceptibility category, i.e. susceptible or resistant. Moreover, they would be unlikely to provide the information MacGowan & Bennett call for. Our point was that if these clinical tests could be carried out by determining MICs with appropriate ranges of antibiotic concentrations, the resulting data could be used as a form of surveillance for detecting reduced susceptibility. The second method of undertaking surveillance by susceptibility testing is one for which surveillance itself is the primary objective. In studies of this type, much of the information alluded to by MacGowan & Bennett can be gathered as part of the process. To reiterate, the message we attempted to convey was that in-vitro antimicrobial susceptibility tests are essential for the purpose of monitoring changes in susceptibility to antibacterial agents. However, in order to do so, the tests must be performed according to uniform methods.

*Tel: 49-931-201-5160; Fax: 49-931-201-3445; E-mail: [email protected] Sir, Verocytotoxin-producing strains of Escherichia coli (VTEC), also referred to as Shiga toxin (Stx)-producing E. coli (STEC) or enterohaemorrhagic E. coli (EHEC), are associated with a range of illnesses, including mild diarrhoea, haemorrhagic colitis and the haemolytic– uraemic syndrome (HUS). Most VTEC strains belong to serotype O157, with the remainder belonging to serotypes other than O157, such as O26, O111 and O103.1 The management of patients with VTEC infections and the sequelae of these infections continues to be non-specific and largely empirical. The diarrhoeal phase is self-limiting and the role of early antibiotic therapy in the prevention of HUS is still unclear.1 A further complication is the isolation of VTEC strains exhibiting resistance to commonly used antibiotics.2–4 The aims of the present study were to determine the in-vitro susceptibilities of VTEC strains isolated in Germany in 1996 from patients with infections caused by these organisms and from healthy subjects and to characterize those strains that exhibited resistance. The 166 VTEC strains, which were isolated from 61 patients with HUS and 64 with diarrhoea and from 41 healthy individuals, have been described previously.5 Susceptibility to ampicillin, piperacillin, cefotaxime, ceftazidime, gentamicin, tetracycline, trimethoprim– sulphamethoxazole, ofloxacin, ciprofloxacin, chloramphenicol, imipenem and streptomycin was determined by the disc diffusion method 6 and the MICs for strains found to be resistant to one or more of these agents were determined by the Etest method (AB Biodisk, Solna, Sweden); susceptibility categories were allocated according to breakpoints recommended by the National Committee for Clinical Laboratory Standards.6 Conjugation experiments were performed by a standard filter mating technique as described previously,7 with E. coli DH5 (nalidixin-resistant) as the recipient. Of the 166 strains, 19 were resistant to at least one of the antibiotics tested; none of the isolates was resistant to

548

Correspondence cefotaxime, ceftazidime, gentamicin, ofloxacin, ciprofloxacin or imipenem. The antibiotic resistance patterns, the Shiga toxin genotypes and the diseases produced by the 19 strains are shown in the Table. Six strains were resistant to one antibiotic, six to two, and seven to four or more. All but two strains were resistant to tetracycline. In all cases, resistance was high-level, with MICs ranging from 24 to 256 mg/L. Interestingly, only one strain exhibiting resistance belonged to serotype O157, the other 18 strains belonging to non-O157 serotypes. Resistance was more frequently associated with Stx1 production (13 of 19 strains, 68.4%) than with Stx2 production (three of 19, 15.8%) or production of both toxins (three of 19, 15.8%). All 19 resistant strains were used in the conjugation experiments and, as 17 of these were resistant to tetracycline, this antibiotic was used as a selective marker for the transconjugants. Only four strains transferred tetracycline resistance by conjugation and, in two of these, resistance to ampicillin and piperacillin was transferred with that to tetracycline. This study has demonstrated that 11.4% of VTEC strains isolated from symptomatic patients and healthy subjects were resistant to at least one of the antibiotics tested. Only one of the 78 (1.3%) E. coli O157 strains exhibited resistance, compared with 18 of 88 (20.5%) nonO157 strains. It is unlikely that resistance was secondary to

the administration of antibiotics to patients with diarrhoea as most VTEC-associated diseases occur in children up to 5 years of age in whom tetracycline is contraindicated. Because most of the antibiotic-resistant strains were non-O157 serotypes, which are isolated from cattle more frequently than O157 strains, it might be assumed that the resistance phenotypes had originated in cattle. However, the use of tetracycline as a growth promoter was prohibited in Germany 20 years ago. None the less, ampicillin and tetracycline resistance have been detected in high frequencies amongst Enterobacteriaceae isolated from farm animals and, as resistance plasmids often carry multiple resistance genes, it may be that tetracycline resistance has persisted throughout the past 20 years as the result of selective pressures induced by the administration of other antibiotics. Antibiotic resistance in VTEC strains has both clinical and epidemiological implications. Resistant strains would have an advantage over other Enterobacteriaceae colonizing the gastrointestinal tracts of cattle given antibiotics either for veterinary reasons or as growth promoters, thereby increasing the proportion of such strains in these animals. As Enterobacteriaceae are known to be able to transfer genes from species to species, these VTEC strains may represent a resistance gene pool. This being the case, periodic surveillance of the antibiotic susceptibilities of these bacteria would be an important

Table. Features and antibiotic resistance patterns of VTEC strains isolated in Germany in 1996 Strain

Serotype

stx genotype

Disease

Resistance pattern

4992 6037 4417 5380 5727 5721 8574 6153 5236

O157:H7 O111:H– O111:H– O111:H– O111:H2 O111:H2 O26:H11 O26:H11 O26:H11

1, 2 1, 2 1 1 1 1 1 1 1

diarrhoea HUS HUS diarrhoea diarrhoea diarrhoea diarrhoea diarrhoea diarrhoea

O26:H11

1

diarrhoea

5720 3204 7140 7793 7792 3985

O26:H– O8:H– O8:H– O84:H– O84:H– O145:H–

2 1 1 1 1 2

HUS AC diarrhoea diarrhoea HUS HUS

7395 5021 7879

O118:H– Orough:H– O152:H4

1,2 2c 1

diarrhoea AC AC

tetracycline tetracycline, streptomycin tetracycline tetracycline, streptomycin tetracycline tetracycline, streptomycin tetracycline, ampicillin, piperacillin, chloramphenicol tetracycline, ampicillin, piperacillin, chloramphenicol tetracycline, ampicillin, piperacillin, trimethoprim–sulphamethoxazole, chloramphenicol, streptomycin tetracycline, ampicillin, piperacillin, chloramphenicol, streptomycin tetracycline, ampicillin, piperacillin, streptomycin tetracycline, trimethoprim–sulphamethoxazole tetracycline, streptomycin tetracycline, trimethoprim–sulphamethoxazole tetracycline, trimethoprim–sulphamethoxazole tetracycline, ampicillin, piperacillin, chloramphenicol, streptomycin tetracycline, ampicillin, piperacillin, streptomycin streptomycin tetracycline

8399

Abbreviations: HUS, haemolytic–uraemic syndrome; AC, asymptomatic carrier.

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Correspondence measure in helping to control the spread of resistant pathogens.

References 1. Griffin, P. M. (1995). Escherichia coli O157:H7 and other enterohemorrhagic Escherichia coli. In Infections of the Gastrointestinal Tract (Blaser, M. J., Smith, P. D., Ravdin, J. I., Greenberg, H. B. & Guerrant, R. L., Eds), pp. 739–62. Raven Press, New York. 2. Farina, C., Goglio, A., Conedera, G., Minelli, F. & Caprioli, A. (1996). Antimicrobial susceptibility of Escherichia coli O157 and other enterohaemorrhagic Escherichia coli isolated in Italy. European Journal of Clinical Microbiology and Infectious Diseases 15, 351–3. 3. Ratnam, S., March, S. B., Ahmed, R., Beazanson, G. S. & Kasatiya, S. (1988). Characterization of Escherichia coli serotype O157:H7. Journal of Clinical Microbiology 26, 2006–12. 4. Kim, H. H., Samadpour, M., Grimm, L., Clausen, C. R., Besser, T. E., Baylor, M. et al. (1994). Characteristics of antibiotic-resistant Escherichia coli O157:H7 in Washington State, 1984–1991. Journal of Infectious Diseases 170, 1606–9. 5. Bockemühl, J., Karch, H. & Tschäpe, H. (1997). Infektionen des Menschen durch enterohämorrhagische Escherichia coli (EHEC) in Deutschland, 1996. Bundesgesundheitsblatt 6, 194–7. 6. National Committee for Clinical Laboratory Standards. (1990). Performance Standards for Antimicrobial Disc Susceptibility Tests: Approved Standard M2-A4. NCCLS, Villanova, PA. 7. Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

In-vitro activities of cefepime and other -lactam antibiotics against clinical isolates from a Colombian teaching hospital J Antimicrob Chemother 1998; 42: 550–552 Salim Mattar*, Liliana Sánchez, Dalis Pérez, Alvaro Arango, Renata Parodi and Claudia Muelle Unidad de Microbiologia Especial, Departamento de Microbiologia, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia *Fax: 57-1-285-0503; E-mail: [email protected] Sir, Gaps in the spectra of third-generation cephalosporins and increasing numbers of reports of resistance to these agents amongst aerobic Gram-negative bacilli (AGNB), particularly Klebsiella pneumoniae, Enterobacter spp. and Pseudomonas aeruginosa, have prompted a search for new compounds to which these pathogens are susceptible.1 Cefepime is a ‘fourth-generation’ cephalosporin that possesses a broad spectrum of activity that includes

AGNB resistant to third-generation cephalosporins, aztreonam and aminoglycosides and methicillin-susceptible staphylococci.2,3 In this study, we compared the in-vitro activity of cefepime with those of five other -lactams against clinical isolates recovered from patients at a Colombian teaching hospital. The 82 non-replicate isolates, which were identified by standard laboratory techniques, included the following: Escherichia coli (11 strains); Citrobacter freundii (ten); P. aeruginosa (ten); Enterobacter spp. (ten); Klebsiella spp. (ten); Serratia marcescens (five); Acinetobacter spp. (five); Proteus spp. (four); Staphylococcus aureus (nine); and coagulase-negative staphylococci (CNS) (eight). The antibiotics studied were cefepime, cefotaxime, cefoperazone/ sulbactam (2:1 ratio), imipenem, ceftazidime and aztreonam. MICs were determined by the Etest method according to the manufacturer’s instructions and susceptibility categories were allocated according to breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS).4 The susceptibilities of the isolates to the six antibiotics are summarized in the Table. Cefepime was the most active agent overall against the AGNB, 92% of the isolates being susceptible compared with 91% susceptible to imipenem, 82% to cefoperazone/sulbactam, 78% to ceftazidime, 75% to aztreonam and 74% to cefotaxime. Cefepime was also the most active antibiotic against the P. aeruginosa isolates, 100% of which were susceptible. These results show a greater degree of susceptibility than those of Fekete et al.5 who reported that only 70% of their P. aeruginosa isolates were susceptible. The difference is probably accounted for by the fact that cefepime has not been available in Colombia for a sufficiently long period for resistant strains to have been selected. Alternatively, the small number of strains examined by us may not accurately reflect the susceptibilities of Colombian isolates to this agent. Cefepime exhibited superior or equal activity to all of the other agents tested against the E. coli, C. freundii, Acinetobacter spp., S. marcescens and Proteus spp. isolates. The only agent which was more active than cefepime was imipenem against the Klebsiella and Enterobacter spp. isolates (70% and 90% respectively versus 100% and 100% respectively). Of the Grampositive bacteria, 100% were susceptible to cefepime, cefotaxime and imipenem, 94% to cefoperazone/ sulbactam and 64% to ceftazidime. Four strains of S. aureus, four of P. aeruginosa, three of Enterobacter spp. and two of Acinetobacter spp. were isolated from blood cultures obtained from patients with nosocomial infections. Cefepime was the most active agent against these pathogens, 100% of which were susceptible, compared with 87% which were susceptible to imipenem, 83% to cefoperazone/sulbactam and 75% to ceftazidime. The high percentages of resistance to third-generation cephalosporins amongst the AGNB clinical isolates in this study are likely to be the result of excessive and

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Correspondence inappropriate prescribing of these drugs; the ability to obtain antibiotics without prescription in Colombia is a major contributing factor. Periodic surveys of the antibiotic resistance patterns of clinical isolates in developing countries such as ours are necessary in order to monitor trends and to facilitate empirical therapy. Making better use of antibiotics is clearly a priority for the future but, in the meantime, the results of this small study suggest that cefepime represents a suitable choice as empirical monotherapy of patients with serious infections caused by multidrug-resistant AGNB or when the aetiology is unknown.

References 1. Holloway, W. & Palmer, D. (1996). Clinical applications of a new parenteral antibiotic in the treatment of severe bacterial infections. American Journal of Medicine 100, Suppl. 6A, 52S–9S. 2. Segreti, J. & Levin, S. (1996). Bacteriologic and clinical applications of a new extended-spectrum parenteral cephalosporin. American Journal of Medicine 100, Suppl. 6A, 45S–51S. 3. Thornsberry, C. & Yee, Y. C. (1996). Comparative activity of eight antimicrobial agents against clinical bacterial isolates from the United States, measured by two methods. American Journal of Medicine 100, Suppl. 6A, 26S–38S. 4. National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA. 5. Fekete, T., Tumah, H., Woodwell, J., Satishchandran, V., Truant, A. & Axelrod, P. (1996). Comparative susceptibilities of Klebsiella species, Enterobacter species, and Pseudomonas aeruginosa to 11 antimicrobial agents in a tertiary-care university hospital. American Journal of Medicine 100, Suppl. 6A, 20S–5S.

In-vitro activity of DU-6859a against methicillin-resistant Staphylococcus aureus isolates with reduced susceptibilities to vancomycin J Antimicrob Chemother 1998; 42: 552–553 Mayumi Tanakaa*, Naoya Wadaa, Seiko Mori Kurosakaa, Megumi Chibaa, Kenichi Satoa and Keiichi Hiramatsub a

New Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd, 16-13 Kitakasai 1-Chome, Edogawa-ku, Tokyo 134-8630; bDepartment of Bacteriology, Juntendo University, Tokyo, Japan *Tel:

81-3-3680-0151; Fax:

81-3-5696-8344.

Sir, A methicillin-resistant strain of Staphylococcus aureus, Mu50, exhibiting reduced susceptibility to vancomycin

(VISA), was isolated in Japan in 1996.1,2 The strain was also resistant to various other antibiotics, including the quinolones.3 The present study was undertaken to evaluate the in-vitro activity of DU-6859a, a novel investigative quinolone, against VISA strain Mu50, its presumptive precursor strain Mu3 (hetero-VISA) and its in-vitro derivative, Mu3-8R, which was selected following single passage of Mu3 on agar containing vancomycin at a concentration of 8 mg/L. 2 We also determined the nucleotide sequences of the quinolone-resistance determining regions (QRDRs) of the genes encoding type II topoisomerases in these strains. Of the antibiotics studied, the quinolones—DU-6859a, levofloxacin, ciprofloxacin, sparfloxacin and tosufloxacin —were synthesized in New Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd; the others (vancomycin, minocycline, arbekacin and oxacillin) were obtained from their respective manufacturers. MICs were determined by an agar dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 4 with Brain Heart Infusion agar (Difco Laboratories, Detroit, MI, USA) being substituted for Mueller–Hinton agar. The QRDRs of grlA (the gene encoding subunit A of topoisomerase IV) and gyrA (the gene encoding subunit A of DNA gyrase) were amplified by the polymerase chain reaction (PCR). The resulting DNA was analysed by single strand conformational polymorphism (SSCP) according to a method described previously5 and/or sequenced with commercially available kits (AutoLoad Solid Phase Sequencing Kit, Pharmacia Biotech, Tokyo, Japan, or Cycle sequencing method, New England Biolabs, Beverly, MA, USA) and a fluorescence sequencer (Pharmacia Biotech, Tokyo, Japan).6 Substitution at codon 432 of grlB (which encodes topoisomerase IV subunit B) was detected by agarose gel electrophoresis of amplified DNA following treatment with HinfI, as described previously.6 The MICs of the antibiotics tested for the three MRSA strains and the methicillin-susceptible S. aureus control strain, FDA 209-P, are shown in the Table. The MRSA strains (Mu50, Mu3 and Mu3-8R) were resistant to minocycline, arbekacin, oxacillin, levofloxacin, ciprofloxacin, sparfloxacin and tosufloxacin on the basis of breakpoints recommended by the NCCLS;4 only the MICs of DU-6859a were 1.0 mg/L. The mutations in grlA and gyrA were identical in the three strains, presumably reflecting their clonal relatedness.2 There was a TCC TTC mutation in codon 80 of grlA and a TCA TTA mutation in codon 84 of gyrA, resulting in Ser Phe and Ser Leu substitutions respectively. No substitution was detected in codon 432 of grlB. The pattern of amino acid substitutions described is the one most commonly recognized in Japan5 in association with quinolone resistance.6,7 The results of this small study suggest that DU-6859a would be effective therapy in patients with infections

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Correspondence caused by VISA strains. This drug is currently undergoing Phase III studies in Japan and Phase II studies in Europe.8

References 1. Hiramatsu, K., Hanaki, H., Ino, T., Yabuta, K., Oguri, T. & Tenover, F. C. (1997). Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibilty. Journal of Antimicrobial Chemotherapy 40, 135–6. 2. Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y., Hori, S. et al. (1997). Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350, 1670–3. 3. Tenover, F. C., Lancaster, M. V., Hill, B. C., Steward, C. D., Stocker, S. A., Hancock, G. A. et al. (1998). Characterization of staphylococci with reduced susceptibility to vancomycin and other glycopeptides. Journal of Clinical Microbiology 36, 1020–7. 4. National Committee for Clinical Laboratory Standards. (1990). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Second Edition: Approved Standard M7A2. NCCLS, Villanova, PA. 5. Wong, T., Tanaka, M. & Sato, K. (1998). Detection of grlA and gyrA mutations in 344 Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 42, 236–40. 6. Tanaka, M., Onodera, Y., Uchida, Y., Sato, K. & Hayakawa, I. (1997). Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV purified from Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 41, 2362–6. 7. Yamagishi, J.-I., Kojima, T., Oyamada, Y., Fujimoto, K., Hattori, H., Nakamura, S. et al. (1996). Alterations in the DNA topoisomerase IV grlA gene responsible for quniolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 40, 1157–63. 8. Sato, K., Hoshino, K., Tanaka, M., Hayakawa, I. & Osada, Y. (1992). Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates. Antimicrobial Agents and Chemotherapy 36, 1491–8.

In-vitro activity of a novel penem, Men 10700, against meningococci and gonococci, and the effect of a cysteine-containing supplement J Antimicrob Chemother 1998; 42: 553–555 J. M. T. Hamilton-Miller* and Saroj Shah Department of Medical Microbiology, Royal Free Hospital School of Medicine, London NW3 2QG, UK *Tel:

44-171-794-0500; Fax:

44-171-435-9694.

Sir, Men 10700 is a new penem antibiotic with high in-vitro potency against Gram-positive cocci and many commonly 553

Correspondence encountered Gram-negative pathogens.1 We report here its activity compared with several other antibiotics against pathogenic Neisseria spp., and also our finding that penems, like carbapenems,2 are largely inactivated in the presence of cysteine-containing supplements, such as Vitox (Unipath, Basingstoke, UK), Isovitalex (BBL/ Difco, Moseley, UK) and CVA (Gibco-BRL, Paisley, UK), routinely used when testing gonococci. Media were obtained from Unipath. Twenty strains of Neisseria meningitidis and 40 of Neisseria gonorrhoeae (14 of the latter produced -lactamase) were grown overnight at 37°C on GC Agar Base containing 5% lysed horse blood and 1% Vitox in 6% CO2. Bacterial growth from these plates was suspended in Mueller–Hinton broth to an OD625nm of 0.15–0.2, and 1 L volumes were deposited on to plates containing doubling dilutions of antibiotic. Plates were read after overnight incubation at 37°C in 6% CO2. Staphylococcus aureus Oxford was used as a reference strain. The antibiotics used are shown in the Table. The medium used for the susceptibility testing was GC Agar Base with 5% lysed horse blood either by itself (for testing Men 10700 and imipenem against N. meningitidis), or supplemented with either 1% glucose and 2.5% yeast

hydrolysate (for testing Men 10700 and imipenem against N. gonorrhoeae) or 1% Vitox (for all other tests). Time–kill experiments were carried out with two strains of gonococci incubated alone and in the presence of 2 and 4 MICs of Men 10700, cefotaxime and imipenem, using ANM medium3 supplemented with 2.5% yeast dialysate. Counts were made at intervals on GC Agar Base containing 5% lysed horse blood and 1% Vitox, and the times required for 99.9% kill were read by interpolation from plots of viable count against time. It was found necessary to test penems in the absence of cysteine in order to maintain their full activity. Preliminary experiments showed that Vitox increased the MICs of Men 10700 and ritipenem (another investigational penem) against S. aureus by 8- to 16-fold (from 0.06 mg/L to 0.5 or 1 mg/L); MICs of Men 10700 against a panel of 20 gonococci were similarly 8- to 16-fold lower when the GC Agar Base medium was supplemented with glucose and yeast extract rather than Vitox. Vitox did not interfere with the MICs of any of the other antibiotics tested, and MICs for the control strain, S. aureus, were as expected.4 Results (Table) show that Men 10700 is as active as imipenem against gonococci (whether or not they produced -lactamase), and slightly more active against

Table. Activity of Men 10700 and comparators against 40 strains of N. gonorrhoeae (14 of which produced -lactamase) and 20 strains of N. meningitidis MIC parameter (mg/L) Antibiotic

Species

Men 10700

N. gonorrhoeae

Imipenem

N. meningitidis N. gonorrhoeae

Cefotaxime

N. meningitidis N. gonorrhoeae

Cefepime

N. meningitidis N. gonorrhoeae

Ceftriaxone

N. meningitidis N. gonorrhoeae

Ciprofloxacin

N. meningitidis N. gonorrhoeae

Co-amoxiclav

N. meningitidis N. gonorrhoeae

Spectinomycin

N. meningitidis N. gonorrhoeae

-Lactamase production + – – + – – + – – + – – + – – + – – + – – + –

range

I50

I90

0.03–0.25 0.008–0.25 0.015–0.13 0.03–0.25 0.016–0.25 0.015–0.25 0.004–0.13 0.001–0.13 0.004–0.015 0.008–0.13 0.008–0.13 0.008–0.03 0.001–0.06 0.001–0.03 0.001–0.004 0.002–0.016 0.002–0.13 0.004 0.25–1 0.03–1 0.06–0.5 8–128 8–16

0.06 0.053 0.014 0.06 0.05 0.02 0.004 0.012 0.048 0.011 0.012 0.007 0.002 0.0034 0.001 0.003 0.002 0.003 0.5 0.18 0.053 5.6 6

0.12 0.19 0.03 0.12 0.15 0.06 0.054 0.07 0.012 0.055 0.095 0.015 0.012 0.022 0.002 0.007 0.006 0.004 0.85 0.75 0.13 7.8 9

554

geometric mean 0.085 0.08 0.025 0.09 0.076 0.038 0.013 0.018 0.005 0.022 0.032 0.011 0.005 0.003 0.002 0.004 0.004 0.004 0.64 0.25 0.09 9.8 8.9

Correspondence meningococci. Meningococci were two to three times more sensitive to all the compounds tested, except ciprofloxacin (which was equally active against both species). The times required for 99.9% kill of the two strains of gonococci by Men 10700, imipenem and cefotaxime at two or four times their respective MICs ranged from 3.3 to 4.3 h. Thus, these compounds resembled ofloxacin, pefloxacin and spectinomycin in their rates of killing,5 and were more rapidly bactericidal than rifampicin.6 We conclude that Men 10700 has the potential to be an effective drug against gonococcal and meningococcal infections. Like imipenem and meropenem, penems are unstable in the presence of sulphydryl groups so, when carrying out susceptibility testing using any antibiotic in either of these classes, supplements such as Vitox, Isovitalex and CVA should be avoided.

Acknowledgements We thank Anna King for supplying some of the -lactamase-producing strains of N. gonorrhoeae, and Dr M. De Luca of Menarini Ricerche for valuable discussions and financial support.

References 1. Hamilton-Miller, J. M. T. & Shah, S. (1997). In-vitro microbiological assessment of a new penem, Men 10700. Journal of Antimicrobial Chemotherapy 39, 575–84. 2. Jones, R. N., Gavan, T. L., Thornsberry, C., Fuchs, P. C., Gerlach, E. H., Knapp, J. S. et al. (1989). Standardization of disk diffusion and agar dilution susceptibility tests for Neisseria gonor rhoeae: interpretive criteria and quality control guidelines for ceftriaxone, penicillin, spectinomycin and tetracycline. Journal of Clinical Microbiology 27, 2758–66. 3. Hafiz, S. & McEntergart, M. G. (1976). Prolonged survival of Neisseria gonorrhoeae in a new liquid medium. British Journal of Venereal Diseases 52, 381–3. 4. Working Party of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–47. 5. Crenn, Y., Meyran, M., Samson, T. & Cavallo, J. D. (1994). Bactericidal activity of six antibiotics against Neisseria gonorrhoeae. Journal of Antimicrobial Chemotherapy 33, 855–60. 6. Hamilton-Miller, J. M. T., Bruzzese, T., Nonis, A. & Shah, S. (1996). Comparative anti-gonococcal activity of S-565, a new rifamycin. International Journal of Antimicrobial Agents 7, 247–50.

In-vitro activity of a combination of two oral -lactams (cefpodoxime and amoxycillin) against Streptococcus pneumoniae isolates with reduced susceptibilities to penicillin J Antimicrob Chemother 1998; 42: 555–557 David M. Johnson and Ronald N. Jones* Medical Microbiology Division, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242, USA *Corresponding author. Tel: 1-319-356-2990; Fax: 1-319-356-4916; E-mail: [email protected]

Sir, Streptococcus pneumoniae is one of the most important causes worldwide of upper and lower respiratory tract infections. Although strains of pneumococci with reduced susceptibilities to penicillin (MICs 0.12 mg/L) have compromised therapy of patients with closed space infections such as meningitis and acute otitis media,1 efforts to treat patients with non-meningeal infections caused by pneumococci exhibiting intermediate susceptibility or resistance to penicillin with high dosages of penicillins or newer cephalosporins (cefotaxime or ceftriaxone) have generally been successful. In-vitro studies have evaluated the activities of oral cephalosporins, fluoroquinolones and -lactam/ -lactamase inhibitor combinations against pneumococci with reduced susceptibilites to penicillin.1–3 Both initial4 and subsequent studies1–3 reported that the MIC90 of cefpodoxime for S. pneumoniae (0.12 mg/L) was two- to 64-fold lower than those of other orally administered cephems such as cefixime, cefaclor and cefadroxil. Of the penicillins, amoxycillin, with or without clavulanic acid, has been shown to exhibit activity that is equal or superior to that of penicillin,1–3 thereby ensuring that its role as therapy of patients with infections caused by S. pneu moniae is preserved. In this study, we have evaluated the in-vitro activity of a combination of cefpodoxime proxetil (the orally active ester of cefpodoxime), a methoxyiminoaminothiazolyl third-generation cephalosporin, and amoxycillin against S. pneumoniae isolates with reduced susceptibilities to penicillin. The strains studied were 25 non-replicate clinical isolates of S. pneumoniae from the University of Iowa College of Medicine collection; 13 strains exhibited intermediate susceptibility to penicillin (MICs 0.12–1 mg/L) and 12 were resistant (MICs 2 mg/L). The antibiotics tested were cefpodoxime (Pharmacia–Upjohn, Kalamazoo, MI, USA), amoxycillin (SmithKline Beecham Pharmaceuticals, King of Prussia, PA, USA) and penicillin

555

Correspondence (Sigma Chemical Co., St Louis, MO, USA). Synergy studies were performed by a chequerboard microbroth dilution technique according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS);5 the medium was 3% lysed horse blood (PML Microbiologics, Wilsonville, OR, USA) and the trays were stored at –70°C until used. The final suspension contained 5 105 cfu/L and S. pneumoniae ATCC 49619 was included as a control with each test. MICs (taken as the lowest concentration giving total inhibition of growth) of each antibiotic alone and in combination were read after incubation for 24 h at 35°C in an atmosphere containing 5% CO2. The fractional inhibitory concentration index (FICI) for each isolate was calculated according to the following formula:

MIC of amoxycillin in combination MIC of amoxycillin alone

MIC of cefpodoxime in combination MIC of cefpodoxime alone FIC of amoxycillin FIC of cefpodoxime FIC FICI The results were interpreted as follows: synergy, FICI 0.5; partial synergy, 0.5 FICI 1; addition, FICI 1; indifference, 1 FICI 4; and antagonism, FICI 4. The MICs of cefpodoxime and amoxycillin, alone and in combination, and the FICIs for the 25 isolates are summarized in the Table. Overall, 12 strains (eight of 13 pencillin-intermediate and four of 12 penicillin-resistant) exhibited synergy (one strain) or partial synergy (11

Table. In-vitro activities of amoxycillin and cefpodoxime alone and in combination against 25 strains of S. pneumoniae with reduced susceptibilities to penicillin MIC (mg/L) Bacterium

amoxycillin

Penicillin-intermediate (n 13) 102 0.12 019-204 0.12 023-124 0.12 019-202 0.12 020-121 0.12 001-025 0.12 113 0.25 014-293 0.25 004-433 0.5 020-123 0.5 011-087 0.5 124 1 141 2 Penicillin-resistant (n 12) 025-406 1 025-414 1 125 1 127 1 019-187 1 003-363 1 001-022 1 020-120 2 001-031 2 138 4 089 4 004-001 4

cefpodoxime 0.12 0.5 0.5 0.5 2 0.12 0.5 0.12 1 1 1 1 2 1 1 2 2 2 2 2 1 2 4 4 16

amoxycillin/cefpodoxime in combination

FICI

Interactive category

0.015/0.015 0.06/0.25 0.06/0.015 0.06/0.12 0.06/0.12 0.06/0.06 0.12/0.12 0.12/0.015 0.25/0.5 0.5/1 0.5/1 0.12/0.5 0.5/1

0.250 1.000 0.531 0.750 0.563 1.000 0.750 0.625 1.000 2.000 2.000 0.625 0.750

synergic additive partially synergic partially synergic partially synergic additive partially synergic partially synergic additive indifferent indifferent partially synergic partially synergic

0.5/0.12 0.25/0.5 0.5/1 0.5/1 0.5/1 0.5/1 0.5/1 0.5/1 1/0.12 2/2 4/4 2/4

0.625 0.750 1.000 1.000 1.000 1.000 1.000 1.250 0.563 1.000 2.000 0.750

partially synergic partially synergic additive additive additive additive additive indifferent partially synergic additive indifferent partially synergic

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Correspondence strains). Although interpretative criteria for cefpodoxime when tested alone against pneumococci with reduced susceptibilities to penicillin have not been published to date, 11 of the 25 strains would be categorized as resistant to cefpodoxime if the criteria recommended for parenteral -lactams, such as ceftriaxone and cefotaxime (susceptible, MICs 0.5 mg/L; resistant, MICs 2 mg/L), are used.5 However, in the presence of amoxycillin, the MICs for three of these isolates were markedly reduced (fourfold in one case and 16-fold in two). Of the 14 strains that were either of intermediate susceptibility or resistant to amoxycillin (MICs 1 mg/L), ten, including two of the six resistant strains (MICs 2 mg/L), were susceptible to this agent (MICs 0.5 mg/L) when combined with cefpodoxime at a concentration 1 mg/L. The percentage of pneumococcal isolates exhibiting intermediate susceptibility or resistance to penicillin has increased dramatically in recent years. Antibiotics that are suitable as treatment of patients with infections caused by these organisms include novel -lactams and some of the older penicillins such as amoxycillin. Previous in-vitro studies have shown cefpodoxime and amoxycillin each to be more active than cefixime, cefadroxil and cefaclor.1–4 In the present study, the MICs of either amoxycillin or cefpodoxime or a combination of the two for 88% of pneumococcal isolates exhibiting reduced susceptibility to penicillin were 0.5 mg/L; included amongst these were three strains classified as resistant to amoxycillin (MICs 2 mg/L). Recently, Chavanet et al.6 similarly reported in this journal synergy between a potent parenteral cephalosporin (ceftriaxone) and amoxycillin against S. pneumoniae strains with reduced susceptibilities to both penicillin and third-generation cephalosporins. In conclusion, this and previous in-vitro studies1–3 have demonstrated the potent activities of cefpodoxime and amoxycillin against one of the principal bacterial causes of community-acquired respiratory tract infections. As antibiotics are often prescribed without knowledge of either the pathogen or its susceptibility in these clinical settings, there is a need for clinical trials comparing single agents with combinations of -lactams, particularly in regions where pneumococci with reduced susceptibilities to penicillin are common.

References 1. Jones, R. N., Pfaller, M. A., Doern, G. V., Verhoff, J., Jones, M., Sader, H. S. et al. (1997). Initial report of a longitudinal, international antimicrobial surveillance study (SENTRY): alarming resistance rates in monitored sites (68 medical centers) in the USA, Canada, South America and Europe. In Proceedings of the ThirtySeventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 1997. Abstract E-109, p. 133. American Society for Microbiology, Washington, DC. 2. Pankuch, G. A., Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1995). Activities of oral and parenteral agents against pencillin-

susceptible and -resistant pneumococci. Antimicrobial Agents and Chemotherapy 39, 1499–504. 3. Frampton, J. E., Brogden, R. N., Langtry, H. D. & Buckley, M. M. (1992). Cefpodoxime proxetil. A review of its antibacterial activity, pharmacokinetic properties and therapeutic potential. Drugs 44, 889–917. 4. Jones, R. N. & Barry, A. L. (1988). Antimicrobial activity and disk diffusion susceptibility testing of U-76,253A (R-3746), the active metabolite of the new cephalosporin ester, U-76,252 (CS-807). Antimicrobial Agents and Chemotherapy 32, 443–9. 5. National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA. 6. Chavenet, P., Dalle, F., Delisle, P., Duong, M., Pechinot, A., Buisson, M. et al. (1998). Experimental efficacy of combined ceftriaxone and amoxycillin on penicillin-resistant and broadspectrum cephalosporin-resistant Streptococcus pneumoniae infection. Journal of Antimicrobial Chemotherapy 41, 237–46.

Antimicrobial interactions of trovafloxacin and extended-spectrum cephalosporins or azithromycin tested against clinical isolates of Pseudomonas aeruginosa and Stenotrophomonas maltophilia J Antimicrob Chemother 1998; 42: 557–559 David M. Johnson, Ronald N. Jones* and Michael A. Pfaller Medical Microbiology Division, University of Iowa College of Medicine, Iowa City, Iowa, USA *Corresponding author. Department of Pathology, C606 GH, University of Iowa College of Medicine, Iowa City, IA 52242, USA. Tel: 1-319-356-2990; Fax: 1-319-356-4916. Sir, Pseudomonas aeruginosa and Stenotrophomonas malto philia are aerobic Gram-negative non-fermentative bacilli that can lead to serious infections, especially in immunosuppressed or neutropenic patients and burn victims.1–4 Both species are intrinsically resistant to several antimicrobial agents by various mechanisms, such as low membrane permeability, efflux mechanisms, or the production of -lactamase enzymes.3,4 Resistance to one or more classes of antimicrobial agents often develops during the course of single-agent chemotherapy.2–4 Because of the difficulty in treating infections caused by these organisms, combination drug therapy is often necessary. Recently, studies have been performed to investigate the activity of fluoroquinolones combined with other agents against these

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Correspondence bacteria.1,2,4,5 In this study, trovafloxacin was combined with azithromycin (potentially active against virulence factor production), cefepime, cefoperazone and ceftazidime using the chequerboard assay and tested against recent clinical isolates of P. aeruginosa and S. maltophilia from several geographic locations.3 Cefepime was obtained from Bristol-Myers Squibb (Wallingford, CT, USA), ceftazidime from Glaxo– Wellcome, Inc. (Research Triangle Park, NC, USA), and azithromycin, cefoperazone and trovafloxacin from Pfizer Inc. (Groton, CT, USA). A total of 83 organisms (40 P. aeruginosa, including isolates from cystic fibrosis patients, and 43 S. maltophilia) were tested. All were recent clinical isolates and included strains from the University of Iowa College of Medicine (Iowa City, IA, USA), Brazil (Dr Helio Sader, São Paulo, Brazil) and Switzerland (Dr Reno Frei; Basle, Switzerland). Each

strain was tested with the chequerboard dilution assay method in cation-adjusted Mueller–Hinton broth microdilution trays (PML Microbiologics, Wilsonville, OR, USA). The organisms were stored in sterile distilled water and then subcultured twice before each strain was inoculated into a tray at a final concentration of 5 105 cfu/mL. The trays were then incubated for 16–20 h in ambient air at 35°C. After incubation, the trays were examined to determine the MIC for each drug.6 All tests were performed and susceptibility data analysed according to the recommended methods issued by the National Committee for Clinical Laboratory Standards (NCCLS).6 The susceptibility of a non-fermentative bacillus quality control strain (P. aeruginosa ATCC 27853) was tested on each day of testing. Ceftazidime and cefepime were the most potent agents against P. aeruginosa (70% susceptible) while only trova-

Table. Results of antimicrobial interaction (synergy) studies for trovafloxacin tested with azithromycin, cefepime, cefoperazone or ceftazidime against P. aeruginosa and S. maltophilia clinical strains Interaction categoryb Organism (n)/co-drug P. aeruginosa (40) azithromycin

cefepime

cefoperazone

ceftazidime

S. maltophilia (43) azithromycin

cefepime

cefoperazone

ceftazidime

Significance of resultsa

synergy

partial synergy

additive

indifferent

indeterminant

significant insignificant total significant insignificant total significant insignificant total significant insignificant total

0 0 0 6 0 6 6 1 7 4 0 4

0 1 1 23 2 25 18 0 18 24 1 25

0 2 2 5 0 5 8 2 10 7 1 8

0 0 0 0 1 1 1 1 2 2 0 2

0 37 37 2 1 3 0 3 3 1 0 1

significant insignificant total significant insignificant total significant insignificant total significant insignificant total

0 0 0 22 0 22 21 0 21 20 1 21

1 3 4 14 2 16 16 0 16 15 1 16

0 5 5 0 0 0 1 0 1 2 0 2

0 0 0 0 0 0 0 0 0 0 0 0

2 32 34 3 2 5 3 2 5 3 1 4

a A significant interaction produces MIC results within the susceptible or intermediate range for both drugs, e.g. achievable levels in humans at usually administered doses. b The drug interaction definitions were as follows. Synergy, four-fold or more decrease in the MIC of both drugs; partial synergy, a four-fold or more decrease in one drug and a two-fold decrease in the co-drug; additive, a two-fold decrease in the MIC of both drugs; indifference, no significant change in the MIC of either drug; indeterminate, MICs above or below the relevant dilution schedules utilized.

558

Correspondence floxacin, ceftazidime and cefoperazone (84%, 77% and 65% susceptible, respectively) demonstrated any significant activity against S. maltophilia (data not shown). Azithromycin had very limited activity against both species (MIC50 16 mg/L). When combined with trovafloxacin, cefepime and ceftazidime were equally potent, producing enhanced activity interactions (synergy or partial synergy) against 70% of P. aeruginosa, while the combination of cefoperazone and trovafloxavin was active against 60% of the isolates (Table). All three cephalosporins were potent against S. maltophilia ( 80% synergy or partial synergy) when combined with trovafloxacin. Cefoperazone and cefepime combinations with trovafloxacin were slightly more active than ceftazidime. Importantly, synergy was demonstrated at clinically achievable concentrations of each of these antimicrobial agents.5 Azithromycin combined with trovafloxacin yielded only one significant interaction (partial synergy) against an S. maltophilia strain, but was inactive as a co-drug against P. aeruginosa. However, azithromycin reduced pigment production in all P. aeruginosa strains at concentrations of 2 mg/L (data not shown). In conclusion, the results of this and previous studies1,2,4,5 show that several drug combinations have potent activity against P. aeruginosa and S. maltophilia. Interestingly, although macrolides have been shown to be clinically effective with long-term low-dose use in chronic lung infections, 3 this study found that azithromycin alone or in combination with trovafloxacin had essentially no measurable activity by routine standardized susceptibility tests6 against P. aeruginosa or S. maltophilia. Clinical studies to determine the potential therapeutic applications for these cited drug combinations against these tested species, especially in cases of chronic infections (i.e. cystic fibrosis), are certainly warranted.

References 1. Visalli, M. A., Bajaksouzian, S., Jacobs, M. R. & Appelbaum, P. C. (1997). Comparative activity of trovafloxacin, alone and in combination with other agents, against gram-negative nonfermentative rods. Antimicrobial Agents and Chemotherapy 41, 1475–81. 2. Howe, R. A. & Spencer, R. C. (1997). Macrolides for the treatment of Pseudomonas aeruginosa infections? Journal of Antimicrobial Chemotherapy 40, 153–5. 3. Klepser, M. E., Patel, K. B., Nicolau, D. P., Quintiliani, R. & Nightingale, C. H. (1995). Comparison of the bactericidal activities of ofloxacin and ciprofloxacin alone and in combination with ceftazidime and piperacillin against clinical strains of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 2503–10. 4. Denton, M. & Kerr, K. G. (1998). Microbiological and clinical aspects of infection associated with Stenotrophomonas maltophilia. Clinical Microbiology Reviews 11, 57–80. 5. Visalli, M. A., Bajaksouzian, S., Jacobs, M. R. & Appelbaum,

P. C. (1998). Synergistic activity of trovafloxacin with other agents against gram-positive and -negative organisms. Diagnostic Microbiology and Infectious Disease 30, 61–4. 6. National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

Emergence in vivo of resistance to ampicillin in a clinical isolate of Enterococcus hirae J Antimicrob Chemother 1998; 42: 559–561 Rosana Massaa, Carlos Bantarb, Marta Molleracha, Federico Nicolab, Barbara E. Murrayc, Jorgelina Smayevskyb and Gabriel Gutkinda* a

Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, and bLaboratorio de Microbiología, Centro de Educación Médica e Investigaciones Clínicas, Buenos Aires, Argentina; c Division of Infectious Diseases, University of Texas Medical School, Houston, TX, USA *Corresponding author. Departamento de Microbiología, Inmunología y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 954, 1113 Buenos Aires, Argentina. Fax: +54-1-962-5341; E-mail: [email protected] Sir, Enterococci are increasingly being identified as important causes of severe infections, such as endocarditis and septicaemia, and of urinary tract infections in hospitalized patients and renal transplant recipients. Their intrinsic resistance to many antibiotics limits the number of agents available for use as treatment; this problem is compounded by the capacity of these bacteria to acquire resistance during therapy.1 Fontana et al.,2 while investigating Enterococcus hirae R40, a penicillin-resistant mutant selected in vitro from E. hirae ATCC 9790 (formerly Streptococcus faecium ATCC 9790), were the first to propose the basis of intrinsic penicillin resistance amongst enterococci, namely increased production of the low-affinity penicillin-binding protein (PBP), PBP 5; the degree of penicillin resistance is proportional to the amount of protein produced. Klare et al.3 have recently emphasized the feasibility of selecting resistant mutants that overproduce low-affinity PBPs by serial passage in the presence of increasing concentrations of penicillin, in a manner that simulates exposure to standard dosages of -lactams in vivo. To the best of our

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Correspondence knowledge, no clinical isolate of E. hirae that exhibits reduced susceptibility to penicillins has been reported. In this study, we have characterized such a strain, as well as the penicillin-susceptible parent strain. A strain of E. hirae with reduced susceptibility to penicillins (EHR) (MICs of ampicillin and penicillin, 4 and 8 mg/L respectively) was isolated from the urine of a patient with persistent bacteriuria after treatment for 10 days with ampicillin in a total daily dosage of 1.5 g. The penicillin-susceptible parent strain (EHS) (MICs of ampicillin and penicillin 0.5 mg/L) was isolated before ampicillin therapy was initiated. Both strains were identified by standard laboratory techniques4 and identification was confirmed by hybridization with a DNA probe for a muramidase-2 gene from an E. hirae clone.5 Identical patterns were obtained with EHR and EHS following resolution of SmaI-digested DNA by pulsed-field gel electrophoresis performed according to methods described previously.6,7 A penicillin-resistant laboratory mutant (EHM) (MICs of ampicillin and penicillin, 16 and 32 mg/L respectively) was selected following serial passage of EHS in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) containing increasing concentrations of penicillin (0.5–4 mg/L); E. hirae R40 was used as a reference strain. Although -lactamase-producing enterococci have been identified previously, no -lactamase activity was detected in EHR, EHS or EHM by the chromogenic cephalosporin method with nitrocefin (Oxoid, Basingstoke, UK) as the substrate.8 As the emergence of high-level resistance to penicillin in enterococci has principally been associated with alterations to the PBPs, we analysed the PBPs of EHR, EHS and EHM with 125I-labelled penicillin X as the radiotracer. Penicillin X (p-hydroxybenzylpenicillin) was synthesized, purified and iodinated as described previously.9 Membranes, which were prepared as described elsewhere,10 were incubated at 37ºC for 1 h in screwcapped microcentrifuge tubes containing varying concentrations of 125I-labelled penicillin X. Labelling was interrupted by transferring the reaction tubes to an ice bath and by simultaneously adding cold penicillin X at a concentration of 200 mg/L. The proteins were solubilized by adding sample buffer (60 mM Tris–HCl, pH 6.8, containing 2% sodium dodecyl sulphate, 5% mercaptoethanol, 0.002% bromophenol blue and 10% glycerol) and boiling for 3 min. Following centrifugation at 13,500g for 1 min, the proteins in the supernatants were separated by SDS–PAGE at 50 V. The gels were fixed and stained with 0.25% Coomassie Brilliant Blue R in acetic acid– methanol–water (10:40:50, by volume) and decolorized by successive solvent changes. They were then dried in vacuo and exposed to Kodak X-ray film with two intensifying screens at –70ºC for approximately 15 days. The magnitude of the signals attributable to PBP 5 that were detected in EHM and R40 with 125I-labelled

Figure. Western blots of the membrane proteins of EHS, EHR, EHM and E. hirae R40 (reference strain).

penicillin X were greater than those detected in EHR and EHS, but there were no marked differences between EHR and EHS in terms of their PBPs (data not shown). Western blotting with a polyclonal antibody to the PBP 5 of E. hirae R40 was performed as described previously.11 As shown in the Figure, the signals for PBP 5 in the membrane proteins of R40 and EHM were greater than those in the membrane proteins of EHS and EHR. Furthermore, no novel PBP with atypical electrophoretic mobility, including PBP 3r described in E. hirae S185r by Piras et al.,11 was identified in EHR or EHM by PBP analysis or by Western blotting with polyclonal antibody to PBP 3r (data not shown). We have shown that the investigations carried out in the present study are capable of detecting overproduction of a low affinity PBP in the control strain, R40, and in the in-vitro-derived mutant EHM, thereby eliminating this mechanism as the basis of penicillin resistance in EHR. Therefore, resistance in this isolate may be attributable to a slight, indeed undetectable, decrease in the affinities of -lactams for one or more PBPs or to an unidentified mechanism. In the patient from whom the resistant strain was isolated, the administration of nitrofurantoin sterilized the urine, confirming that resistance to ampicillin was, at least in part, the reason for the persistence of the strain of E. hirae during treatment with this antibiotic.

Acknowledgements This work was supported, in part, by grants from UBACYT, Argentina, to M. M. and G. G. and from CONICET, Argentina, to G. G. We gratefully acknowledge the valuable technical contribution of T. Coque and K. V. Singh. We also thank J. Coyette for providing the anti-PBP 5 and anti-PBP 3r antibodies and E. hirae R40, and O. Mascaretti and C. Borchetti for their collaboration in the synthesis of penicillin X.

References 1. Murray, B. E. (1990). The life and times of the Enterococcus. Clinical Microbiology Reviews 3, 46–65.

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Correspondence 2. Fontana, R., Cerini, R., Longoni, P., Grossato, A. & Canepari, P. (1983). Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin. Journal of Bacteriology 155, 1343–50. 3. Klare, I., Rodloff, A. C., Wagner, J., Witte, W. & Hakenbeck, R. (1992). Overproduction of a penicillin-binding protein is not the only mechanism of penicillin resistance in Enterococcus faecium. Antimicrobial Agents and Chemotherapy 36, 783–7. 4. Facklam, R. R. & Sahm, D. F. (1995). Enterococcus. In Manual of Clinical Microbiology, 6th edn (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 308–14. American Society for Microbiology, Washington, DC. 5. Chu, C. P., Kariyama, R., Daneo-Moore, L. & Shockman, G. D. (1992). Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae. Journal of Bacteriology 174, 1619–25. 6. Murray, B. E., Singh, K. V., Heath, J. D., Sharma, B. R. & Weinstock, G. M. (1990). Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. Journal of Clinical Microbiology 28, 2059–63. 7. Miranda, A., Singh, K. V. & Murray, B. E. (1991). DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool. Journal of Clinical Microbiology 29, 2752–7. 8. Murray, B. E. & Mederski-Samoraj, B. (1983). Transferable -lactamase. A new mechanism for in vitro penicillin resistance in Streptococcus faecalis. Journal of Clinical Investigation 72, 1168–71. 9. Masson, J. M. & Labia, R. (1983). Synthesis of a 125Iradiolabeled penicillin for penicillin-binding proteins studies. Analytical Biochemistry 128, 164–8. 10. Coyette, J., Ghuysen, J.-M. & Fontana, R. (1980). The penicillin-binding proteins in Streptococcus faecalis ATCC 9790. European Journal of Biochemistry 110, 445–6. 11. Piras, G., el Kharroubi, A., van Beeumen, J., Coeme, E., Coyette, J. & Ghuysen, J. M. (1990). Characterization of an Enterococcus hirae penicillin-binding protein 3 with low penicillin affinity. Journal of Bacteriology 172, 6856–62.

Relationship between mutations in the coding and promoter regions of the norA genes in 42 unrelated clinical isolates of Staphylococcus aureus and the MICs of norfloxacin for these strains J Antimicrob Chemother 1998; 42: 561–563 Franz-Josef Schmitza,b*, Britta Hertela, Basia Hofmanna, Sibylle Scheuringc, Jan Verhoefb, A. C. Fluitb, Hans-Peter Heinza, Karl Köhrerc and Mark E. Jonesb a

Institute for Medical Microbiology and Virology, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany; bEijkman-Winkler Institute for Clinical Microbiology, Utrecht University, Utrecht, The Netherlands; cMolekularbiologisches Zentrallabor im Biologisch-Medizinischen Forschungszentrum, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany *Correspondence address: Institute for Medical Microbiology and Virology, Heinrich-HeineUniversität Düsseldorf, Universitätsstrasse 1, Geb. 22.21, 40225 Düsseldorf, Germany. Tel: 49-2132-72040; Fax: 49-2132-72040. Sir, Fluoroquinolone resistance in Staphylococcus aureus, especially methicillin-resistant strains (MRSA), undermines the use of these drugs as effective therapy of patients with staphylococcal infections. This resistance is mediated by mutations in the following genes: the gyrA and gyrB genes which encode DNA gyrase; the grlA and grlB genes which encode DNA topoisomerase IV; and the norA gene (present in all S. aureus isolates) which encodes the drug efflux pump, NorA.1–4 A point mutation in the norA gene at base position 1085 and other unidentified mutations, together with increased norA expression, have all been proposed as conferring resistance to fluoroquinolones. In addition, it has been speculated that mutations in the potential regulatory protein binding site (8 bp inverted repeat) upstream of norA, which includes the –10 region of the putative promoter, mediate resistance to these antibiotics.1–4 Ng et al.3 identified a single nucleotide change 89 bp upstream from the putative ATG start codon and suggested that this mutation leads to increased levels of norA mRNA transcription. It is unclear whether mutations in the coding and/or promoter regions of norA contribute to reduced susceptibility to quinolones amongst clinical isolates with concomitant mutations in grl and gyr. In the present study, we sequenced the coding and promoter regions of norA in 42 randomly selected, unrelated, clinical isolates of S. aureus from eight different countries and attempted to

561

Correspondence correlate these data with the MICs of norfloxacin for the isolates. Thirty-one methicillin-resistant and 11 methicillinsusceptible strains of S. aureus were isolated in Germany (27 strains), Japan (three), Brazil (three), Switzerland (two), Sri Lanka (two), Spain (two), UK (two) or Hungary (one). These particular strains were selected because they represented all combinations of mutations in gyr and grl that were associated with reduced susceptibility to quinolones in 116 S. aureus strains.5 We have previously characterized grlA, grlB, gyrA and gyrB in these 42 isolates and demonstrated their contributions to decreased susceptibility to various quinolones.5 Norfloxacin was obtained from Sigma Chemical Co. (St Louis, MO, USA) and MICs were determined by a microbroth dilution method according to guidelines issued by the National Committee for Clinical Laboratory Standards.6 Oligonucleotide primers were selected on the basis of published norA DNA sequences.2,4 The primer sequences flanking the promoter region were 5 TATGATCAATCCCCTTTAT-3 (positions 269–287) and 5 -CTACCAGTTAATCCCAAATCTT-3 (positions 589–610) and those flanking the coding region were 5 -GGTCATTATTATATTCAGTTGTTG-3 (positions 1298–1321) and 5 -GTAAGAAAAACGATGCTAAT-3 (positions 1716–1735). Four independent PCRs and sequencing reactions were performed as described previously.5 The mutations in the coding and promoter regions of norA in the 42 isolates and the corresponding MICs of norfloxacin are shown in the Table. In 39 of the 42 isolates, the sequences of the norA coding region were either identical or closely related to the sequence reported by Yoshida et al. 2 In three isolates, base pair mutations that did not result in amino acid changes (Table) were associated with undetectable decreases in susceptibility. In

14 of the 28 norfloxacin-resistant isolates, Gly291 Asp substitutions were detected; similar substitutions were not identified in the 14 norfloxacin-susceptible isolates. As this mutation was present in only 50% of norfloxacin-resistant strains, it is unlikely that it was responsible for the increased MICs. Moreover, the same substitution has been identified in a partial sequence in a quinolone-susceptible strain, the implication being that it does not play an important role in norA-mediated quinolone resistance.7 In 36 of the 42 isolates sequenced, we found promoter region sequences identical to those previously described by Yoshida et al.,2 suggesting that this region is highly genetically stable. From the corresponding MIC data, it seems that base pair substitutions in six of the 42 S. aureus isolates played little or no role in resistance to the quinolones, the MICs for these strains ranging from 0.5 to 128 mg/L (Table). The MICs for four of these six strains ranged from 0.5 to 2 mg/L—values that are equal to or lower than those for wild-type strains without mutations in any of the gene loci contributing to fluoroquinolone resistance. In summary, the sequence of norA has been shown here to be highly conserved in 42 randomly selected clinical isolates of S. aureus. No particular mutation in norA is clearly associated with quinolone resistance, although in 14 of the 28 norfloxacin-resistant isolates (but in none of the 14 norfloxacin-susceptible isolates) there was a Gly291 Asp substitution in NorA. Changes in the NorA structure seem to have little or no effect on susceptibility to the quinolones, although it is possible that mutations in norA might contribute to reduced susceptibility when they occur in conjunction with mutations in the grl and gyr genes. However, to date, we have not found any isolates that would allow us to test this hypothesis. Finally, expression of norA may be regulated by an unidentified gene elsewhere on the chromosome.1,3

Table. Mutations and consecutive amino acid changes in the coding and promoter regions of norA in 42 S. aureus isolates and the corresponding MICs of norfloxacin Mutations coding region

No. of isolates with MICs (mg/L) of: promoter region

Type Aa Type A Type A, Gly291 Asp Type A Type A, Gly291 Asp Type A, T388 A Type A, A1389 T; Type B, A389 T; T1395 C; C1464 T G445 T; T498 A Type A, T1395 C, C1464 T Type B, A356 T, A389 T Type Bb Type B, A389 T Type B, C1738 T Type B, A389 T a

0.5

1

2

2

4

4

4

8

16

32

64

128

2

1 5

4 2

5 1 1

256 1 5

1 1 1

1 1

Sequence of the coding and promoter regions of the norA gene described by Yoshida et al.2 Sequence of the coding and promoter regions of the norA gene described by Kaatz et al.4 Isolates were designated as Type A or Type B according to whichever they most resembled. Type A and Type B differ by 60 bp in the coding region and 25 bp in the promoter region sequenced.

b

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References 1. Kaatz, G. W. & Seo, S. M. (1995). Inducible norA-mediated multidrug resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 2650–5. 2. Yoshida, H., Bogaki, M., Nakamura, S., Ubukata, K. & Konno, M. (1990). Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. Journal of Bacteriology 172, 6942–9. 3. Ng, E. Y. W., Trucksis, M. & Hooper, D. C. (1994). Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrobial Agents and Chemotherapy 38, 1345–55. 4. Kaatz, G. W., Seo, S. M. & Ruble, C. A. (1993). Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 37, 1086–94. 5. Schmitz, F.-J., Hofmann, B., Hansen, B., Scheuring, S., Lückefahr, M., Klootwijk, M. et al. (1998). Relationship between ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin (BAY 12-8039) MICs and mutations in grlA, grlB, gyrA and gyrB in 116 unrelated clinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 41, 481–4. 6. National Committee for Clinical Laboratory Standards. (1993). Methods for Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Third Edition: Approved Standard M7-A3. NCCLS, Villanova, PA. 7. Ohshita, Y., Hiramatsu, K. & Yokota, T. (1990). A point mutation in the norA gene is responsible for quinolone resistance in Staphylococcus aureus. Biochemical and Biophysical Research Communications 172, 1028–34.

Lowering of plasma valproic acid concentrations during concomitant therapy with meropenem and amikacin J Antimicrob Chemother 1998; 42: 563–564 B. J. G. De Turcka, M. W. Diltoera*, P. J. W. W. Cornelisa, V. Maesb, H. D. M. Spapena, F. Camua and L. P. Huyghensa a

Critical Care Department and bClinical Chemistry, Akademisch Ziekenhuis, Vrije Universiteit Brussel, Laarbeeklaan 101, B1090 Brussels, Belgium

*Corresponding author: Tel: 32-2-477-5178; Fax: 32-2-477-5179; E-mail: [email protected] Sir, Meropenem, a carbapenem launched in 1989, has a broad spectrum of activity that includes -lactamase-producing organisms. Compared with imipenem/cilastatin, meropenem has a lower affinity for the -butyric acid (GABA) receptor and thus a lower potential for inducing seizures;

this makes the latter drug, even at higher dosages, suitable for the treatment of patients with infections of the central nervous system.1,2 Because of in-vitro synergy between amikacin and meropenem, combinations of the two antibiotics have been used as treatment of nosocomial infections in critically ill patients.3 We report here our observations of decreases in the plasma concentrations of valproic acid to sub-therapeutic levels in two adult neurosurgical patients during concomitant therapy with meropenem and amikacin. The first patient, a 65 year old female, was admitted to the intensive care unit after suffering a subdural haemorrhage. Following insertion of an external ventricular drain to relieve obstructive hydrocephaly, she was given valproic acid 1200 mg as a continuous iv infusion over 24 h. Fluconazole was administered for oral candidosis which was diagnosed on the fourth post-operative day and suspected nosocomial pneumonia was treated with cefuroxime 1.5 g tds for 7 days. When an aerobic Gram-negative bacillus was isolated from blood cultures and bronchial secretions, the antibiotic therapy was changed to iv ciprofloxacin 400 mg tds. This was subsequently changed to meropenem 1 g tds and amikacin 15 mg/kg od when the pathogen was identified as Enterobacter aerogenes. Plasma valproic acid concentrations were measured once daily with a fluorescence polarization immunoassay (FPIA) on Axsym (Abbott, Hoofddorp, The Netherlands). The concentrations of this drug were maintained at therapeutic levels (50–100 mg/L) by the administration of total daily doses ranging from 1200 mg to 1600 mg. However, on the day after therapy with meropenem and amikacin was initiated, the valproic acid concentration fell to a subtherapeutic level despite the dosage of this drug having been supplemented (Figure). After 3 days of sub-therapeutic concentrations, the valproic acid was replaced as anti-epileptic prophylaxis with phenytoin 100 mg bd or tds, the concentrations of which remained at therapeutic levels without the need to adjust the dosage. The second patient, a 57 year old female, was given valproic acid as anti-epileptic prophylaxis following the clipping of multiple cerebral aneurysms; on the ninth postoperative day, iv phenytoin 100 mg tds was added to the valproic acid. The patient subsequently developed lobar pneumonia accompanied by thrombocytopenia, leucocytosis and fever. Cefuroxime 1.5 g tds was commenced as empirical therapy, but soon after was changed to coamoxiclav. As the patient’s clinical status deteriorated, amikacin and erythromycin were added. On the assumption that the infection might be caused by Pseudomonas aeruginosa, the co-amoxiclav was replaced with ceftazidime, and fluconazole was administered for both vulval candidosis and suspected fungal pneumonia. After 6 days, the phenytoin was inadvertently discontinued. When Klebsiella pneumoniae and P. aeruginosa were identified as the aetiological agents of the pneumonia, meropenem and amikacin were administered and all other anti-

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Figure. Temporal relationship between valproic acid dosage (—)/plasma concentration ( ) and the concomitant administration of other drugs in patient 1.

microbial agents were withdrawn. Despite supplementing the dosage of valproic acid, we observed that the plasma concentration of this drug fell from 44 mg/L to 5 mg/L within 24 h of starting the meropenem. The plasma elimination half-life of valproic acid, which is normally 15 h, was calculated by analysing samples of blood obtained after withdrawing the anti-epileptic drug and was found to be only 4 h. Both of the patients described in the present report survived and neither developed seizures. Although multiple other drugs were administered concomitantly, the plasma concentrations of valproic acid fell only after therapy with meropenem and amikacin was initiated. However, the meropenem was regarded as the likely cause as no such effect had been observed when amikacin was administered in its absence. Meropenem has been prescribed with many other different drugs, but the only adverse interaction reported to date has been with probenecid,4 although low serum concentrations of valproic acid were also recently described in three children receiving concurrent treatment with another carbapenem, panipenem/betamipron.5 While the mechanism of this interaction has not been determined, the speed of the decline in the levels of the anti-epileptic drug is not consistent with enzyme induction. Interference with protein binding is also unlikely as valproic acid is highly protein bound, whereas both meropenem and amikacin have low protein binding capacities. A possible explanation, which has been suggested for panipenem/betamipron,5 is that meropenem accelerates the renal excretion of valproic acid. While meropenem itself has a low propensity for causing seizures, the administration of this drug to epileptic patients being treated with valproic acid may be

associated with serious side effects as the result of the former causing the plasma concentrations of the latter to fall to sub-therapeutic levels. It is strongly recommended, therefore, that, at the very least, the plasma concentrations of valproic acid in such patients should be monitored and, preferably, that anti-epileptic prophylaxis or therapy be changed to an alternative drug, such as phenytoin.

References 1. Norrby, S. R., Newell, P. A., Faulkner, K. L. & Lesky, W. (1995). Safety profile of meropenem: international clinical experience based on the first 3125 patients treated with meropenem. Journal of Antimicrobial Chemotherapy 36, Suppl. A, 207–23. 2. Schmutzhard, E., Williams, K. J., Vukmirovits, G., Chmelik, V., Pfausler, B., Featherstone, A. & the Meropenem Meningitis Study Group. (1995). A randomised comparison of meropenem with cefotaxime or ceftriaxone for the treatment of bacterial meningitis in adults. Journal of Antimicrobial Chemotherapy 36, Suppl. A, 85–97. 3. Ferrara, A., Grassi, G., Grassi, F. A., Piccioni, P. D. & Gialdroni Grassi, G. (1989). Bactericidal acitivity of meropenem and interactions with other antibiotics. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 239–50. 4. Bax, R. P., Bastain, W., Featherstone, A., Wilkinson, D. M., Hutchison, M. & Haworth, S. J. (1989). The pharmacokinetics of meropenem in volunteers. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 311–20. 5. Nagai, K., Shimizu, T., Togo, A., Takeya, M., Yokomizo, Y, Sakata, Y. et al. (1997). Decrease in serum levels of valproic acid during treatment with a new carbapenem, panipenem/betamipron. Journal of Antimicrobial Chemotherapy 39, 295–6.

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Pharmacokinetics, safety and anti-human immunodeficiency virus (HIV) activity of hydroxyurea in combination with didanosine J Antimicrob Chemother 1998; 42: 565–566 Roberto Luzzatia*, Giovanni Di Perria, Doris Fendta, Dunia Ramarlia, Giampietro Broccalib and Ercole Conciaa a

Institute of Immunology and Infectious Diseases, University of Verona, Verona; bB.T. Biotecnica, Varese, Italy *Correspondence address: Divisione Clinicizzata di Malettie Infettive, Ospedale Civile Maggiore, Piazzale Stefani 1, 37126 Verona, Italy. Tel: 39-45-807-3295; Fax: 39-45-834-0223; E-mail: [email protected] Sir, Hydroxyurea is a hydroxamate compound that directly inhibits DNA synthesis as a result of its effect on ribonucleotide reductase, thereby decreasing the intracellular pool of deoxyribonucleotides. During the last 30 years, hydroxyurea has been used widely for treating patients with malignancies, especially chronic myelogenous leukaemia and myeloproliferative disorders.1 More recently, a combination of low-dosage hydroxyurea and didanosine (ddI) was shown to exhibit synergic activity against the human immunodeficiency virus (HIV) in vitro2 and, in a preliminary study, in vivo.3 In contrast to currently available, highly active antiretroviral therapies, this combination exerts its antiviral effect on both activated and quiescent human lymphocytes2 which constitute an important reservoir of latent virus. Steady-state plasma concentrations of hydroxyurea presumably adequate to suppress HIV replication have been detected in HIV-infected individuals receiving this agent as monotherapy or in combination with zidovudine.4 The aim of the present study was to investigate the plasma pharmacokinetics of hydroxyurea when administered in combination with ddI, both by the oral route, to HIV-infected patients. In addition, the safety and antiviral activity of this regimen were evaluated over a 24 week period. Subjects with CD4 T-lymphocyte counts of 200– 500/mm3 were included in the study. Other eligibility criteria were as follows: neutrophil count 1 109/L; 9 platelet count 100 10 /L; haemoglobin concentration 10 g/dL; serum creatinine concentration 1.5-fold greater than the upper limit of normal; serum transaminase and alkaline phosphatase concentrations 5-fold greater than the upper limits of normal; and serum amylase concentration within the normal range. Nine patients (seven males and two females) with a mean age of 34.7 years (range 27–48 years) received hydroxyurea

500 mg bd and ddI 200–300 mg bd. The diseases of seven patients were classified as CDC stage A2 and those of two as stage B2. At entry, the median CD4 count was 285/mm3 (range 230–488/mm3) and the median plasma HIV RNA load (as determined by a Quantiplex HIV RNA 2.0 assay, Chiron, Emeryville, CA, USA) was 3500 copies/mL (range 1000–415,000 copies/mL). All patients were ddI-naive, although seven had previously been treated with zidovudine. Written consent was obtained from all patients and the study was approved by the local research ethics committee. Samples of blood for determination of hydroxyurea concentrations were obtained 1, 2, 4, 6, 8 and 12 h after the first dose. In addition, pre-dose steady-state plasma concentrations were determined after 20–24 weeks of treatment. The hydroxyurea concentrations were determined by high-performance liquid chromatography with electrochemical detection; the limit of quantification was 0.006 mmol/L.5 Hydroxyurea plasma concentration–time data were evaluated by a non-compartmental model with an integrated computer system for pharmacokinetic analysis (Siphar, version 4.0, Micropharm International, SARL, Paris, France). The baseline characteristics of the patients and singledose pharmacokinetic parameters are summarized in the Table. Hydroxyurea was rapidly absorbed from the gastrointestinal tract and the mean ( S.D.) peak plasma concentration was 0.203 (0.063) mmol/L—twice the concentration (0.1 mmol/L) previously shown in an invitro study to result in complete suppression of HIV when used in combination with ddI.2 The mean ( S.D.) trough plasma concentration of hydroxyurea at steady state was 0.031 (0.006) mmol/L, a value that exceeded the concentration (0.0085 0.003 mmol/L) previously found in HIVpositive patients receiving hydroxyurea and zidovudine twice daily;4 the difference may be attributable to different rates of metabolism and/or interactions between hydroxyurea and zidovudine or ddI. The hydroxyurea/ddI regimen was well tolerated by the subjects, all of whom completed the course. One patient experienced an episode of neutropenia (0.7 109 neutrophils/L), but the count recovered when the daily dosage of hydroxyurea was halved for 2 weeks. The median increases in the CD4 counts from baseline were 15, 24 and 48 cells/mm3 at 4, 12 and 24 weeks respectively. The failure of the CD4 count to increase more dramatically in this study, as well in others,3 may have been accounted for by the cytostatic effect of hydroxyurea,1 particularly in patients with advanced HIV infection. The median decline in the plasma HIV RNA load from baseline was 2800 copies/mL at 4 weeks and this was maintained at 12 and 24 weeks. This reduction in viral load is similar to that reported by others in a study of ddIexperienced patients who were treated with ddI and hydroxyurea.3 Interestingly, this regimen led to long-term suppression of viral replication, with negligible HIV levels in plasma and lymphoid tissue being maintained for as

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Correspondence Table. Baseline characteristics of patients and single-dose pharmacokinetics of hydroxyurea Patient no.

Age (years)

Weight (kg)

Cmax (mmol/L)

Tmax (h)

AUC0– (mmol.h/L)

t½ (h)

1 2 3 4 5 6 7 8 9 Mean S.D.

40 48 36 30 38 27 32 34 28 34.7 6.2

81 87 80 71 70 49 68 52 90 72 13.5

0.191 0.140 0.185 0.162 0.192 0.308 0.164 0.313 0.171 0.203 0.063

1 2 1 2 2 1 2 1 1 1.44 0.52

0.726 0.748 0.831 0.633 0.777 1.136 1.044 1.554 0.702 0.906 0.293

2.54 2.15 3.08 2.37 2.2 2.17 3.52 3.21 2.44 2.63 0.51

long as 12 months after the treatment of two patients with high baseline CD4 counts was discontinued.6 This study has shown that combination therapy with hydroxyurea and ddI leads to substantial decreases in the plasma viral load and is generally well tolerated by patients with advanced HIV infection. The pharmacokinetic properties of hydroxyurea reported here suggest that this inexpensive drug, at current dosages, warrants further evaluation in controlled clinical trials.

References 1. Donehower, R. C. (1992). An overview of the clinical experience with hydroxyurea. Seminars in Oncology 19, Suppl. 9, 11–9. 2. Malley, S. D., Grange, J. M., Hamedi-Sangsari, F. & Vila, J. R. (1994). Synergistic anti-human immunodeficiency virus type 1 effect of hydroxamate compounds with 2 ,3 -dideoxyinosine in infected resting human lymphocytes. Proceedings of the National Academy of Sciences of the USA 91, 11017–21.

3. Montaner, J. S. G., Zala, C., Conway, B., Raboud, J., Patenaude, P., Rae, S. et al. (1997). A pilot study of hydroxyurea among patients with advanced human immunodeficiency virus (HIV) disease receiving chronic didanosine therapy: Canadian HIV Trials Network Protocol 080. Journal of Infectious Diseases 175, 801–6. 4. Villani, P., Maserati, R., Regazzi, M. B., Giacchino, R. & Lori, F. (1996). Pharmacokinetics of hydroxyurea in patients infected with human immunodeficiency type I. Journal of Clinical Pharmacology 36, 117–21. 5. Luzzati, R., Fendt, D., Ramarli, D., Parisi, S., Broccali, G. P. & Concia, E. (1997). Pharmacokinetics (PK), safety and antiviral activity of hydroxyurea (HU) in combination with didanosine (ddI) in HIV-infected individuals. In Program and Abstracts of the ThirtySeventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, 1997. Abstract A-6, p. 2. American Society for Microbiology, Washington, DC. 6. Vila, J., Nugier, F., Bargués, G., Vallet, T., Peyramond, D., Hamedi-Sangsari, F. et al. (1997). Absence of viral rebound after treatment of HIV-infected patients with didanosine and hydroxycarbamide. Lancet 350, 635–6.

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