Characterization of ceftriaxone-resistant Enterobacteriaceae: a molticentre study in 26 French hospitals

Transcription

1 Journal of Antimicrobial Chemotherapy (99), 9-0 Characterization of ceftriaxone-resistant Enterobacteriaceae: a molticentre study in French hospitals F. W. Goldstein*, Y. Pean*, A. Rosato', J. Gertner*, L. Gutmann' and the Vigil'Roc Study Group* "Laboratoire de Microbiologie Midicale, Hdpital Saint-Joseph, 7 rue Pierre Larousse, 77 Paris Cidex ; b Laboratoire de Bactiriologie, Hdpital International de I'Universite de Paris, Boulevard Jourdan, 70 Paris and 'Laboratoire de Microbiologie Medicate, Hdpital Broussais, 9 rue Didot, 77 Paris Cidex, France During a multicentre study performed in French hospitals, 7 (%) of 90 Enterobacteriaceae isolated, mainly Enterobacter spp., Serratia spp., Citrobacter spp. and Klebsiella spp. were classified as ceftriaxone resistant on the basis of an MIC > mg/l or the presence of an extended-spectrum /Mactamase. Extended-spectrum /Mactamase was present mainly in Klebsiella pneumonlae ( strains, 0-%) and very rarely in Escherichia coll, Proteus mirabilis, Klebsiella oxytoca, Citrobacter spp. and Enterobacter spp. The extended-spectrum /Mactamases conferred low-level resistance to ceftriaxone in nearly 0% of the strains harbouring them, emphasizing the need for routine testing for the presence of these enzymes. Among transconjugants three types of extended-spectrum /Mactamase were identified. Those resembling TEM- were the most common, but TEM-, and SHV- were also found. Clavulanate and to a lesser extent sulbactam inhibited all the extended-spectrum /Mactamases encountered in this study. Introduction Acquired resistance to extended-spectrum cephalosponns was reported soon after their introduction into clinical usage and was related to the hyperproduction of a chromosomal cephalosporinase (Sanders, 97). More recently, plasmid-mediated extended-spectrum /Mactamases inactivating extended-spectrum cephalosponns and B. Ferre (Centre Hospitaller, 90-Orsay); B. Hacquard (Centre Hospitalier, 770-Lagny); R. Bonete (Hdpital Saint-Michel, 770-Parls); F. Evreux (Hflpital J. Monod, 790-Montivillien); P. Lemaitre (Centre Hospitalier General, 009-Creil); J. C. Cartron (Centre Hospitalier General, 07-Dreux); O. Khallouf (Centre Hospitaller Rene Dubos, 90-Cergy-Pontoise); F. Canii (Centre Hospitalier, 900-Tourcoing); M. Bouuard (Centre Hospitaller, 07 Verdun); A. Trevoux (Centre Hoipltalier Regional, 0- Mulhouie); C. Cattoen (Centre Hospitalier General, 9-Valendennes); G. Wdsse (Hdpital S. S. M., 700-Frymong Merlebeach); V. Larroque (C. H. G. Antoine Gayraud, 0-Carca«sonne); E. Borderon (Hdpital General, 000-Orleani); J. Le Garzic (Centre Regional de Geriatric -Chantepie); M. Laureni (Centre Hospitaller, 9-Cholet); A. Romaru (Centre Hospitalier General, 790-Niort); B. Barbier (Centre Hospitaller General, 00-Chalons-sur-Marne); T. Meley (Clinique Mutualiste de la Crolx de l'orme, 00-Salnt-Etienne; Dr Malpotte (Hdpital du Petit Paris, 0-Grasse); R. Sanchez (Centre Ho*pitalier, 000-Perigueux); B. Birrere (Hopital de Font Prf, 0-Toulon; B. Tourrand (Centre Hospitalier General, 00-Alei); J. L. Reffay (Centre Hoipitalier General, 07-Sete); D. Pressac (Centre Hospitalier General, 00-Tulle); A. Marmonier (Centre Hospitalier, 707-Le Mam). Downloaded from at Pennsylvania State University on September, /9/ $0.00/0 99 The British Society for Antimicrobial Chemotherapy

2 9 F. W. Goldstein et al. monobactams have been reported from Germany, (Knothe et al., 9; Kliebe et al., 9) France (Sirot et al., 97; Jarlier et al., 9; Kitzis et al., 9; Thabaut et al., 9; Legrand et al., 99; Arlet et al., 990) and also other countries (Jacoby & Medeiros, 99). The prevalence of strains harbouring extended-spectrum /J-lactamases varies considerably from one hospital to another and may be underestimated since many strains fall within the susceptible MIC range. The aim of this study was to determine the prevalence of resistance to ceftriaxone and other /Mactams in clinical isolates collected over a month period from general hospitals in France and investigate the role of extended-spectrum /J-lactamases in ceftriaxone resistance. Collection of bacterial strains Material and methods All non-duplicate Enterobacteriaceae isolated from clinical specimens by participating centres (see title page) from April to July 99 were included in the investigation. All centres performed their own identification using API0E. Three reference strains, Escherichia coli ATCC 9 (a susceptible strain), Klebsiella pneumoniae CF 0 (a TEM- producing strain (Sirot, 97)) and E. coli RM (a high-level cephalosporinase producer, from the St Joseph Hospital, Paris), were also included for comparative purposes. Susceptibility testing and data interpretation All participating laboratories performed disc susceptibility testing on Mueller-Hinton agar (Diagnostic Pasteur, Marnes-la-Coquette, France) by the WHO method (WHO, 977). The antibiotics tested were ampicillin or amoxycillin, amoxycillin plus clavalanate (co-amoxiclav), ticarcillin, cephalothin, cefoxitin, cefixime, ceftazidime, aztreonam, ceftriaxone, and cefamandole. MICs on all ceftriaxone-resistant isolates were determined at the St Joseph Hospital by the agar dilution method using Mueller-Hinton agar. Inocula were 0'cfu/spot and the MICs were read after h incubation at 7 C. Susceptibility breakpoint zone diameters and corresponding MICs, as recommended by the French Antibiogram Committee (Acar et al., 99) were as follows: ampicillin > 7 mm ( < mg/l), amoxycillin > mm ( < mg/l), ticarcillin > mm ( < mg/l), cephalothin > mm ( < mg/l), co-amoxiclav > mm ( < mg/l), cefoxitin > mm ( < mg/l), cefixime mm ( < mg/l), ceftriaxone and ceftazidime > mm ( < mg/l), aztreonam > mm ( mg/l), cefamandole > mm ( < mg/l). Extended-spectrum /Mactamases were detected by the double diffusion method (Sirot et al., 99) demonstrating a synergic effect between the association co-amoxiclav and either ceftriaxone, ceftazidime or aztreonam. This test was performed by all centres with all the centres' isolates. The test was scored positive when at least one of the antibiotics showed an enhanced zone. Strains classified as susceptible by disc testing but demonstrating a syngergic effect as above were reclassified as resistant. All ceftriaxone-resistant strains were sent to the St Joseph Hospital where their identify was confirmed and their MICs were determined as defined above. Downloaded from at Pennsylvania State University on September, 0 Collection, storage and analysis of the data Information concerning all strains (specimens, departments, inhibition zone diameters etc) were collected on a notebook computer (Psion MC 00, Aware, Paris, France) by

3 Cefbiaxone-resiftant Enterobacterlaceae 97 each participating centre and at the end of the study the data were transferred electronically to a central data base (Paradox 0, Borland Velizy, France) on a PC. Transfer ability studies Attempts to transfer ceftriaxone resistance were performed at the St Joseph Hospital with all the strains harbouring an extended-spectrum /Mactamase. Transconjugants were prepared by mixing 0- ml of an overnight Mueller-Hinton broth culture of each donor with the recipient strain E. coli K, J - Azide R Rif* on Mueller-Hinton agar (Diagnostics Pasteur, Marnes-la-Coquette, France). After h incubation at 7 C the culture was spread on a Drigalski agar (Diagnostics Pasteur) plate containing ceftriaxone 0- mg/l and sodium azide 00 mg/l as counterselecting agents. At least four different colonies selected from each mating were tested for antibiotic susceptibility. The experiment was repeated three times if initial results were negative. Isoelectric focusing and P-lactamase assays Analytical isoelectric focusing of /Mactamase extracts obtained after ultrasonic-disruption of cells was performed at the St Joseph Hospital in polyacrylamide gels according to the method of Matthew et al. (97), and visualized with nitrocefin (Glaxo, France). The pi was determined for the enzymes from out of the transconjugants. Strains collected Results During the -month study period, 90 strains of Enterobacteriaceae were isolated with a highly variable frequency: 7 strains (70-%) were recovered from urine, 0 (-%) from blood, 9 (-7%) from pus, (-9%) from the respiratory tract and 99 (-7%) from other clinical specimens. Susceptibility testing Quality control results of the ceftriaxone inhibition zone diameters for the three reference strains showed good agreement between the centres. E. coli ATCC 9, K. pneumoniae CF 0 and E. coli RM, mean (S.D.) zone sizes were - (-0)mm, - (-)mm and 9- (-)mm, respectively. Overall, 9% of the 90 strains tested were fully susceptible to ceftriaxone. The percentage of susceptible strains according to the species and antibiotics tested are shown in Table I. Unexpectedly a small percentage of Klebsiella spp. were sensitive to ampicillin or amoxycillin. Some of these strains have been misidentified by the supplying laboratory but the data have not been omitted since we have rarely encountered strains of K. pneumoniae not harbouring SHV- and fully susceptible to ampicillin or amoxycillin (Goldstein, F. W., unpublished data). Major differences in the prevalence of ceariaxone resistance were observed between the centres with 0---% of the strains being ceftriaxone resistant. Greater than % ceariaxone resistance was observed in Klebsiella spp., Enterobacter spp., Serratia spp. and Citrobacter spp. (Tables I & II). CeAriaxone-resistant strains and especially K. pneumoniae producing an extended- Downloaded from at Pennsylvania State University on September, 0

4 Organism E.coli P. mirabilis lndok+proteus K. pnaononiae K. oxytoca Enterobacter spp. Serratia spp. Citrobacter spp. Salmonella spp. Shigella spp. Othere* Total TaUe L Antibiotic susceptibility of Entcrobactcriaceae isolated from French hospitals Number tested AMP AMO TIC % of susceptible strains' CF AMC FOX CFM CAZ ATM CRO 7 O I(H> from at Pennsylvania State University on September, AMP, Amptrillin; AMO, amoxycillin; TIC ticarciffin; CF, ccphalothin; AMC co-amondav, FOX, ccfoxitin; CFM, ce&ume; CAZ, ceftazidime; ATM, aztreonam; CRO, ceftriaxone. "Susceptibility was defined as imminimdrri by the French Antibiogram Committee (Acar el at., 99). *Hafnia, Proridatda tpp. and other isolates which could not be dearly danificd.

5 Ceftrlaxone-reslstaiit Eoterobacterlaceae 99 Table II. Total number of ceftriaxone-resistant strains and presence of an extended-spectrum /Mactamase detected by the double diffusion test method Number (%) Total ceftriaxone' Number (%) Overall (%) Species isolates resistant with ESBLA* resistant K. pneumoniae 7 (9) (9-) 9- K.oxytoca 09 7() (-9) P. mirabuis 0 (0-) 7 (0-) 0- E. coli 70 9 (0-) (0-0) 0- Citrobacter spp. (-9) (-) -9 Enterobacter spp. (-) (0-) - Others 7 (-) 0(0) - Total 90 7() (0-9) MIC > mg/l; ESBLA, extended-spectrum /7-lactamase detected by the method of Matthew et al. (97). spectrum /Mactamase were isolated most often from urine (% resistant) and from the respiratory tract (9% resistant) and mainly from patients in intensive-care units (% resistant) or surgical wards (% resistant). The total number of ceftriaxone-resistant strains and strains producing an extendedspectrum /Mactamase are shown in Table II. Ceftriaxone resistance due to the production of extended-spectrum /Mactamase was important only in K. pneumoniae, with (9-%) strains producing the enzyme. Among the participating laboratories, ten had no extended-spectrum /Mactamase-producing K. pneumoniae and for the others the incidence ranged from to %. However, 0% of the K. pneumoniae with extendedspectrum /Mactamase were isolated in one centre; these strains were of a single biotype and resistance pattern (cefoxitin, ciprofloxacin, aminoglycosides, sulphonamide, trimethoprim) and were very likely derived from a single strain. Ninety-six per cent of the strains harbouring an extended-spectrum /Mactamase were also tobramycin, netilmicin and amikacin resistant. Thirty-one of the strains could transfer their ceftriaxone resistance into E. coli K and all the transconjugants also aquired resistance to tobramycin, netilmicin and amikacin. Characterization of the extended-spectrum fi-lactamases The origin and characteristics of the extended-spectrum /Mactamase studied in E. coli K transconjugants are shown in Table III. Twelve out of the transconjugants studied had an enzyme similar to TEM- (pi ), six others and enzyme similar to TEM- (pi -) and three transconjugants an enzyme similar to SHV- (pi 7-) Tables IV and V show the MICs of six antibiotics for those strains of K. pneumoniae and other Enterobacteriaceae which produced an extended-spectrum /Mactamase. The MIC range of ceftriaxone for K. pneumoniae was - mg/l with % of the strains being susceptible (MIC < mg/l) despite the presence of an extended-spectrum /Mactamase; -% of the K. pneumoniae strains harbouring an extended-spectrum /Mactamase were cefoxitin resistant (MIC > mg/l). For the other Enterobacteriaceae harbouring an extended-spectrum /J-lactamase, the MIC range of ceftriaxone was 0-0- mg/l. Table VI shows the MICs of Downloaded from at Pennsylvania State University on September, 0

6 00 F. W. Goldstein et at. Table HI. Enzyme isoelectric point (pi) and MICs of ceftriaxone, ceftazidime and aztreonam for clinical isolates and E. coli K, transconjugants harbouring extended-spectrum /Mactamases Species E. coli K J - Az R Rif* Pi MIC of clinical isolates (mg/l) CRO CAZ ATM MIC of transconjugants (mg/l) CRO CAZ ATM K. pneumoniae E. coli C. freundii E. cloacae P. mirabilis K. pneumoniae K. pneumoniae CRO, Ceftriaxone; CAZ, ceftazidime; ATM, aztreonam. : : ' X \ ceftriaxone in presence of /Mactamase inhibitors. In presence of clavulanate mg/l all the strains except two Enterobacter cloacae were inhibited by < 0 mg/l of ceftriaxone. Sulbactam mg/l was less effective; in presence of sulbactam all the strains of K. pneumoniae susceptible to cefoxitin (MIC =$ mg/l) were inhibited by mg/l of ceftriaxone while the cefoxitin-resistant strains (MIC > mg/l) had ceftriaxone MICs of 0-- mg/l. Discussion In the study reported here ceftriaxone, ceftazidime and aztreonam (susceptibility breakpoints mg/l) had the best overall activity among the /Mactam antibiotics tested against Enterobacteriaceae isolates from French hospitals. Against E. coli, ceftriaxone was more effective than co-amoxiclav, older cephalosporins and cefixime. Ceftriaxone resistance was marked in K. pneumoniae and due to the production of extended-spectrum /Mactamase. Among Enterobacter, Serratia or Citrobacter spp. resistance was presumably due to the production of large amounts of cephalosponnase since only a few strains produced extended-spectrum /Mactamase. In 9% of strains harbouring an extended-spectrum /Mactamase, ceftriaxone resistance was associated with resistance to tobramycin, netilmicin, amikacin and other antimicrobial agents, restricting the choice of alternative therapeutic agents. Downloaded from at Pennsylvania State University on September, 0

7 Table IV. MICs of /?-lactam antibiotics for strains of K. pneumoniae harbouring an extended-spectrum /J-lactamase Ceftriaxone Ceftazidime Aztreonam Cefoxitin Cefamandole Amoxycillin-clavulanate* "Clavulanate was used at a fixed concentration of mg/l. Breakpoint MIC (mg/l) MIC % > (mg/l) resistant Table V. MICs of 0-lactam antibiotics for 7 strains of Enterobacteriaceae* harbouring an extended-spectrum /Mactamase <O ownloaded from at Pennsylvania State University on September, Breakpoint MIC (mg/l) MIC % > (mg/l) resistant Ceftriaxone Ceftazidime Aztreonam Cefoxitin Cefamandole Amoxycillin-clavulanate' 'C. freundii two strains; K. oxyloca four strains; P. mirabilis seven strains; E. coli two strains; E. cloacae two strains. *Clavulanatc was used at a fixed concentration of mg/l. s

8 0 F. W. Goldstein et al. Table VI. MICs of ceftriaxone in combination with a fixed concentration of clavulanate ( mg/l) or sulbactam ( mg/l) for Enterobacteriaceae harbouring an extended-spectrum /Mactamase No of MIC (mg/l) strains Ceftriaxone + clavulanate ( mg/l) K. pneumoniae Other species' 7 Ceftriaxone + mg/l sulbactam () K. pneumoniae Other species 7 'C. freundii two strains; K. oxytoca four strains; P. mirabilis seven strains; E. coli two strains; Enterobacter cloacae two strains. When present, extended-spectrum /Mactamases were often associated with only lowlevel ceftriaxone resistance with 9-7% of the strains having an MIC of < mg/l indicating clinical susceptibility. However, previous clinical studies have demonstrated that such strains are resistant in vivo, emphasizing the need for a routine testing for the presence of extended-spectrum /Mactamases (Brun-Buisson et al., 97; Jarlier et al., 9). As reported previously (Jacoby & Medeiros, 99; Sirot et al., 99) local epidemiological factors and antibiotic policies may explain the wide variation between hospitals in the incidence of resistance seen in this study; % of the strains harbouring an extended-spectrum /Mactamase came from a single centre and excluding the data from this centre would shift the incidence of strains harbouring an extended-spectrum /Mactamase from 0-9 to 0-%. Clavulanate was a better extended-spectrum /Mactamase inhibitor than sulbactam. This was particularly marked in K. pneumoniae with decreased susceptibility to cefoxitin which may be related, in the absence of an additional /Mactamase, to a porindeficient outer membrane (Gutmann et al., 99; Pangon et al., 99). An enzyme with the same pi (Brun-Buisson et al., 97; Jarlier et al., 9) as TEM- was the most predominant extended-spectrum /Mactamase isolated as has previously been demonstrated in French studies (Sirot et al., 97; Jarlier et al., 9; Kitzis et al., 9; Gutmann et al., 99). /?-Lactamases with a pi of - reassembling TEM- and a pi of 7- reassembling SHV- were also isolated (Jacoby & Medeiros, 99). In this large multicentre study, involving more than 9000 isolates of Enterobacteriaceae the most significant finding is that after more than 0 years of intensive use, more than 9% of strains are still fully susceptible to extended-spectrum cephalosporins and aztreonam. Such encouraging results should not, however, underestimate the potential risk of resistant strains or species in special clinical settings and emphasizes the need for frequent epidemiological studies of antimicrobial resistance patterns. Downloaded from at Pennsylvania State University on September, 0 Acknowledgements This study was supported by Produits Roche, France. We thank Monique Boda for secretarial assistance.

9 Ceftriaxone-reslstant Enterobacteriaceae 0 References Acar, J., Bergogne-Berezin, E., Chabbert, Y., Ouzel, R., Courtieu, A., Courvalin, P. et al. (99). Communique 99 dc Comite du rantibiogramme dc la Societe Francaise de Microbiologie. Pathologie Biologie 9, Arlet, G., Sanson-le Pors, M. J., Rouveau, M., Fournier, G., Marie, O., Schlemmer, B. et al. (990). Outbreak of nosocomial infections due to Klebsiella pneumoniae producing SHV- beta-lactamase. European Journal of Clinical Microbiology and Infectious Diseases 9, Brun-Buisson, C, Legrand, P., Philippon, A., Montravers, F., Ansquer, M. & Duval, J. (97). Transferable enzymatic resistance to third-generation cephalosporins during nosocomial outbreak of multiresistant Klebsiella pneumoniae. Lancet ii, 0. Gutmann, L., Ferre, B., Goldstein, F. W., Rizk, N., Pinto-Schuster, E., Acar, J. F. et al. (99). SHV-, a novel SHV-type /Mactamase that hydrolyzes broad-spectrum cephalosporins and monobactams. Antimicrobial Agents and Chemotherapy, 9-. Jacoby, G. A. & Medeiros, A. A. (99). More extended-spectrum /Mactamases. Antimicrobial Agents and Chemotherapy, Jarlier, V., Nicolas, M.-H., Fournier, G. & Philippon, A. (9). Extended-spectrum /Mactamases conferring transferable resistance to newer /Mactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Reviews of Infectious Diseases 0, 7-7. Kitzis, M. D., Billot-KJein, D., Goldstein, F. W., Williamson, R., Tran Van Nhieu, G., Carlet, J. et al. (9). Dissemination of the novel plasmid-mediated /Mactamase CTX-, which confers resistance to broad-spectrum cephalosporins, and its inhibition by /Mactamase inhibitors. Antimicrobial Agents and Chemotherapy, 9. Kliebe, C, Nies, B. A., Meyer, J. F., Tolxdorff-Neutzling, R. M. & Wiedmann, B. (9). Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy, 0-7. Knothe, H., Shah, P., Krcmery, V., Antal, M. & Mitsuhashi, S. (9). Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection, -7. Legrand, P., Fournier, G., Bure, A., Jarlier, V., Nicolas, M. H., Deere, D. et al. (99). Detection of extended broad-spectrum /Mactamases in Enterobacteriaceae in four French hospitals. European Journal of Clinical Microbiology and Infectious Diseases, 7-9. Matthew, M., Harris, A. M., Marshall, M. J. & Ross, G. W. (97). The use of analytical isoelectric focusing for detection and identification of /Mactamases. Journal of General Microbiology, 9-7. Pangon, B., Bizet, C, Bure, A., Pichon, F., Philippon, A., Regnier, B. et al. (99). In vivo selection of a cephamycin-resistant, porin-deficient mutant of Klebsiella pneumoniae producing a TEM- /Mactamase. Journal of Infectious Diseases 9, 00-. Sanders, C. C. (97). Chromosomal cephalosporinases responsible for multiple resistance to newer /Mactam antibiotics. Annual Review of Microbiology, 7-9. Sirot, D., Goldstein, F. W., Soussy, C. J., Courtieu, A. L., Husson, M. O., Lemozy, J. et al. (99). Resistance to cefotaxime and seven other /Mactams in members of the family Enterobacteriaceae: a -year survey in France. Antimicrobial Agents and Chemotherapy, 77-. Sirot, D., Sirot, J., Labia, R., Morand, A., Courvalin, P., Darfeuille-Michaud, A. et al. (97). Transferable resistance to third-generation cephalosporins in clinical isolates of Klebsiella pneumoniae: identification of CTX-, a novel /Mactamase. Journal of Antimicrobial Chemotherapy 0, -. Thabaut, A., Acar, J., AJlouch, P., Arlet, G., Berardi-Grassias, L., Bergogne-Berezin, E. et al. (990). Frequence et distribution des betalactamase chez 79 souches de Klebsiella pneumoniae isolees en France entre 9 et 9. Pathologie Biologie, 9-. WHO Expert Committee on Biological Standardization. (977). WHO Twenty-eighth report, pp. 9-. Technical Report Series 0. WHO. Geneva. Downloaded from at Pennsylvania State University on September, 0 (Received February 99; accepted May 99)