Study of the comparative activity of piperacillin / tazobactam with currently available antibiotics against 8206 aerobic isolates

Division of Microbiology, Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia; Department of Microbiology, Mount Sinai and Princess Margaret Hospitals, Toronto, Ontario; Department of Microbiology and Infectious Diseases, Hôpital Maissoneuve-Rosemont, Montréal, Québec; Microbiology and Public Health, University of Alberta Hospital, Edmonton, Alberta; Department of Microbiology, Sunnybrook Health Sciences Centre, Toronto, Ontario; Wyeth-Ayerst Canada Inc, North York, Ontario Correspondence: Dr K Forward, Division of Microbiology, Queen Elizabeth II Health Sciences Centre, 1278 Tower Road, Halifax, Nova Scotia B3H 2Y9. Telephone 902-473-4109, fax 902-473-4432, e-mail kforward@ac.dal.ca Received for publication May 30, 1996. Accepted September 30, 1996 KR FORWARD, DE LOW, M LAVERDIERE, et al. Study of the comparative activity of piperacillin/tazobactam with currently available antibiotics against 8206 aerobic isolates. Can J Infect Dis 1997;8(3):147-153.

T he recent approval of a new antimicrobial such as piper- acillin-tazobactam (PT) provides an opportunity to examine the susceptibility profiles of this and other available antibiotics and to study the emergence of antibiotic resistance in different settings and among different genera in a cross-Canada survey.
Tazobactam is a beta-lactamase inhibiting penicillanic acid sulphone derivative which, when combined with piperacillin, expands its spectrum of activity to include most strains of Staphlococcus aureus and a number of Gram-negative bacilli producing beta-lactamases of Bush group two (1,2).The spectrum of activity of PT is the broadest of drugs in their class and typically approaches that of imipenem (2,3).We undertook to compare the activity of PT with other commonly used antibiotics against aerobic Gram-negative bacilli and Gram-positive cocci from across Canada and from a variety of clinical sources.We also screened Escherichia coli and Klebsiella pneumoniae for susceptibility patterns typical of strains producing extended-spectrum beta-lactamases (ESBLs).Because a large number of commonly occurring species were tested, we were able to compare the activity of piperacillin, PT and ticarcillinclavulanate (TC) at different body sites, in university and community hospitals and in different regions of the country.

MATERIALS AND METHODS
Study design: Sixty-one laboratories in 10 Canadian provinces each tested up to 150 clinical strains from hospitalized patients.To ensure representative surveillance patterns, only one strain of each species per patient was included in the study, and strains were collected consecutively.Strains were tested only if they met the criteria for clinical significance established in individual laboratories.Included in the analysis were isolates from blood, lower respiratory tract, skin and soft tissue, intra-abdominal and female pelvic sites.Urinary, fecal, upper respiratory and vaginal isolates were not included.Also excluded from analysis were methicillin-resistant S aureus, anaerobes, Gram-positive bacilli, Gram-negative cocci and fastidious aerobic Gram-negative bacilli.Coagulase-negative staphylococci were included for analysis only if they were recovered from multiple specimens collected from known sterile sites at different times.Susceptibility testing: Disk susceptibility testing was performed in accordance with National Committee for Clinical Laboratory Standards (NCCLS) documents M2-A5 and MS1 00-S5 (4,5).To ensure optimal standardization of test results, centres were obliged to use disks (Becton Dickinson Laboratories, Maryland) of the same batch number provided by the study co-ordinator.Organisms were included as controls as appropriate for the organisms and agents tested.These included S aureus ATCC 25923, E coli ATCC 25922, E coli ATCC 35218, Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853.Strains of Klebsiella species and E coli that exhibited decreased susceptibility to third-generation cephalosporins were referred centrally and screened by one of the researchers for ESBLs using both the double disk diffusion method and E test ESBL strips (AB Biodisk, Solona, Sweden) (6).Inhibition of ESBL by clavulanic acid reduces the minimum inhibitory concentration (MIC) of ceftazidime when the two are tested together.A ratio of the ceftazidime MIC without and with clavulanic acid of 16 or more was interpreted as indicating ESBL production.Data analysis: Organisms were coded by species, hospital type (community or teaching) and geographic region (Atlantic, Quebec, Ontario, Western).The clinical site of infection was recorded.Results were collected on a standardized data collection form.Results were interpreted using NCCLS interpre-Étude de l'activité comparative de la pipéracilline/tazobactam et des antibiotiques actuellement utilisés contre 8 206 isolats aérobies OBJECTIFS : Comparer l'activité de la pipéracilline/tazobactam et de la pipéracilline et d'autres antibiotiques administrés par voie parentérale contre des bacilles gram-négatifs aérobies et des cocci gram-positifs isolés, provenant de plusieurs centres canadiens et déterminer la prévalence de la résistance associée aux céphalosporinases à spectre élargi.MÉTHODES : Soixante-et-un laboratoires ont participé à l'étude.Des techniques de diffusion ont été utilisées, conformément aux normes du National Committee for Clinical Laboratory Standards.Des antibiogrammes ont été effectués sur 8 206 isolats.On a procédé à un dépistage d'Escherichia coli et de Klebsiella pneumoniae présentant une sensibilité moindre aux céphalosporines de troisième génération pour y déceler la présence de bêtalactamases à spectre élargi (BLSE).RÉSULTATS : La pipéracilline/tazobactam a été efficace contre 92 % des souches, la pipéracilline, contre 81 % et la ticarcilline-acide clavulanique, contre 88 %.Peu de différences ont été observées quant à la sensibilité relative des souches provenant des hôpitaux communautaires ou universitaires, provenant de différents sites anatomiques ou de différentes régions du pays.Les bacilles gram-négatifs aérobies testés tendaient à être plus sensibles à tous les agents, comparativement à un récent rapport d'une étude américaine similaire.Quarante-trois pour cent seulement des Enterococcus faecium ont été sensibles à l'ampicilline et 42 % à la pipéracilline avec et sans tazobactam.Seules deux souches entérococciques ont été résistantes à la vancomycine et 19 présentaient des zones de taille intermédiaire.Parmi les dix souches de E. coli et les huit souches de K. pneumoniae présentant une sensibilité réduite aux céphalosporines à large spectre, une seule a présenté une activité BLSE typique.CONCLUSIONS : Les cocci gram-positifs aérobies et les bacilles gram-négatifs restent très sensibles aux nombreux antibiotiques actuellement sur le marché.Ces résultats confirment l'activité à large spectre de la pipéracilline/tazobactam et indiquent que le mode de sensibilité est assez uniforme, peu importe le site de l'infection, tant dans les hôpitaux universitaires que communautaires et d'un bout à l'autre du pays.
tive criteria and, where none were available, criteria for related species were applied.P values from Fishers exact test were calculated with m-1 degrees of freedom for the comparison of m proportions.Multiple comparisons were performed for the overall statistically significant results (P≤0.05).Because there were six pairwise comparisons among four proportions in Tables 3 and 5, using a 5% level of significance implies that some of comparisons were likely to be significant by chance alone.To maintain the overall P≤0.05 a fixed significance level, the Bonferroni method was applied as follows: P was divided by the number of pairwise comparisons and its result was then used as a cut-off point of significance level.For this case, dividing P=0.05 by 6 yields 0.083, which was used as a cut-off point of significance level for multiple comparisons in supplementary Tables 3 and 5.

RESULTS
The 61 participating laboratories submitted susceptibility results on 8026 strains, including 1240 P aeruginosa, 1069 E coli, 974 S aureus, and 649 Enterococcus species.Table 1 lists the results of testing of aerobic Gram-negative bacilli, the number of strains of each group and the proportion of strains tested and susceptible to each antibiotic tested.Of 5132 aerobic Gram-negative rods tested, 93% were susceptible to ceftaz-idime, 92% to PT, 92% to gentamicin, 88% to TC and 81% to piperacillin.Against E coli, PT was active against 98% of strains compared with 74% for piperacillin alone, 92% for TC, 97% for gentamicin and 99% for each of the third-generation cephalosporins tested.
Of K pneumoniae, 96% were susceptible to PT compared with 95% for TC and 82% for piperacillin alone.The third-generation cephalosporins were each active against more than 99% of strains.PT was the most active of the agents tested against P aeruginosa.Ninety-six per cent of strains were susceptible to PT, while 95% of strains were susceptible to ceftazidime, piperacillin and gentamicin.Acinetobacter species were considerably more susceptible to both PT and TC than to piperacillin alone.Only 54% of strains were susceptible to piperacillin, whereas more than 90% of strains were susceptible to the beta lactam-inhibitor combinations.Overall, only Stenotrophomonas maltophilia was usually resistant to PT.
Table 2 shows results of testing of aerobic Gram-positive cocci.Of the E faecalis strains that were speciated, 99.8% were susceptible to PT and piperacillin compared with 42% and 43% of E faecium strains, respectively.Only two vancomycin-resistant enterococci were identified.Of S aureus, 99% were susceptible to PT, whereas 72% were susceptible to piperacillin and 87% were susceptible to erythromycin.Rank order based upon the percentage of all aerobic Gram-negative bacilli inhibited Tables 3,4 and 5 show the proportion of strains submitted from different body sites, hospital type and geographic regions.A total of 2215 strains were submitted from the four western provinces, 2876 from Ontario, 2196 from Quebec, and 739 from the four Atlantic provinces.

DISCUSSION
Piperacillin in combination with tazobactam has been shown to be effective in the treatment of a variety of infections in a variety of settings, including intra-abdominal sepsis infections associated with febrile neutropenia, skin and soft tissue infections, and nosocomial pneumonia (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).It is likely that this drug will be used widely for the treatment of polymicrobial infections caused by piperacillin-susceptible organisms and organisms producing one of the of beta-lactamase susceptible to tazobactam.Among these are beta-lactamases of the Bush-Jacoby-Medeiros group 2 that include both the conventional TEM and SHV enzymes and the emerging extended-spectrum beta-lactamases (TEM-3 to TEM-26 and SHV-2 to SHV-5).Also included among the beta-lactamases inhibited by tazobactam are those of S aureus, Haemophilus influenzae, Moraxella catarrhalis and Bacteroides fragiles.Beta-lactamases of the Bush-Jacoby-Medeiros group 1, including the inducible cephalosporinases of Enterobacter species, Citrobacter species, Serratia marcescens and P aeruginosa, are not inhibited by either tazobactam or other currently available lactamase inhibitors.Tazobactam is, however, a less potent inducer of group 1 lactamases than other beta-lactamases (19).
Several of the antibiotics tested, including PT, gentamicin and ceftazidime, were highly active against the 5132 strains of aerobic Gram-negative bacilli tested, each inhibiting more than 90% of isolates.The addition of tazobactam to piperacillin increased the proportion of E coli susceptible from 74% to 98%; of K pneumoniae from 82% to 96%; and of Klebsiella oxytoca from 84% to 96%, reflecting the prevalence of group 2 beta-lactamases in this group of organisms.
Of interest, we did observe occasional differences in the susceptibilities of commonly occurring aerobic Gram-negative bacilli among the four regions of Canada.Klebsiella species were generally more resistant to piperacillin with or without tazobactam and to TC in the Atlantic provinces.Ontario strains  were less susceptible than Quebec and Western region strains.Not enough is known about patterns of antibiotic usage across the country to provide an explanation for this observation.Generally, aerobic Gram-negative bacilli isolates from different body sites had similar levels of susceptibility to piperacillin with and without tazobactam and to TC. Intra-abdominal isolates of Enterobacter species were more likely to be resistant to TC than skin and soft tissue strains (P<0.005).Respiratory tract isolates of enterobacter were also significantly less susceptible to TC than skin and soft tissue isolates.Both intra-abdominal isolates and respiratory tract isolates of enterobacter were more resistant than blood culture isolates to TC.The reasons for these observations may relate to the difficulty eradicating organisms from these and the frequent need for prolonged antibiotic courses.
Few differences were seen between the susceptibilities of aerobic Gram-negative bacilli in community and teaching hospitals.P aeruginosa isolates recovered in teaching hospitals were more likely to be resistant to piperacillin with or without tazobactam or to TC.In many other pairwise comparisons, other aerobic Gram-negative bacilli from teaching hospitals were slightly less susceptible than community hospital strains; however, these differences were generally small in magnitude.It is not clear whether these differences relate to patient or hospital characteristics, but likely are a reflection of both.
We screened seven isolates of K pneumoniae and six strains of E coli with reduced susceptibility to third-generation cephalosporins for ESBLs using two methods.We identified one strain from a hospital in Toronto that demonstrated the typical susceptibility profile of ESBLs extended-spectrum beta-lactamases.This finding is consistent with other recently published surveys of Canadian strains (20).This is in sharp contrast to the experience in many other countries where both endemic and epidemic strains are frequently encountered (21)(22)(23)(24)(25).
Of interest, each of the aerobic Gram-negative bacilli tested in sufficient numbers was more susceptible in Canada than those tested in the United States national survey of PT activity published by Baron and Jones (3).Table 6 is a compilation of American data from Baron and Jones (3) and Canadian data from this study.Both the current study and the American study used fresh clinical isolates; frozen stock cultures were not tested.Both studies used only one isolate of the same species from each patient to avoid the bias that might occur from multiple testing of the same strains.Neither study used urinary or fecal isolates, and the number of frequently occurring species was limited.In both cases, NCCLS methodology and interpretive criteria were applied.In almost all antibioticorganism combinations compared in Table 6, a larger proportion of Canadian strains were susceptible.The exceptions were usually in those combinations where the species were intrinsically resistant, eg, ceftriaxone versus P aeruginosa and cefazolin versus Enterobacter species or for those combina-  It is likely that the differences that we observed in the relative susceptibility of Canadian and American isolates to the antibiotics tested are real.The delay in the appearance in Canada of ESBL-producing organisms, vancomycin-resistant enterococci, methicillin-resistant S aureus and penicillin-resistant S pneumoniae have been noted by others (26)(27).It has been suggested that more stringent controls on antimicrobial usage in hospitals and well developed infection control practices may contribute to these differences.It has also been suggested that Canada's widely scattered population may prevent transmission between geographic regions; however, this explanation seems improbable in our highly mobile society.Because the data by Baron and Jones were not analyzed by region or hospital type, we were not able to examine whether the American and Canadian differences might in some way be explained by differences in the proportion of isolates from each setting.Although we did not share isolates between the 61 Canadian and American laboratories, it is unlikely that there are enough systemic differences in the way that NCCLS methods are applied to account for the differences between countries.The relative roles played by each of these factors and whether other factors are responsible for the observed differences have yet to be studied in a systematic fashion.

CONCLUSIONS
PT is highly active against commonly occurring aerobic Gram-negative rods and Gram-positive cocci.Fairly uniform activity was seen between geographic regions within Canada between different body sites and between community and teaching hospitals.Canadian isolates were generally more susceptible to PT and to the other agents tested than were American strains tested using similar methods.
Differences between the susceptibility patterns in American and Canadian hospitals should be studied further to understand better the factors contributing to the lower levels of resistance in Canada.

TABLE 2 Comparative activity of piperacillin-tazobactam and other parenterally administered antibiotics against 2074 Gram-positive organisms isolated at 61 Canadian hospitals Percentage of strains susceptible to Organism # Tested Piperacillin- tazobactam Piperacillin Vancomycin Erythromycin
*As determined by the oxacillin disk screening method

TABLE 3 Comparative activity of piperacillin-tazobactam and other parenterally administered antibiotics against aerobic Gram-negative bacilli isolated from different body sites Percentage of strains susceptible to
IA Intra-abdominal; Resp Lower respiratory tract; SST Skin and soft tissue

TABLE 4 Comparative activity of piperacillin-tazobactam and other parenterally administered antibiotics against aerobic Gram-negative bacilli isolated in community and teaching hospitals Percentage of strains susceptible to
*Fishers Exact Test

TABLE 5 Comparative activity of piperacillin-tazobactam and other parenterally administered antibiotics against aerobic Gram-negative bacilli isolated in four Canadian regions
*Fishers exact test.Atlan Atlantic; Ont Ontario; Que Quebec; West Western provinces tions where resistance was rare in either country, eg, E coli or Proteus mirabilis versus PT.

TABLE 6 Comparative activity of aerobic Gram-negative bacilli in Canada (Can) and the United States (US)*
*American data from reference 3 with permission; † Fishers exact test.PT Piperacillin-tazobactamMembers of