J Turnidge, J Bell, ML Beach, RN Jones*,

1. Extended Spectrum -Lactamase (ESBL) Phenotypes In Enteric Bacilli from the Asia-Pacific Region: Report from the SENTRY Antimicrobial Surveillance Program Escherichia coli (87) Klebsiella pneumoniae (138) Proteus mirabilis (2) 1998 1999 1998 1999 1998 1999 29 12 32 24 1 - 7 8 30 23 - - 4 2 6 2 1 - 20 5 18 3 - - Australia 200 201 202 203 109 139 90 109 0 1 (0.7) 1 (1.1) 0 39 31 35 48 0 0 0 7(14.6) 12 15 8 13 0 0 0 0 China 204 208 209 210 153 34 18 44 25 (16.3) 10 (29.4) 4 (22.2) 11 (25.0) 69 5 19 42 10 (15.5) 2 (40.0) 7 (36.8) 14 (33.3) 12 0 0 6 0 0 0 0 Species/nation/medical center (no. isolates) No. of isolates (%) resistant to: Japan 205 206 207 30 17 48 0 0 2 (4.2) 23 18 41 3 (13.0) 1 (5.5) 1 (2.4) 3 3 2 1(33.3) 0 0 Cefoxitin Ciproa Amika Genta Tobraa Tetraa TMP/SMX Taiwan 214 215 216 The Philippines 211 55 8 12 126 20 (15.9) 5 (10.0) 2 (25.0) 0 30 4 7 134 52 (38.8) 3 (10.0) 0 1(14.3) 14 4 0 1 1(7.1) 0 0 0 Singapore 212 77 4 (5.2) 64 25 (39.1) 10 0 South Africa 213 34 2 (5.9) 34 12 (35.3) 8 0 China 209 210 18 44 4 (22.2) 11 (25.0) 19 42 7 (36.8) 14 (33.3) 18 14 2 (11.1) 5 (35.7) Taiwan 214 215 55 8 5 (9.1) 2 (25.0) 30 4 3 (10.0) 0 6 4 1(16.7) 1 (25.0) The Philippines 211 126 20 (15.9) 134 52 (38.8) 49 2 (4.1) Singapore 212 77 4 (5.2) 64 25 (39.1) 18 2 (11.1) South Africa 213 34 2 (5.9) 34 12 (35.3) 12 2 (16.7) J Turnidge, J Bell, ML Beach, RN Jones*, SENTRY Participants Group (Asia-Pacific). Women’s and Children’s Med Ctr, Adelaide, Australia; Univ of Iowa, Iowa City, IA; The JONES Group/JMI Laboratories, N. Liberty, IA Poster #293 Ronald N. Jones, M.D. The JONES Group / JMI Laboratories 345 Beaver Kreek Centre, Suite A, North Liberty, Iowa 52317 Phone: 319-665-3370 Fax: 319-665-3371 ronald-jones@jmilabs.com ABSTRACT Background: ESBL-production among various Enterobacteriaceae presents a serious threat to continued clinical efficacy of newer -lactams. Regional and national differences in rates of occurrence have been cited, but limited results have been reported from the Asia- Pacific area and S. Africa. Methods: The SENTRY Program monitored 17 laboratories (seven nations) in 1998-99 via the referral of strains to a regional monitor. E. coli (EC; 1103), Klebsiella spp. (KSP; 643), P. mirabilis (PM; 111) and Enterobacter spp. (EBS; 283) were screened for ESBL phenotypes using NCCLS methods and criteria. All isolates were clinical infections including bacteremias, pneumonia and UTI. At least one ESBL strain was detected at each participating site. Results: The ESBL-phenotype rates overall among species were: EC (7.9%), KSP (22.7%), PM (1.8%) and EBS (5.3%). Several phenotypic strains were not CA inhibitable (cefoxitin MIC, 32 g/ml), consistent with an amp C patterns thus corrected ESBL rates were 3.6, 12.8 and 0.9 for EC, KSP and PM. Highest EC ESBL rates were in Hong Kong (8.5%) and mainland China (8.3%), while EC amp C patterns were highest in mainland China (17.7%) and The Philippines (9.5%). In KSP, ESBLs were highest in Singapore (34.4%) a mainland China (22.7%) and amp C’s in the Philippines (29.9%) and China (12.1%). S. Africa ESBL rates were 2.9 and 38.2% for EC and KSP. ESBL transfer into EBS was observed in Singapore (11.1%), China (20.6%), The Philippines (4.1%), Taiwan (18.2%) and S. Africa (16.7%); into PM in Japan and The Philippines. Conclusions: Widely different patterns of ESBL-phenotypes have emerged in the Asia-Pacific Region. Rates vary from rare isolations in Australia (0.2-5.2%) to alarmingly elevated occurrences in Singapore, The Philippines, mainland China and S. Africa. Phenotypic patterns and preferred substrates for detection also vary indicating variable enzyme types (TEM, SHV, OXA, CTX-M, etc.). Continued monitoring and local interventions appear prudent. MATERIALS AND METHODS – CON’T TABLE 1. Occurrence and trends in the Asia Pacific Region for isolates having an ESBL phenotypea(SENTRY Program, 1998-99) TABLE 4: Distribution of the preferred -lactam substrate for detection of ESBL isolates in the Asia Pacific region TABLE 6: Antimicrobial activity of selected antimicrobial agents against ESBL-producing isolates collected by SENTRY Antimicrobial Surveillance Program in the Asia-Pacific region, 1998-99 ESBL phenotype criteria Isolates of Klebsiella spp., P. mirabilis, and E. coli with elevated MIC results ( 2 g/ml) for ceftazidime and/or ceftriaxone and/or aztreonam were considered as possible ESBL-producing phenotypes according to NCCLS criteria. Characterization of the ESBL phenotype The production of an ESBL was confirmed by using the agar dilution technique. Agar plates containing - lactam substrates (ceftriaxone, cefotaxime, ceftazidime, and cefepime) with and without clavulanate (2 g/ml) were used. The variations of the MIC for the -lactam alone and for -lactam/clavulanate combination were compared. A reduction of the -lactam MIC of more than two log2 dilutions (> four-fold) in the presence of clavulanate indicated ESBL production (see NCCLS criteria). E. coli K. pneumoniae P. mirabilis Species (No. of isolates) Occurrence by site of infection Country/substrate No. ESBL % detected No. ESBL % detected No. ESBL % detected Year Blood Respiratory SSTIb Urine Australia Aztreonam Ceftriaxone Ceftazidime 2 1 1 100.0 50.0 50.0 6 7 7 85.7 100.0 100.0 -a -a -a -a -a -a Antimicrobial agents Escherichia coli (87) Klebsiella pneumoniae (134) g/ml g/ml China Aztreonam Ceftriaxone Ceftazidime 47 36 39 94.0 72.0 78.0 32 30 30 97.0 91.0 91.0 -a -a -a -a -a -a MIC50/90 % susc.a MIC50/90 % susc.a Piperacillin/Tazobactam Cefoxitin Imipenem Ciprofloxacin Amikacin Gentamicin Tobramycin Tetracycline 8/>64 32/>32 0.25/0.5 >2/>2 2/32 8/>16 8/>16 >8/>8 70.1 25.3 100.0 34.5 86.5 24.1 31.0 23.0 2/>16 16/>32 0.25/1 1/>2 8/32 16/>16 16/>16 16/>16 87.3 43.5 100.0 55.8 84.1 41.3 27.5 46.3 60.0 60.0 100.0 Japan Aztreonam Ceftriaxone Ceftazidime 2 1 2 100.0 50.0 100.0 3 3 5 1 1 0 100.0 100.0 aThe ESBL phenotype was defined according to the NCCLS (2001) criteria. bSSTI, skin and soft tissue. Taiwan Aztreonam Ceftriaxone Ceftazidime 6 5 7 85.7 71.4 100.0 3 4 4 75.0 100.0 100.0 -a -a -a -a -a -a RESULTS The Philippine Aztreonam Ceftriaxone Ceftazidime 17 13 18 85.0 65.0 90.0 50 49 52 96.2 94.2 100.0 0 0 1/1 0.0 0.0 100.0 • E. coli, K. pneumoniae and P. mirabilis accounted for more than 25% of all bacterial samples isolated from the Asian-Pacific region. The ESBL phenotype was more commonly observed among K. pneumoniae (24.5%) than E. coli (7.9%) or P. mirabilis isolates (1.8%). • Isolates of E. coli exhibiting an ESBL phenotype were more frequently isolated from blood (47.1%), followed by urine (28.7%) and respiratory (17.2%) sites (Table 1). This pattern was not the same among the K. pneumoniae isolates, where isolates exhibiting an ESBL phenotype were isolated almost equally from blood (40.6%) and respiratory (38.4%) sites. • Among the 87 possible ESBL-producing E. coli, 57.5% of them were isolated by the centers in China, followed by The Philippines (23.0%) and Taiwan (8.0%) centers. Approximately one-half of the Chinese isolates were collected from a single center located in Hong-Kong. • Among the 564 K. pneumoniae, 138 isolates were classified as possible ESBL-producers. Over one-third (38.0%) of them were collected from The Philippine center followed by China (24.0%) and the Singapore (18.1%) centers. The local frequency of occurrence of the ESBL phenotype varied widely among the Asian-Pacific centers; highest in the Chinese (15.5%-40%), Philippine (38.8%), Singaporean (39.1%) and South African (35.3%) centers. • Among the 111 P. mirabilis isolates, only two demonstrated an ESBL-phenotype; the Japanese isolate was confirmed as an ESBL producer, but this strain occurred in a center with the lowest prevalence rates of ESBL phenotypes among E. coli and K. pneumoniae. • The spread of plasmids encoding ESBL to species that usually produce chromosomal Amp C enzymes, such as Enterobacter spp. and Serratia marcescens, has been observed in some institutions. Seven of 17 medical centers evaluated in this study had Enterobacter isolates exhibiting the ESBL phenotype (Table 3). These isolates showed high MICs for cefepime (16 g/ml) that decreased at least eight-fold in the presence of clavulanate. The local frequency of occurrence of the ESBL phenotype among Enterobacter spp. varied from 4.1% (The Philippines) to 35.7% (one of the Chinese medical centers). The spread of ESBL to Enterobacter species could not be directly related to the high prevalence of ESBL-producing K.pneumoniae and/or E. coli. • Different ESBLs hydrolyze -lactams to varying degrees, substrate specificity (Table 4). Aztreonam was the preferred substrate for the detection of the ESBL phenotype among the E. coli, followed by ceftazidime; whereas among the K. pneumoniae ESBL phenotypes, ceftazidime was the best substrate, followed by aztreonam and ceftriaxone. ESBLs derivatives from the CTX-M genes generally hydrolyze aztreonam and ceftriaxone at higher rates than ceftazidime indicating that such enzymes may be prominent among the Chinese isolates. • The ciprofloxacin resistance rates were very high among isolates collected from China, Singapore, and The Philippines (Table 5). Co-resistances to tobramycin, gentamicin, tetracyclines, and trimethoprim/sulfamethoxazole were common throughout isolates collected from several centers. Resistance to cefoxitin was observed in 47(54.0%) E. coli and 64 (45.7%) K. pneumoniae isolates exhibiting ESBL phenotype. This pattern was mainly observed among E. coli and K. pneumoniae isolated from the Chinese and The Philippines centers, respectively. Interestingly, 23 (48.9%) of 47 E. coli and 47 (70.1%) of 67 K. pneumoniae were inhibited by clavulanic acid, confirming the production of ESBL. All the E. coli strains resistant to cefoxitin (MICs 32 g/ml) isolated from Australia, Taiwan, Japan, and China (medical center 209) showed confirmatory tests negative for ESBL production, probably indicating the production of Amp C enzymes. On the other hand, all cefoxitin-resistant E. coli strains isolated from China (medical center 208), Singapore, and South Africa showed positive confirmatory tests for ESBL production. ESBL-producing isolates could be resistant to cefoxitin due to other mechanisms of resistance, such as loss or decrease of outer membrane proteins. • The best spectrum against ESBL-producing isolates was obtained with the carbapenems (Table 6). Against ESBL-producing E. coli, amikacin (susceptibility of 86.5%) was the second best choice followed by piperacillin/tazobactam (susceptibility of 70.1%), while against ESBL-producing K. pneumoniae, the opposite was observed with more than 87% of the isolates susceptible to piperacillin/tazobactam. TABLE 2. Comparison of ESBL phenotype occurrence rates among the Asia Pacific medical centers (SENTRY Program, 1998-99). Singapore Aztreonam Ceftriaxone Ceftazidime 4 4 4 100.0 100.0 100.0 23 24 24 92.0 96.0 96.0 -a -a -a -a -a -a aBreakpoints of susceptibility defined by NCCLS (2001). South Africa Aztreonam Ceftriaxone Ceftazidime 2 2 2 100.0 100.0 100.0 12 12 12 100.0 100.0 100.0 -a -a -a -a -a -a • CONCLUSIONS • The number of ESBL phenotype isolates detected in E. coli and Klebsiella pneumoniae by the SENTRY Program varied widely among the Asia-Pacific medical centers. Most of them had a high frequency of occurrence of the ESBL phenotype comparable only to those reported by us in Latin American medical centers. • The antimicrobial resistance patterns observed among these isolates confirm that the therapeutic options for treatment of infections caused by ESBL pathogens is very limited, and only a few drugs such as carbapenems constitute a safe option. • The results presented by this report emphasise the importance of regional antimicrobial surveillance studies such as the SENTRY Antimicrobial Surveillance Program in combating and controlling antimicrobial resistance. E. coli K. pneumoniae P. mirabilis Country/medical center no. Total no.of isolates No. of ESBL (%) Total no.of isolates No. of ESBL (%) Total no.of isolates No. of ESBL (%) All nations Aztreonam Ceftriaxone Ceftazidime 80 62 73 92.0 71.3 84.0 129 129 134 93.5 93.5 97.1 1 1 1 50.0 50.0 50.0 INTRODUCTION aESBL-producing Proteus mirabilis isolates were not detected. Among Gram-negative bacteria, the production of -lactamases is the most important mechanism of resistance to -lactam agents. Members of the family Enterobacteriaceae, commonly express plasmid-encoded -lactamases which confer resistance to penicillins but not to extended-spectrum cephalosporins. In 1983, a novel group of enzymes, subsequently named extended-spectrum -lactamases (ESBL), was detected among Serratia marcescens and Klebsiella pneumoniae in Europe. This group of enzymes has mainly arisen from genes coding common plasmid- mediated enzymes, such as TEM-1 or -2 and SHV-1, which possess minor gene point mutations. The ESBLs are capable of hydrolyzing many -lactams (except the carbapenems) and most of them are inhibited by the clinically available -lactamase-inhibitors. ESBL-producing clinical strains have been isolated from many parts of the world. However, their frequency of occurrence varies widely. The identification of ESBL-producing isolates is important since the activity of the extended-spectrum cephalosporins may not be accurately predicted from standardized susceptibility tests. In 1999, the NCCLS published methods for screening and confirming the presence of ESBLs in K. pneumoniae, K. oxytoca, and Escherichia coli. Considering that ESBL’s can have different preferred -lactam substrates, and confirmatory tests demand time and additional cost, it is important to establish locally which substrate must be tested for optimal detection of such isolates. The objective of this evaluation was to describe in detail the frequency of occurrence, the preferred substrate, and the co-resistance patterns of the ESBL-producing isolates collected from medical centers in the Asia-Pacific (also South Africa) region through the SENTRY Antimicrobial Surveillance Program (1998-99). TABLE 5: Co-resistance phenotypes observed among the ESBL-producing isolates by nation and medical center. REFERENCES Escherichia coli Australia 201 (1) 202 (1) 1(100.0) 0 0 0 0 0 0 0 0 1(100.0) 0 1(100.0) 0 1(100.0) Bush, K., Jacoby, G.A., Medeiros, A.A. (1995) A functional classification scheme for -lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39,1211-1233. Cormican, M.G., Marshall, S.A., Jones, R.N. (1996) Detection of Extended-spectrum -lactamase (ESBL)-producing strains by Etest ESBL screen. J Clin Microbiol 34,1880-1884. Jacoby, G.A. (1997) Extended-spectrum -lactamases and other enzymes providing resistance to oxyimino--lactams. Inf Dis Clin North Am11,875-887. Knothe, H., Shah, P., Kremery, V., Antal, M., Mitsuhashi, S. (1983) Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection 11, 315-317. Martinez-Martinez, L., Hernandez-Alles, S., Alberti, S., Tomas, J.M., Benedi, V.J., Jacoby, G.A. (1996). In vivo selection of porin-deficient mutants of K. pneumoniae with increased resistance to cefoxitin and expended-spectrum cephalosporins. Antimicrob Agents Chemother40,342-348. National Committee for Clinical Laboratory Standard. (2001). Performance standards for antimicrobial susceptibility testing; eleventh informational supplement M100-S9. NCCLS, Wayne, PA. National Committee for Clinical Laboratory Standards (NCCLS). (2000a). Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically, fifth edition. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, PA. Rice, L.B., Yao, D.C., Klimm, K., Eliopoulos, G.M., Moellering, R.C. Jr. (1991) Efficacy of different -lactams against an extend-spectrum -lactamases-producing Klebsiella pneumoniae strain in the rat intra-abdominal abscess model. Antimicrob Agents Chemother 34,2193-2199. Sader, H.S., Pfaller, M.A., Jones, R.N., Doern, G.V., Gales, A.C., Winokur, P.L., Kugler, K.C. (1999) Bacterial Pathogens Isolated from Patients with Bloodstream Infections in Latin America, 1997: Frequency of Occurrence and Antimicrobial Susceptibility Patterns from the SENTRY Antimicrobial Surveillance Program. Braz J Infect Dis3,97-110. Winokur, P.L., Canton, R., Casellas J.M., Legakis, N. (2001) Variations in the prevalence of strains expressing an extended-spectrum beta-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin Infect Dis32(Suppl2),94-103. China 204 (25) 208 (10) 209 (4) 210 (11) 12(48.0) 4(40.0) 3(75.0) 10(90.9) 16(64.0) 9(90.0) 4(100.0) 10(90.9) 3(12.0) 1(100.0) 0 1(9.1) 18(72.0) 10(100.0) 4(100.0) 8(72.7) 13(52.0) 9(90.0) 4(100.0) 7(63.6) 20(80.0) 8(80.0) 3(75.0) 8(72.7) 4(16.0) 1(10.0) 3(75.0) 8(72.7) Japan 207 (2) 1(50.0) 1(50.0) 0 1(50.0) 1(50.0) 0 0 Taiwan 214 (5) 215 (2) 2(40.0) 0 2(40.0) 0 0 1(50.0) 3(60.0) 2(100.0) 2(40.0) 2(100.0) 4(80.0) 2(100.0) 4(80.0) 2(100.0) The Philippine 211 (20) 12(60.0) 12(60.0) 4(20.0) 13(65.0) 15(75.0) 15(75.0) 18(90.0) TABLE 3. Comparison of ESBL occurrence rates among E. coli and K.pneumoniae isolates in those institutions having ESBL production in Enterobacter spp. Singapore 212 (4) 1(25.0) 3(75.0) 0 4(100.0) 4(100.0) 4(100.0) 4(100.0) MATERIALS AND METHODS South Africa 213 (2) 1(50.0) 0 0 2(100.0) 2(100.0) 2(100.0) 2(100.0) Klebsiella pneumoniae Bacterial Strains A total of 1,103 Escherichia coli, 643 Klebsiella spp., and 111 Proteus mirabilis isolates were collected from diverse body sites by the SENTRY Program between January 1998 and December 1999 in the Asian-Pacific region (17 medical centers). The medical centers were distributed throughout 14 cities in seven countries: Australia (four centers), China (four centers, including a site located in Hong Kong), Japan (three centers), Taiwan (three centers), The Philippines (one center), Singapore (one center), and South Africa (one center). Because there is only one African site included in the SENTRY Program, its data was analyzed with the Asia-Pacific Region. Only one isolate per patient was evaluated in this study. Susceptibility Testing The isolates were identified to the species level by the participating center and sent to the monitoring laboratory (Women and Children’s Hospital, Adelaide, Australia) for identification confirmation and reference susceptibility testing. Antimicrobial susceptibility testing of the possible ESBL-producers was performed by reference broth microdilution method as described by the National Committee for Clinical Laboratory Standards (NCCLS). Quality control (QC) was performed using E. coli ATCC 25922, S. aureus ATCC 29213, and P. aeruginosa ATCC 27853. All QC results were within published ranges. Australia 203 (7) 2(28.6) 1(14.3) 1(14.3) 5(71.4) 6(85.7) 5(71.4) 2(28.6) E. coli K. pneumoniae P. mirabilis China 204 (10) 208 (2) 207 (7) 210 (4) 7(70.0) 1(50.0) 1(14.3) 6(42.9) 5(50.0) 2(100.0 2(28.6) 11(78.6) 1(10.0) 0 3(42.9) 5(35.7) 7(70.0) 1(50.0) 5(71.4) 5(35.7) 8(80.0) 2(100.0) 5(71.4) 5(35.7) 8(80.0) 2(100.0) 2(28.6) 4(28.6) 6(60.0) 2(100.0) 1(14.3) 5(35.7) Country/medical center no. Total no.of isolates No. of ESBL (%) Total no.of isolates No. of ESBL (%) Total no.of isolates No. of ESBL (%) Japan 205 (3) 206 (1) 207 (1) 0 1(100.0) 1(100.0) 0 0 0 0 0 0 0 0 0 0 0 0 3(100.0) 1(100.0) 1(100.0) 0 1 0 Taiwan 214 (3) 216 (1) 2(66.6) 0 0 0 2(66.6) 0 3(100.0) 0 3(100.0) 0 1(33.3) 1(100.0) 2(66.6) 0 The Philippines 211 (52) 40(77.0) 20(38.4) 4(7.7) 24(46.1) 34(65.4) 30(57.6) 44(84.0) Singapore 212 (25) 3(12.0) 7(100.0) 6(24.0) 17(68.0) 19(76.0) 10(40.0) 14(56.0) South Africa 213 (12) 0 0 0 12(100.0) 12(100.0) 4(33.3) 5(41.7) aCipro = ciprofloxacin; Amik = amikacin; Gent = gentamicin; Tobra = tobramycin; Tetra = tetracycline; and TMP/SMX = trimethoprim/sulfamethoxazole. A156-24