Molecular Mechanisms of Carbapenem Resistance in Enterobacter cloacae Clinical Isolates from Korea and Clinical Outcome

Size: px

Start display at page:

Download "Molecular Mechanisms of Carbapenem Resistance in Enterobacter cloacae Clinical Isolates from Korea and Clinical Outcome"

Duane Park
6 years ago
Views:

1 Annals of Clinical & Laboratory Science, vol. 42, no. 3, 2012 Available online at Molecular Mechanisms of Carbapenem Resistance in Enterobacter cloacae Clinical Isolates from Korea and Clinical Outcome Yangsoon Lee 1, 2, Heekyeong Choi 3, Jong Hwa Yum 4, Girung Kang 1, Il Kwon Bae 1, Seok Hoon Jeong 1, and Kyungwon Lee 1 1 Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine; 2 Department of Laboratory Medicine, National Health Insurance corporation Ilsan Hospital; 3 Department of Infectious Diseases, Wonju Christian Hospital; 4 Department of Clinical Laboratory Science, Dong-eui University; Seoul, Korea Abstract. We investigated the molecular mechanisms of carbapenem resistance in clinical isolates of Enterobacter cloacae and their clinical characteristics. Nonreplicable E. cloacae isolates were recovered from six cancer patients and one patient with liver cirrhosis at a tertiary-care hospital in Korea between 2002 and Two patients who were considered to have a true infection caused by these microorganisms have died. All isolates produced AmpC β-lactamases, including ACT-1, ACT-2, MIR-3 and DHA-1, and CTX-M- or SHV-type extended-spectrum β-lactamase. Two isolates produced plasmid-borne VIM-2 carbapenemase. All probes specific for bla AmpC genes hybridized with I-CeuI chromosomal fragments were also recognized by a probe specific for 16S rdna, suggesting a chromosomal location. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a major outer membrane protein, OmpF, was absent in all isolates. PFGE of XbaI-digested DNA were considered to be unrelated. The results of our study suggest that the chromosomal AmpC β-lactamase with impermeability in E. cloacae clinical isolates implicated in reduced carbapenem susceptibility. Although carbapenem-resistant E. cloacae isolates were isolated in a few patients in our study, the clinical outcomes were grave. Therefore, the patients colonized or infected by carbapenemresistant E. cloacae isolates should gain attention of antibiotic therapy. Introduction Enterobacter cloacae has recently emerged as an important nosocomial pathogen which harbors inducible chromosomal AmpC β-lactamase. Carbapenems are the drug of choice in the treatment of serious infections caused by producing extended-spectrum β-lactamase (ESBL) E. cloacae. The emergence of carbapenem-resistant E. cloacae is of great concern due to limited antimicrobial treatment options. Moreover, clinical outcomes of the patients infected by carbapenem resistant Enterobacteriaceae are known to be grave [1-3]. Marchaim et al. [2] reported that infections caused by KPC-2-producing-Enterobacter strains were associated with increased mortality (33%). Falcone et al. [4] reported that patents with infections caused Address correspondence to Seok Hoon Jeong; Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 250 Seongsanno, Seodaemungu, Seoul , Korea; tel: ; fax: ; kscpjsh@yuhs.ac by VIM-1-producing E. cloacae had high risk factors such as relapse of infection and prolonged duration of antibiotic therapy. Carbapenem resistance in E. cloacae isolates has arisen via Ambler classes A or B carbapenem-hydrolyzing β-lactamases. NMC-A and IMI-1 are chromosomally encoded class A carbapenemases, which are first identified from E. cloacae clinical isolates in 1990 and 1984, respectively [5, 6]. KPC- 2 or -3 producer E. cloacae clinical isolates were reported in the USA in 2003 [1]. Class B carbapenemase such as VIM-1, VIM-2, and IMP-1 have been identified in E. cloacae clinical isolates since the first report of plasmid-borne VIM-2 in an E. cloacae KU680 from Korea in 2000 [7-10]. Carbapenem resistance in E. cloacae has also been attributed to AmpC enzymes or ESBLs combined with impermeability [11, 12]. Reduction in expression level of outer membrane proteins (OMPs), such as OmpC and OmpF, was involved with impermeability in E. cloacae [12-14] /12/ by the Association of Clinical Scientists, Inc.

2 282 Annals of Clinical & Laboratory Science, vol. 42, no. 3, 2012 Table 1. Clinical characteristics and outcomes of the patients carrying carbapenem-resistant E. cloacae Patient Sex/Age Depart Comorbidity Duration of Charlson Previous use Isolation Previous Clinical illness Treatment (years) ment hospital stay comorbidity of carbapenem site(s) E. cloacae regimens/ before acquisition score or cephalosporin isolation outcome in days (duration in days) 1 M/67 Medical Non small 21 8 IPM (16) Sputum No Pneumonia CPZ, TZP, CIP, ICU cell lung cancer, SXT, VA/ Recent steroid use Failure due to radiation pneumonitis 2 M/49 Surgical HTN, CBD 10 6 CEF (10) Peritoneal Yes Intraabdominal CPZ, CEF, ISE ward cancer s/p fluid infection /Improve Whipple operation 3 M/56 Surgical DM, s/p liver 69 5 CTX (8), ward transplantation ZOX (10), Bile Yes Colonization LZD, SXT/ ND due to cirrhosis CPZ (15), ETP (2) 4 F/59 Surgical HTN, DM, CRO (83), Bile Yes Colonization AN, LVX, VA/ ICU s/p liver CPZ (15), ND transplantation ZOX (15), due to HCC MEM (16) 5 F/8 Pediatric ALL 1 2 No Urine No Colonization ISE,TEC, SXT/ ward ND 6 F/1 Pediatric ALL 18 2 CFP (12) Urine Yes Colonization CEF, ISE/ ND ward 7 F/62 Medical NHL, 66 4 CPZ (30) Throat, Yes Oral infection CPZ, ISE, AN, ward Do-not- sputum, TEC/ Failure resuscitate state oral Abbreviations: M, male; F, female; HTN, hypertension; ALL, acute lymphoblastic leukemia; DM, diabetes mellitus; HCC, hepatocellular carcinoma; URI, upper respiratory infection; IPM, imipenem; ETP, ertapenem; MEM, meropenem; CTX, Cefotaxime; ZOX, ceftizoxime; CPZ, cefoperazone-sulbactam; CRO, ceftriaxone; CFP, cefpiran; TZP, piperacillin-tazobactam; CIP, ciprofloxacin; SXT, Trimethoprim-sulfamethoxazole; VA, vancomycin; CEF, cefpiramide; ISE, isepamicin; LZD, linezolid; AN, amikacin; LVX, levofloxacin; TEC, teicoplanin; ND, not detected.

3 The aims of this study were to investigate the carbapenem resistant mechanisms in clinical isolates of E. cloacae from a tertiary-care hospital in Korea and present the clinical characteristics of patients infected or colonized by these organisms. Materials and Methods Patients and bacterial isolates. Clinical isolates of carbapenem resistant E. cloacae were recovered from seven inpatients of a tertiary-care hospital in Korea between 2002 and The isolates were identified using conventional biochemical methods and the Vitek 2 system (biomérieux, Marcy l Etoile, France). Clinical features of seven patients were reviewed through electronic medical records. E. cloacae ATCC13047 was used as a reference strain. Antimicrobial susceptibility testing. Antimicrobial susceptibilities were determined by disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines [15]. The modified Hodge test and the imipenem and EDTA-sodium mercaptoacetic acid double-disk synergy (IEDDS) test were performed on MacConkey agar plates as previously described for the screening of carbapenemases and metallo-βlactamases (MBLs), respectively [16]. MICs of imipenem and meropenem were determined using the E-test (Becton Dickinson, Sparks, MD) on Mueller-Hinton agar plates with or without a fixed concentration of clavulanic acid (CA; 4 mg/l; Sigma-Aldrich Corporation, St. Louis, MO) as an inhibitor of ESBL, aminophenyl boronic acid (APB; 300 mg/l; Sigma-Aldrich Corporation) as an inhibitor of AmpC β-lactamase, or Phe-Arg-βnaphthylamide (PAβN; 40 mg/l; Sigma-Aldrich Corporation) as an efflux pump inhibitor. PCR experiments for β-lactamase genes. Detection of genes coding for AmpC β-lactamases, ESBLs, and classes A and B carbapenemases was performed by PCR amplification with primers described previously [17, 18], PCR products were subjected to direct sequencing with an automatic DNA sequencer (model 3730xl; Applied Biosystems, Weiterstadt, Germany). SDS-PAGE for OMPs. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was carried out to investigate alterations in OMPs as previously described [19]. Briefly, bacterial cells Carbapenem-resistant E. cloacae were disrupted by ultrasonic disintegration and the supernatants were treated with 0.1 M sodium carbonate. After incubation on ice for 1hr, OMPs were collected by centrifugation at 115,000 g for 2 hr at 4 C and were analyzed by SDS-PAGE on a Mini-PROTEAN 3 Cell apparatus (Bio-Rad, Hercules, CA). The 10% (wt/vol) polyacryl gels were stained with Coomassie Brilliant Blue. Pulsed-field gel electrophoresis (PFGE). Plugs containing whole genomic DNA of the isolates were digested with XbaI, I-CeuI or S1 nuclease. DNA fragments were separated using PFGE with a CHEF-DRII device (Bio-Rad). The PFGE conditions of XbaI-macrorestriction analysis were 6 V/ cm for 20 hr with pulse times ranging from 0.5 to 60 seconds at 14 C. The pulse times for I-CeuI and S1 nuclease restriction analysis were 9 to 90 seconds. The lambda ladder (Bio-Rad) was used as a DNA size marker. Dendrograms were generated by the unweighted pair group method with arithmetic average method, and DNA relatedness was calculated based on the criteria suggested by Tenover et al. [20]. Southern blotting. The gels with I-CeuI-digested chromosomal DNA and S1 nuclease-treated linearized plasmids were blotted onto nylon membranes (Bio-Rad) and hybridized with probes specific for the bla ACT, bla MIR-3, bla AmpC, and 16S rdna. The probes were obtained via PCR experiments as described above. Probe labeling, hybridization, and detection were performed with the DIG DNA Labeling and Detection kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer s protocols. Results 283 Description of the patients. Clinical characteristics and outcomes of seven patients were presented in Table 1. The median age of the patients was 56 years (range, 1-67), and three patients were male. Four patients had chronic or malignant liver diseases, and three patients had hematologic malignancies. The median Charlson comorbidity score was 5 (range, 2-8). Five patients had been previously colonized with carbapenem-susceptible E. cloacae. They were exposed to the oxyimino-cephalosporins during their prolonged hospital stay (median duration, 64 days; range, )

4 284 Annals of Clinical & Laboratory Science, vol. 42, no. 3, 2012 Table 2. Carbapenems MICs with or without inhibitors and β-lactamase genes for clinical isolates of E. cloacae Strains MICs (mg/l) β-lactamase gene Without inhibitors APB CA PAßN ESBL or MBL AmpC IPM MER IPM MER IPM MER IPM MER YMC 09/5/ CTX-M-57 ACT-2 YMC 08/12/ CTX-M-3, ACT-1 VIM-2 YMC 06/7/ CTX-M-9, ampc 1 SHV-12 YMC 05/10/ NG NG CTX-M-9, ampc 1 SHV-12 YMC 04/8/ CTX-M-3 MIR-3 YMC 03/4/ CTX-M-57, ampc 1 VIM-2 YMC 02/9/ CTX-M-15 ACT-2, DHA-1 1 The bla genes with sequence identical to that of ampc gene of the TR211 strain (Genbank accession number: DQ478700). Abbreviations: APB, aminophenylboronic acid (300 mg/l); CA, clavulanic acid (4 mg/l); PAßN, Phe-Arg-ß-naphthylamide (40 mg/l); IPM, imipenem; MEM, meropenem; ESBL, extended-spectrum β-lactamse; MBL, metallo-β-lactamase; ND, not detected; NG, no growth. before carbapenem-resistant E. cloacae strains were isolated. Three cases were considered to be true infection, while remaining four cases to be simple colonization, judging from CDC/NHSN surveillance definition of health care-associated infection. Only one patient with intra-abdominal infection improved with antimicrobial treatment, while two patients with pneumonia or oral infection died within 30 days of infection. Antimicrobial susceptibilities. All seven isolates were nonsusceptible to piperacillin, amoxicillinclavulanic acid, cefoxitin, ceftazidime, cefotaxime, aztreonam, cefoperazone-sulbactam, and trimethoprim-sulfamethoxazole using disk diffusion assay. They exhibited reduced susceptibilities to imipenem or meropenem (MIC range, mg/l) using E-test. APB lowered imipenem and meropenem MICs 2- to 21-fold and 2- to 32-fold, respectively, while CA and PAβN did not (Table 2). One isolate (YMC 03/4/397) showed positive results with the modified Hodge and the IEDDS tests, suggesting MBL production. Identification of β-lactamase genes. PCR and sequencing experiments detected AmpC β-lactamase genes such as bla ACT-1, bla ACT-2, bla MIR-3 and bladha-1 genes in four isolates. Both bla ACT-2 and bla DHA-1 genes were detected in one isolate (YMC 02/9/932). AmpC β-1actamase genes identical to the sequence of E. cloacae TR211 strain (GenBank accession no. DQ478700) were detected in three isolates. The ESBL genes such as bla SHV-12, bla CTX-M-3, bla CTX-M-9, bla CTX-M-15 and bla CTX- M-57 genes were detected. Two isolates carried both bla CTX-M-57 and bla SHV-12 genes. Two isolates (YMC 03/4/397, YMC 03/4/397)) were found to produce VIM-2 MBL. The bla genes encoding class A carbapenemase including KPC, IMI, GES and SME were not detected in any of the isolates. Locations of β-lactamase genes. In Southern blot assay, probes specific for the bla ACT-1 and the bla ACT-2 hybridized with 450-kb I-CeuI chromosomal fragments from YMC 08/12/3793 and 600- kb from YMC 09/5/165 isolate, respectively. Probes for the bla MIR-3 and the bla DHA-1 were hybridized with 550-kb and 250-kb I-CeuI chromosomal fragments from YMC 04/8/2304 and YMC 02/9/932 isolates, respectively. The unnamed AmpC β-lactamase genes probe hybridized with approximately 350 kb I-CeuI chromosomal fragments in

5 Carbapenem-resistant E. cloacae 285 Figure 1. SDS-PAGE analysis of outer membrane proteins (OMPs) of E. cloacae isolates. Lane M, protein molecular weight size markers of 25, 37 and 50 kda; lanes 1 to 7, seven experimental isolates ; lane C, the control strain E. cloacae ATCC three isolates. However, all were hybridized with S1 nuclease-treated linearized plasmids. The 16S rdna probes were hybridized with the same chromosomal fragments hybridized with the probes specific for all AmpC β-lactamase genes detected. These findings suggest the presence of all AmpC β-lactamase genes detected in our isolates on a chromosome rather than on a plasmid (data not shown). However, the bla VIM-2 probe hybridized with 400 kb S1 nuclease-treated plasmids not with I-CeuI chromosomal fragments, which suggested plasmid location of that gene. SDS-PAGE for OMPs. SDS-PAGE analysis of OMPs revealed that all isolates showed lacked or diminished expression of OmpF porins compared to the reference strain E. cloacae ATCC Two isolates (YMC 09/5/165 and YMC 04/8/2304) lacked both OmpF and OmpC porins. All the isolates retained expression of OmpA porins compared to the control strain (Figure 1). Clonal relatedness. XbaI-digested DNA from all isolates showed genetic similarity of 65% to 85%, and were considered to be unrelated. Discussion Over an eight-year period, seven cases of clinical isolates of E. cloacae exhibiting carbapenem resistance were identified in a tertiary-care hospital in Seoul, Korea. All patients were in immunosuppressant states, undergoing chemotherapy or post-operation. Most of these patients acquired those bacteria during a prolonged hospital stay, and were exposed to the oxyimino-cephalosporins in hospital. Three cases were considered to be true infections while four cases simple colonization. Of these, two of three patients died in spite of appropriate antibiotic treatment. Although carbapenemresistant E. cloacae were isolated in a few patients in our study, the clinical outcomes were grave. Therefore, the patients colonized or infected by carbapenem-resistant E. cloacae isolates should gain attention of antibiotic therapy. Acquired carbapenem resistance is still uncommon among E. cloacae but can arise in the presence of classes A or B carbapenemase and AmpC or ESBLs enzymes combined with impermeability [12, 14]. In our study, carbapenem-resistant E. cloacae clinical isolates were comediated by chromosomally encoded AmpC enzymes and OMPs loss. The expression of OmpF porin of all isolates was greatly decreased based on SDS-PAGE experiments, while that of OmpC porin was decreased in two isolates. The expression of OmpA porins in two isolates lacking both OmpC and OmpF porins was relatively stronger compared to that of the other OmpA porins. It supported that minor porins may express stronger to acquire nutrients for their fitness, when

6 286 Annals of Clinical & Laboratory Science, vol. 42, no. 3, 2012 major porins were greatly diminished [21]. Synergy test with PAβN did not indicate efflux as a major component in carbapenem resistance, although it has been reported to have contributed to ertapenem resistance in clinical isolates of E. cloacae [12]. VIM-2 carrying E. cloacae clinical isolate was rare in our study, and was the second report in the world. In conclusion, the reduced carbapenem susceptibility in clinical isolates of E. cloacae was associated with porin loss combined with chromosomal AmpC β-lactamase. Although the prevalence of carbapenem-resistant E. cloacae and patients infected with these microorganisms are still low, their occurrence needs to be continuously monitored. Acknowledgements This study was supported by a grant (A090971) from the Korea Healthcare Technology R&D Project run by the Ministry for Health, Welfare & Family Affairs of the Republic of Korea. References 1. Bratu, S., D. Landman, R. Haag, R. Recco, A. Eramo, M. Alam, and J. Quale. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch Intern Med 2005; 165: Marchaim, D., S. Navon-Venezia, M. J. Schwaber, and Y. Carmeli. Isolation of imipenem-resistant Enterobacter species: emergence of KPC-2 carbapenemase, molecular characterization, epidemiology, and outcomes. Antimicrob Agents Chemother 2008; 52: Patel, G., S. Huprikar, S. H. Factor, S. G. Jenkins, and D. P. Calfee. Outcomes of carbapenem-resistant Klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies. Infect Control Hosp Epidemiol 2008; 29: Falcone, M., M. L. Mezzatesta, M. Perilli, C. Forcella, A. Giordano, V. Cafiso, G. Amicosante, S. Stefani, and M. Venditti. Infections with VIM-1 metallo-{beta}-lactamaseproducing enterobacter cloacae and their correlation with clinical outcome. J Clin Microbiol 2009; 47: Nordmann, P., S. Mariotte, T. Naas, R. Labia, and M. H. Nicolas. Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli. Antimicrob Agents Chemother 1993; 37: Rasmussen, B. A., K. Bush, D. Keeney, Y. Yang, R. Hare, C. O Gara, and A. A. Medeiros. Characterization of IMI-1 betalactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. Antimicrob Agents Chemother 1996; 40: Chen, L. R., H. W. Zhou, J. C. Cai, R. Zhang, and G. X. Chen. Detection of plasmid-mediated IMP-1 metallo-betalactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate. J Zhejiang Univ Sci B 2009; 10: Ikonomidis, A., N. Spanakis, A. Poulou, S. Pournaras, F. Markou, and A. Tsakris. Emergence of carbapenem-resistant Enterobacter cloacae carrying VIM-4 metallo-beta-lactamase and SHV-2a extended-spectrum beta-lactamase in a conjugative plasmid. Microb Drug Resist 2007; 13: Trevino, M., L. Moldes, L. Martinez-Lamas, C. Varon, and B. J. Regueiro. Carbapenem-resistant Enterobacter cloacae and the emergence of metallo-beta-lactamase-producing strains in a third-level hospital (Santiago de Compostela, NW Spain). Eur J Clin Microbiol Infect Dis 2009; 28: Jeong, S. H., K. Lee, Y. Chong, J. H. Yum, S. H. Lee, H. J. Choi, J. M. Kim, K. H. Park, B. H. Han, S. W. Lee, and T. S. Jeong. Characterization of a new integron containing VIM-2, a metallo- beta-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae. J Antimicrob Chemother 2003; 51: Pages, J. M., C. E. James, and M. Winterhalter. The porin and the permeating antibiotic: a selective diffusion barrier in Gramnegative bacteria. Nat Rev Microbiol 2008; 6: Szabo, D., F. Silveira, A. M. Hujer, R. A. Bonomo, K. M. Hujer, J. W. Marsh, C. R. Bethel, Y. Doi, K. Deeley, and D. L. Paterson. Outer membrane protein changes and efflux pump expression together may confer resistance to ertapenem in Enterobacter cloacae. Antimicrob Agents Chemother 2006; 50: Bennett, J. W., K. Mende, M. L. Herrera, X. Yu, J. S. Lewis, 2nd, B. L. Wickes, J. H. Jorgensen, and C. K. Murray. Mechanisms of carbapenem resistance among a collection of Enterobacteriaceae clinical isolates in a Texas city. Diagn Microbiol Infect Dis 2010; 66: Yigit, H., G. J. Anderson, J. W. Biddle, C. D. Steward, J. K. Rasheed, L. L. Valera, J. E. McGowan, Jr., and F. C. Tenover. Carbapenem resistance in a clinical isolate of Enterobacter aerogenes is associated with decreased expression of OmpF and OmpC porin analogs. Antimicrob Agents Chemother 2002; 46: Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-first Informational Supplement. CLSI document M100-S21. Wayne, PA: CLSI Lee, K., Y. S. Lim, D. Yong, J. H. Yum, and Y. Chong. Evaluation of the Hodge test and the imipenem-edta doubledisk synergy test for differentiating metallo-beta-lactamaseproducing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2003; 41: Lee, H. K., Y. J. Park, J. Y. Kim, E. Chang, S. G. Cho, H. S. Chae, and C. S. Kang. Prevalence of decreased susceptibility to carbapenems among Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii and investigation of carbapenemases. Diagn Microbiol Infect Dis 2005; 52: Song, W., H. Lee, K. Lee, S. H. Jeong, I. K. Bae, J. S. Kim, and H. S. Kwak. CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum beta-lactamase in clinical isolates of Escherichia coli from Korea. J Med Microbiol 2009; 58: Lee, K., D. Yong, Y. S. Choi, J. H. Yum, J. M. Kim, N. Woodford, D. M. Livermore, and Y. Chong. Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 beta-lactamases co-mediated by porin loss. Int J Antimicrob Agents 2007; 29: Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. Interpreting chromosomal DNA restriction patterns produced by pulsedfield gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: Domenech-Sanchez, A., S. Hernandez-Alles, L. Martinez- Martinez, V. J. Benedi, and S. Alberti. Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta-lactam antibiotic resistance. J Bacteriol 1999; 181:

Abstract. Introduction

Abstract. Introduction ORIGINAL ARTICLE BACTERIOLOGY A sensitive and specific phenotypic assay for detection of metallo-blactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic