As presented at the 10th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), May 2000.

Determining Beta-Lactam Resistance among Gram-Negative Isolates in the Phoenix™ Automated Microbiology System. T. WILES, J. HEJNA, M. GOSNELL, C. YU AND V. KENNEDY BD Biosciences • 7 Loveton Circle • Sparks, MD, USA 21152

ABSTRACT

OBJECTIVES: To determine beta-lactam resistance among gram-negative isolates using the Phoenix™ Automated Microbiology System that is currently under development.

METHODS: Phoenix™ susceptibility test results of 210 gram-negative strains that included a challenge set of 110 isolates with known resistance mechanisms and 100 recently acquired clinical isolates were compared to results obtained with a NCCLS recommended Standard Broth Microdilution (SBM) method. Doubling concentrations of ampicillin, amoxicillin, ticarcillin, piperacillin, imipenem and meropenem were tested in both Phoenix™ and SBM.

RESULTS: The overall essential agreement ranged from 91 to 98%. Categorical agreement was from 95 to 100%. OBJECTIVES One very major error was observed with ticarcillin (1%) The beta-lactams are the most utilized and diverse class of all antimicrobial agents. The action of beta-lactams is to inhibit and one with piperacillin (1.3%). No other very major D-alanyl-D-alanine transpeptidase activity by forming stable errors were observed. The major error rates were as follows: esters with the opened lactam ring attached to the –OH group of the enzyme target site. Resistance to beta-lactams in gram- ampicillin (2.1%), amoxicillin (2.5%), ticarcillin (4.0%), negative organisms is often mediated by reduced outer piperacillin (3.1%), imipenem (0.6%) and meropenem (0%). membrane permeability or the production of beta-lactamases. The emergence of antimicrobial resistance among gram-negative CONCLUSIONS: The observations in this study indicate an bacteria, especially high-level resistance to more extended acceptable level of performance in the detection of gram- spectrum beta-lactams, has been reported. The reliable and rapid detection of gram-negative bacterial resistance to beta-lactams is negative resistance to the beta-lactam antibiotics. paramount to a successful therapeutic outcome. The Phoenix™ Automated Microbiology System (BD Biosciences, Sparks, MD, USA) (product currently under Very Major Major development) is intended for the rapid and automated Antibiotic Name n EA % CA % % (n) % (n) identification and susceptibility testing of clinically relevant Aminopenicillins Ampicillin 159 98 97 0 (0) 2.1 (1) bacteria. The emergence of antimicrobial resistance among Amoxicillin 170 95 97 0 (0) 2.5 (1) gram-negative bacteria, especially in species that are frequently Carboxypenicillin isolated from clinical specimens, has placed the emphasis on the Ticarcillin 181 91 95 1.0 (1) 4.0 (3) speed and reliability of automated systems. In the past two Acylureidopenicillin decades, laboratory automation in identification and Piperacillin 184 95 96 1.3 (1) 3.1 (3) susceptibility testing has become more acceptable and allows Carbapenems clinical laboratories to report results with a high degree Imipenem 192 92 99 0 (0) 0.6 (1) of consistency. Meropenem 190 98 100 0 (0) 0 (0) This evaluation investigated the performance of the Phoenix™ System in detecting recognized beta-lactam resistance mechanisms in gram-negative bacteria. The overall performance was based on the accuracy of susceptibility test results generated by the Phoenix™ System as compared to results using the NCCLS recommended test procedures. METHODS

BACTERIAL STRAINS. A total of 110 stock isolates with instructions and incubated for 16–20 h at 35°C in ambient air. known resistance mechanisms and 100 recently acquired fresh Panels were read on the PASCO reader. The NCCLS breakpoints clinical isolates were included in this evaluation. These test were used for categorical interpretation (M100-S10). strains were obtained from the Centers for Disease Control and PHOENIX.™ Phoenix™ panels were manufactured by BD Prevention (Atlanta, GA), La Société Française de Microbiologie Biosciences. Standardized inoculum for each strain was diluted (French National Reference Center Collection, Paris, France), the in Phoenix™ AST Broth, to which one drop of the AST Indicator BD Biosciences (BDB) internal culture collection and fresh clinical had already been added. The final inoculum density was isolates (less than 7 days old from the primary isolation). The approximately 5x105 cfu/ml. The inoculum was poured into the identification of the isolates are listed in Table 1. The characterized Phoenix™ Panel. A panel closure was secured in place and the resistance mechanisms tested in the study are listed in Table 2. inoculated panels were placed into the Phoenix™ instrument. STANDARD BROTH MICRODILUTION (SBM). Frozen Each strain was prepared in duplicate and the panels were microbroth dilution panels manufactured by PASCO (PASCO placed in the Phoenix™ instrument. MICs and categorical Laboratories, BD Biosciences, Sparks, MD) were used as the interpretations (based on the NCCLS breakpoints) were reference. SBM panels were manufactured in doubling dilutions generated by the Phoenix™ instrument. The Phoenix™ System per NCCLS guidelines. The following beta-lactams were tested MICs and categorical interpretations were compared to the in the study: ampicillin (0.5–32 µg/ml), amoxicillin (0.5–32 µg/ml), values obtained from the reference SBM. ticarcillin (1–128 µg/ml), piperacillin (0.5–128 µg/ml), imipenem (0.25–16 µg/ml) and meropenem (0.25–16 mg/ml). All panels were stored at –70°C till ready for use. SBM panels Table 2. ™ were tested in parallel with the Phoenix panels using the same List of Genotypes/Phenotypes Tested inoculum preparation. Panels were inoculated per manufacturer’s Genotype/Phenotype n SHV 1 1 SHV 2 1 Table 1. Species Tested SHV 4 2 Species Total SHV 5 3 Acinetobacter baumannii 4 SHV 6 1 Acinetobacter lwoffii 5 TEM 1 2 Burkholderia cepacia 1 TEM 2 1 Citrobacter amalonaticus 1 TEM 3 2 Citrobacter farmeri 1 TEM 5 2 Citrobacter freundii 2 TEM 6 1 Citrobacter koseri 6 TEM 8 2 Edwardsiella tarda 1 TEM 9 2 Enterobacter aerogenes 4 TEM 10 1 Enterobacter asburiae 1 TEM 11 1 Enterobacter cloacae 5 TEM 12 1 Escherichia coli 68 TEM 24 1 Hafnia alvei 2 TEM 26 1 Klebsiella oxytoca 6 SHV 1, TEM 1 1 Klebsiella pneumoniae ssp ozaenae 1 ANT(2’), OXA 3 1 Klebsiella pneumoniae ssp pneumoniae 27 deletion of OmpC, d OmpF 1 Kluyvera ascorbata 1 ESBL/OSBL 1 Morganella morganii 3 IRT 2, TEM 30 1 Pantoea agglomerans 3 IRT 5, TEM 33 1 Proteus mirabilis 9 IRT 7, TEM 36 1 Proteus vulgaris 3 AmpC 3 Providencia alcalifaciens 2 AmpC derepressed mutants 1 Providencia rettgeri 2 AmpC hyperproducer 1 Providencia stuartii 2 SME 1 1 Pseudomonas aeruginosa 17 TLS 1 Salmonella species 2 basal beta-lactamase 1 Serratia liquefaciens 3 b-lac derepressed mutant 1 Serratia marcescens 3 K1 hyperproduction 3 Serratia odorifera 21 CTX 1 1 Serratia plymuthica 2 OXA 1 1 Shigella boydii 1 OXA 2 1 Shigella flexneri 1 PSE 1 1 Shigella sonne 2 PSE 2 1 Stenotrophomonas maltophilia 4 NMC A 1 Vibrio cholerae 1 Unidentified 160 RESULTS AND DISCUSSIONS

A range of 159 to 192 strains yielded valid results for the antibiotics tested (Table 4). In the study, the previously characterized Extended-spectrum beta-lactamase (ESBL) producing E. coli (13 strains) and Klebsiella species (18 strains) were confirmed using the NCCLS ESBL confirmatory test (M100-S10). The essential and categorical agreements are listed in Table 3 and 4. The average time to results is listed in Table 5. The strain showing a very major error (VME) with piperacillin was previously detected as a Pseudomonas aeruginosa strain resistant to cefepime. However, the strain showing a VME with ticarcillin was a Proteus vulgaris strain with no known resistance mechanism.

Table 5. Average Time to Result Table 3. Essential Agreement of Phoenix™ System with Reference Method for Beta-Lactams Tested

Distribution of Reference MIC Phoenix™ Average Time Species to Result (hour) Drug <=0.25 <=0.5 <=1 <0.5 =1 =2 =4 =8 =16 =32 =64 =128 >16 >32 >128 EA % Achromobacter 15.1 Ampicillin 41 8209736 102 98 Acinetobacter 7.0 Amoxicillin 6 2 11 20 4 2 4 121 95 Burkholderia 11.9 Ticarcillin 21 21 16 385913 8591 Citrobacter 8.2 Piperacillin 15 13 29 13 13 13 7 5 7 69 95 Edwardsiella 8.2 Imipenem 100 30 27 13 10 2 1 9 92 Enterobacter 7.5 Meropenem 156 10 84221 7 98 Escherichia 7.4 1. Number of strains with the indicated reference MIC. Hafnia 8.3 Klebsiella 7.1 Kluyvera 9.7 Morganella 7.7 Table 4. Categorical Agreement of Phoenix™ System with Reference Method Pantoea 9.5 Proteus 7.9 Very Major Major Total Reference N CA Errors Errors Providencia 8.2 Class Name N S I R % N % N % Pseudomonas 13.5 alpha-aminopenicillin Ampicillin 159 47 3 109 97 0 0.0 1 2.1 Salmonella 8.0 alpha-aminopenicillin Amoxicillin 170 40 5 125 97 0 0.0 1 2.5 Serratia 8.3 alpha-carboxypenicillin Ticarcillin 181 75 8 98 95 1 1.0 3 4.0 Shigella 7.2 N-acylureidopenicillin Piperacillin 184 98 10 76 96 1 1.3 3 3.1 Stenotrophomonas 8.9 Carbapenem Imipenem 192 137 6 39 99 0 0.0 1 0.6 Vibrio 8.4 Carbapenem Meropenem 190 134 16 42 100 0 0.0 0 0.0 Average 8.9

CONCLUSION The overall essential agreement ranged from 91 to 98% for the beta-lactams tested. The average time to results of these beta-lactams ranged from 7 to 15 hours depending on the genus tested. The categorical agreement ranged from 95 to 100%. There was only one very major error observed with ticarcillin (1%) and one with piperacillin (1.3%). No other very major errors were observed for the beta-lactams tested. Results from this evaluation indicate the Phoenix™ System is capable of rapidly detecting resistance in gram-negative bacteria with the beta-lactam antibiotics with a mean time to detection at 8.9 h. LR649