Bacteriology-Hygiene, F Paris, France. Université Pierre et Marie Curie-Paris 6,

JCM Accepts, published online ahead of print on 25 September 2013 J. Clin. Microbiol. doi:10.1128/jcm.01936-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Evaluation of LACTA test, a rapid test detecting resistance to third-generation cephalosporins in clinical strains of Enterobacteriaceae. 3 4 5 6 Aurélie Renvoisé 1**, Dominique Decré 2,3**, Rishma Amarsy-Guerle 4, Te-Din Huang 5, Christelle Jost 3, Isabelle Podglajen 5, Laurent Raskine 4, Nathalie Genel 2, Pierre Bogaerts 5, Vincent Jarlier 1 and Guillaume Arlet 2,3,7 *. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Laboratory of Bacteriology-Hygiene, F-75013 Paris, France. Université Pierre et Marie Curie-Paris 6, Laboratory of Bacteriology-Hygiene, F-75013 Paris, France. 2 Université Pierre et Marie Curie-Paris 6, Medical School, Département of Bactériology, F- 75012 Paris, France 3 Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Department of Bacteriology, F-75012 Paris, France 4 Assistance Publique-Hôpitaux de Paris, Hôpital Lariboisière, Laboratory of Bacteriology- Virology-Hygiene, F-75010, Paris, France 5 Université Catholique de Louvain, CHU de Mont-Godinne, Laboratory of Bacteriology, 5530 Yvoir, Belgium 6 Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Microbiologie, F-7515, Paris, France 7 Assistance Publique-Hôpitaux de Paris, Hôpital Tenon, Department of Bacteriology, F- 75020, Paris, France * Corresponding author: guillaume.arlet@tnn.aphp.fr ** AR and DD contributed equally to this work 25 1

26 27 28 Running title: Rapid detection of 3GC resistance in Enterobacteriaceae Key words: rapid, diagnostic test, third-generation cephalosporins, resistance, Enterobacteriaceae, extended-spectrum beta-lactamase, point-of-care 29 2

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Abstract Since decades, third-generation cephalosporins (3GC) are major drugs to treat infections due to Enterobacteriaceae; growing resistance to these antibiotics makes important to rapidly detect such resistance. LACTA test is a chromogenic test developed for detecting within 15 minutes 3GC-resistant isolates from cultures on solid media. A multicenter prospective study conducted in 5 French and Belgian hospitals evaluated the performances of this test on clinical isolates. Based on antibiotic susceptibility testing, strains resistant or intermediate to cefotaxime or ceftazidime were classified as 3GC-resistant and molecular characterization of this resistance was performed. The rate of 3GC-resistance was globally 13.9% (332/2387), 9.4% in Escherichia coli (132/1403), 25.6% in Klebsiella pneumoniae (84/328), 30.3% in species naturally producing inducible AmpC beta-lactamases (109/360), and 5.6% in Klebsiella oxytoca and Citrobacter koseri (7/124). The overall sensitivity and specificity of LACTA test were 87.7% and 99.6% respectively, were 96 and 100% for E. coli and K. pneumoniae and were 67.4% and 99.6% for species naturally producing inducible AmpC beta-lactamase. False negative results were mainly related to 3GC-resistant strains producing AmpC beta-lactamase. Interestingly, the test was positive for all 3GC-resistant extended-spectrum beta-lactamase producing isolates (n=241). Positive predictive value was 97% and would remain 96% for prevalence of 3GC-resistance ranging between 10 and 30%. Negative predictive value was 99% for E. coli and K. pneumoniae and 89% for the species producing inducible AmpC beta-lactamase. In conclusion βlacta test was found to be easy to use and efficient to predict resistance to third-generation cephalosporins, particularly in extended-spectrum betalactamase producing strains. 3

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 Introduction Enterobacteriaceae are one of the most important causes of community and nosocomial acquired infections (1;2). Beta-lactams (mainly extended-spectrum cephalosporins and carbapenems) are widely used to treat such infections (1). However resistance to these molecules has been reported increasingly worldwide and represents a major public health issue. The EARS-net data (http://www.ecdc.europa.eu/en/activities/surveillance/ears-net/pages/index.aspx) in 2011 reported various rates of resistance to third-generation cephalosporins (3GC) for Escherichia coli: from 3% of strains in Sweden to 36.2% in Cyprus, 6% in Belgium and 8.2% in France; and for Klebsiella pneumoniae: from 2.3% of strains in Sweden to 81% in Bulgaria, 13.6% in Belgium and 25.3% in France. Enzymatic resistance to these molecules is related to extendedspectrum beta-lactamases (ESBL), but also to overproduced chromosomal or acquired AmpC beta-lactamases, and to carbapenemases. ESBL-producing Enterobacteriaceae are of growing concern among nosocomial and community-acquired infections (1). Although ESBL producers should not be reported anymore as resistant to all 3GC and aztreonam, even when the strains appear susceptible according to standard breakpoints (3), rapid detection of ESBL producing Enterobacteriaceae is still needed to prompt the implementation of isolation procedures to prevent crosstransmission to other patients and to initiate appropriate antibiotic therapy (4). The detection of beta-lactamases conferring resistance to 3GC is necessary to avoid inappropriate antibiotic use, since multiple studies in a wide range of clinical settings, clinical syndromes, and organisms have shown that failure or delay in adequate therapy results in an adverse mortality outcome (2). For bacteremia, ESBL production was associated with severe adverse outcomes, including higher overall and infection-related mortality, increased length of stay, delay in appropriate therapy, discharge to chronic care and higher costs (5). 4

78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Several phenotypic methods have been proposed for ESBL detection on isolated Enterobacteriaceae strains. The double disk synergy test, the combination disk method and the ESBL Etests have been developed (6). Sensitivities and specificities for these two tests range from 80% to 95% (7;8). Automated phenotypic methods have also been developed (VITEK2 ESBL test, Biomérieux, Marcy l'etoile, France; Phoenix ESBL test, Becton Dickinson, Sparks, MD, USA); they are based on the simultaneous assessment of the antibacterial activity of extended-spectrum cephalosporins, measured alone or in the presence of clavulanate (6). The performances of these systems varied depending upon the species investigated, showing higher sensitivity (80 to 99%) than specificity (50 to 80%) (6-8). Molecular tests for ESBL detection (9)(10)(2) have been developed to reduce time to reporting, but their cost and the need of an experimented staff limit their use. Moreover the observation of new ESBL has limited the interest for these techniques regarding their limited ability to cover the whole range of variants. MALDI-TOF mass spectrometry was recently proposed to detect antibiotic resistance mechanisms (11), but is to date not reliable enough for routine diagnosis (12;13). A new chromogenic test called LACTA test (BLT, Bio-Rad, Marnes-la-Coquette, France) was recently developed for rapid detection (in less than 15 minutes) of Enterobacteriaceae resistant to 3GC from culture on solid media. Preliminary results suggested that this test is reliable for the detection of 3GC resistant Enterobacteriaceae (14). The test was recently evaluated in Pseudomonas aeruginosa to detect resistance to ceftazidime, and was found sensitive (95%) and specific (87%) for this purpose (15). Here we report the result of a prospective multicenter study aiming at evaluating the performances of the BLT to rapidly detect resistance to 3GC in clinical isolates of Enterobacteriaceae. 101 102 Materials and methods 5

103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 Strains. The strains included in the study were consecutive unselected non duplicate strains of Enterobacteriaceae prospectively isolated in 2012 during a period of three months in five different clinical laboratories of French and Belgian teaching hospitals (Figure S1) from clinical specimens of any origins (i.e. not including rectal swabs sampled for systematic screening of multi-resistant bacteria carriers). BLT was performed on primary cultures (or on the first subculture for positive blood cultures) whatever the type of agar medium used, except Mac Conkey agar which is not recommended by the manufacturer. Identification at the species level was performed by MALDI-TOF mass spectrometry analysis or by API 20E system (Biomérieux, Marcy l'etoile, France). βlacta test (BLT). The principle of BLT is based on the cleavage of a chromogenic substrate, HMRZ-86 (chromogenic cephalosporin). This substrate, initially yellow, turns to the red colour when hydrolyzed. The very efficient beta-lactamases hydrolysing HMRZ-86 are ESBL (CTX-M), and carbapenemases (IMP), whereas the activity of overproduced or acquired AmpC is lower (16). In contrast, HMRZ-86 is not hydrolyzed by broad-spectrum beta-lactamases (e.g. SHV-1, TEM-1) or by basal production of AmpC (17). Results were interpreted as recommended by the manufacturer (Bio-Rad, Marnes-la-Coquette, France) both within the first 2 minutes and after 15 minutes, as positive (if colour turned to red or purple) or negative (if colour remained yellow). Results were considered as noninterpretable if the colour turned into orange. Antibiotic susceptibility testing. Antibiotic susceptibility testing (AST) by disk diffusion method was performed the same day as the BLT. AST was performed and interpreted according to CA-SFM 2012 guidelines (inoculum 0.5 McFarland, diluted to 1/10) in the French laboratories (3). The Belgian laboratory (CHU Mont-Godinne, Yvoir, Belgium) used CLSI 2012 guidelines (inoculum 0.5 McFarland) (18). The strains showing intermediate or full resistance were considered together as resistant. For further comparison with BLT, the 6

128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 strains were classified into two groups based on AST results: strains resistant to cefotaxime (CTX) and/or ceftazidime (CAZ) have been defined as 3GC resistant (3GC-R) and strains fully susceptible to CTX and CAZ have been defined as 3GC susceptible (3GC-S). Molecular characterization of beta-lactamases. Beta-lactamases conferring resistance to 3GC were detected by molecular methods for: (a) the strains resistant to 3GC, (b) the strains with discrepant results between BLT and AST, and (c) the strains with noninterpretable results of BLT. DNA preparation and multiplex PCRs, previously developed to detect the most frequent widespread genes encoding the OXA-1-like broad-spectrum betalactamases, ESBL, plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases, were performed as described (19;20). PCR products were purified using the ExoSap purification kit (Illustra, Exostar 1-step, D. Dutscher, Brumath, France) and bidirectional sequencing was performed using BigDye terminator 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Each sequence was aligned using Applied Biosystems SeqScape software v2.7 and then compared with already known beta-lactamase gene sequences by multiple sequence alignment using the BLAST program on Genbank database. For the species naturally producing inducible AmpC (i.e. Enterobacter spp., Serratia spp., Morganella spp., Providencia spp., and Citrobacter freundii), the strains (i) resistant to 3GC and aztreonam but susceptible to cefepime, carbapenems and (ii) with a negative synergy test between 3GC, cefepime or aztreonam and clavulanic acid, were considered as stably producing derepressed AmpC and not producing ESBL (7;21;22) and were not submitted to molecular characterization. Statistic analysis. Performances for BLT were calculated using SISA website (http://www.quantitativeskills.com/sisa/statistics/diagnos.htm). A width of 95% was chosen for confidence interval. 7

153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 Results Characteristics of the strains. A total of 2,387 strains of Enterobacteriaceae were included in the study: 1,403 of E. coli, 328 of K. pneumoniae, 360 of species naturally producing inducible AmpC beta-lactamases (Enterobacter spp., Morganella spp., Providencia spp., Serratia spp., C. freundii ), 172 of P. mirabilis and 124 of species other than K. pneumoniae naturally producing chromosomal class A enzymes inhibited by clavulanic acid (K. oxytoca and C. koseri) (Table 1). Strains were obtained from 1,717 (72%) urines, 238 (10%) skin and soft tissues specimens, 218 (9%) respiratory specimens, 57 (2%) blood cultures, and 157 (7%) various clinical samples. Strains were collected from the following agar media (Bio-Rad, France and Biomérieux, France): 1,447 from Uriselect 4 agar, 282 from Drigalski agar, 250 from CLED agar, 235 from blood agar, 152 from trypticase soy agar, and 21 from chocolate agar. Based on AST, the overall rate of resistance to 3GC was 13.9% (332/2,387), more specifically 9.4% (132/1,403) in E. coli, 25.6% (84/328) in K. pneumoniae, 30.3% (109/360) in species naturally producing inducible AmpC, 0% (0/172) in P. mirabilis, and 5.6% (7/124) in K. oxytoca and C. koseri (Table 1). Results of molecular characterization of beta-lactamases were as follow. ESBLs were found in 114/1,403 E. coli (8.1%) as follows: 105 CTX-M (including 47 CTX-M-15), 4 SHV (-2, -12) and 5 TEM (-15, -19, -24, -52) enzymes; in 77/328 K. pneumoniae (23.5%): 66 CTX-M (including 61 CTX-M-15), 7 SHV (-2, -5, -12) and 4 TEM (-3, -24, -52) enzymes; in 1/172 P. mirabilis (0.6%), CTX-M-15; in 4/124 K. oxytoca and C. koseri (3.2%): 1 CTX-M- 1, 1 SHV (-12) and 2 TEM (-24, -29) enzymes; and in 46/360 species naturally producing inducible AmpC (12.8%): 28 CTX-M (including 26 CTX-M-15), 15 SHV (-12) and 3 TEM (-11, -24) enzymes (Table 1 and Figure S1). Detailed characterization of ESBL enzymes in 8

178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 each center is given in Figure S1. Overall, the proportion of CTX-M-15 among ESBLs was 55.8% (135/242). For the global panel of strains, plasmidic AmpC beta-lactamases were found in 7/1,403 E. coli (6 CMY-2 and 1 DHA-1) and in 7/328 K. pneumoniae (7 DHA-1). No carbapenemase producing strain was detected during the study. Few strains of K. pneumoniae (n=2) and K. oxytoca (n=5), for which BLT yielded discrepant or non interpretable results, were neither producing ESBL nor plasmidic AmpC beta-lactamase, and were considered as overproducing their natural chromosomal beta-lactamase (Table 3) (21;22). Among 109 3GC-R strains of species that naturally produce inducible AmpC, 65 were considered as stably overexpressing this enzyme based on AST (Table 1). Performances of the βlacta test. Resistance to 3GC based on AST results was taken as the gold standard. Consequently, results were considered concordant if 3GC-R strains yielded a positive BLT or if 3GC-S strains yielded a negative BLT. Results were considered discordant in the other cases. Overall, non-interpretable results were observed for 2.4% (57/2,387) of the strains, including 1.4% (20/1,403) in E. coli, 1.8% (6/328) in K. pneumoniae, 0% (0/172) in P. mirabilis, 7.2% (26/360) in species naturally producing inducible AmpC beta-lactamases and 4% (5/124) in K. oxytoca and C. koseri (Table 3). These non-interpretable results were excluded from the concordance analysis. Overall, BLT sensitivity was 87.7% and specificity was 99.6%. Considering each group of species, sensitivity and specificity were 96% and 99.9% for E. coli, 96.3% and 100% for K. pneumoniae, 67.4% and 99.6% for species naturally producing inducible AmpC betalactamases, 100% and 95.5% for K. oxytoca and C. koseri (Table 2). For P. mirabilis (no 3GC resistant strain) specificity was 99.4%. The 8 false positive results observed among the 2,028 3GC-S strains (with interpretable BLT result) included 5 K. oxytoca with a high-level expression of their natural chromosomal enzyme, which were resistant to aztreonam but susceptible to cefotaxime and ceftazidime, 1 CTX-M-15 producing P. mirabilis still 9

203 204 205 206 207 208 209 210 211 212 213 susceptible to 3GC, 1 wild type P. vulgaris, and 1 OXA-1 producing E. coli (Table 3). The 37 false-negative results observed among the 302 3GC-R strains (with interpretable BLT result) included 29 strains overexpressing AmpC beta-lactamases in the species that naturally produce this enzyme, 5 strains producing plasmidic AmpC beta-lactamases (2 CMY-2 producing E. coli and 3 DHA-1 producing K. pneumoniae) and 3 strains of E. coli that did not produce ESBL nor plasmidic AmpC beta-lactamase but that were 3GC-R at low level (Table 3). Importantly, all the ESBL producing strains of the 3GC-R group (n=241), including 114 E. coli, 77 K. pneumoniae, 46 various species naturally producing inducible AmpC betalactamases and 4 others (K. oxytoca and C. koseri), were detected as resistant by BLT (Table 1). 214 215 216 217 218 219 220 221 222 223 224 225 226 227 Discussion BLT, designed to rapidly detect 3GC resistant Enterobacteriaceae isolates, was evaluated on a large set of 2,387 strains prospectively collected in clinical practice during a period of 3 months in 5 teaching hospitals. Compared to routine AST, BLT yielded high specificity, both globally (99.6%) and for each group of species, e.g. 99.9% for E. coli, 100% for K. pneumoniae and 99.6% for species naturally producing inducible AmpC beta-lactamases (Table 2). Sensitivity of BLT was high for E. coli and K. pneumoniae (96%) but lower for the species naturally producing inducible chromosomal AmpC beta-lactamase (67.4%). Interestingly, the proportion of non interpretable results was very low (2.4%) suggesting the test operational in routine practice. False positive results were mainly found in strains producing enzymes known to partly inactivate 3GC, but these strains were classified as 3GC susceptible based on recent guidelines used to interpret AST (3). Positive BLT were found for K. oxytoca isolates with a 10

228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 high-level expression of their natural chromosomally encoded beta-lactamase, as expected and previously reported by Livermore et al. (17), and one P. mirabilis producing CTX-M-15 enzyme was positive using BLT as expected (16;17). In fact, these results are in agreement with some authors who discourage the use of beta-lactamases substrate drugs as therapy (4). The positive predictive value (PPV) of BLT (97%) calculated in this study based on a 14% prevalence of 3GC resistance in Enterobacteriaceae, will remain high in settings with other prevalence rates (e.g. 96%, 98%, and 99% when such prevalence is 10%, 20%, or 30%, respectively). Consequently, the test can predict accurately when 3GC should be excluded from empiric antibiotic treatment for these two major species, several hours or one day before the results of antibiotic susceptibility testing. Sensitivity was high for the two major species of Enterobacteriaceae, i.e. 96% for E. coli and 96.3% for K. pneumoniae. This performance led to high negative predictive values (NPV) for both species, i.e. 99% in the present study where the prevalence of resistance to 3GC were 9.4% and 25.6%, respectively. NPV will remain high within these 2 species even in epidemiological situations where the prevalence of resistance is much higher, e.g. 98% or 97% for prevalences of 30% or 40%. However, due to lower sensitivity of BLT to detect 3GC resistant Enterobacter spp. and Serratia spp. through AmpC stable overproduction (because of lower affinity and activity of hyperproduced AmpC to hydrolyse HMRZ-86 (16)), the NPV was 89% for these species. The overall NPV of 98% in the present study will decrease if the proportion of this latter group among Enterobacteriaceae is higher. However, the place of these opportunistic species of Enterobacteriaceae is generally limited even in nosocomial situation (e.g. 4 to 7% for Enterobacter spp. depending on the sites of infection (23)). Interestingly, all the 3GC resistant ESBL-producing strains of Enterobacteriaceae were found positive using BLT (n=241), a result that became available within minutes (in less than 2 minutes for 167 isolates (69%)). Consequently, BLT is an excellent test to detect 3GC- 11

253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 R Enterobacteriaceae through ESBL production, especially useful in countries where ESBL prevalence is high. In Europe, the proportion of ESBL strains among 3GC-R E. coli and K. pneumoniae is high, ranging from 65 to 100% and 60 to 100%, respectively, depending on the country, based on ECDC 2012 data (23). The test could help to avoid the use of last line antibiotics such as carbapenems in case of negative test. Some remarks on the present results need to be addressed. First, there was no carbapenemase-producing strains in the panel, as expected considering that these organisms are still rare in France as in most European countries (23). However, the spread of carbapenemase-producing Enterobacteriaceae in some countries would justify accurate and rapid tests to detect such strains in addition to 3GC resistant strains (24). Second, the evaluation of the test on P. mirabilis must be held with caution, since no P. mirabilis classified as resistant to 3GC was included in the present study. However 3GC resistance in this species is generally uncommon. Third, the test was evaluated in teaching hospitals with high prevalence of resistant strains. In situation of prevalence of 3GC resistance below 10% (e.g. in some community settings), the PPV could be somewhat lower, e.g. 92% for 5% prevalence of resistance. Last, the study found 2.4% non-interpretable results, mainly in species naturally producing inducible AmpC beta-lactamases (Enterobacter spp. ) (Table 3). Non-interpretable results could limit the interest of BLT in situation of higher prevalence of such species. Nordmann et al have recently proposed a test to specifically detect ESBL producing- Enterobacteriaceae (the ESBL NDP test), relying upon the hydrolysis of the beta-lactam ring of cefotaxime that is inhibited when tazobactam is added to evidence the inhibition of ESBL activity (25). ESBL NDP test was retrospectively evaluated on a collection of 255 strains (from various clinical and geographical origins and previously characterized at the molecular level). The test showed in the published study a specificity of 100% but a sensitivity of 12

278 279 280 281 282 283 284 285 286 287 288 289 290 291 92.6%, missing part of ESBL producing-strains. In the present study, the sensitivity of BLT to detect 3GC-R through ESBL production was 100% as assessed prospectively on 241 consecutive unselected producing strains. Compared to ESBL NDP test, BLT is a commercial test, is less time-consuming (2-15 vs 60 ) and less staff-demanding (1 step vs 2 steps) (25). Moreover, BLT is designed to detect more widely 3GC resistance, a pragmatic endpoint for therapeutical purpose. In conclusion, BLT was proved to be reliable (i) to accurately predict susceptibility to 3GC when the test is negative, and (ii) to accurately predict resistance to 3GC in E. coli and K. pneumoniae when the test is positive, but less accurately in species such as Enterobacter spp. and Serratia spp.. The test is simple to use and could be implemented in laboratories working for general practitioners since 3GC resistance is emerging in E. coli in the community setting, mainly in relation with the spread of ESBLs (2). In the future, studies evaluating the use of BLT directly from specimens (such as urines) in a point-of-care strategy (26) could lead to even reduce further the delay for therapeutical choice. 292 293 294 295 Acknowledgements. Reagents for the βlacta test were provided by Bio-Rad Laboratories, Marnes-la-Coquette, France. We thank Caroline Dallenne and Manette Juvin for their support throughout the study. 296 13

297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 References 1. Coque TM, Baquero F, Canton R 2008 Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill 13:19044. 2. Pitout JD, Laupland KB 2008 Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 8:159-166. 3. Bonnet R, Caron F, Cavallo J, Chardon H, Chidiac CCP, Drugeon H, Dubreuil L, Jarlier V, Jehl F, Lambert T, Leclercq R, Nicolas-Chanoine MH, Plesiat P, Ploy MC, Quentin C, Soussy C, Varon E, Weber P 2012 Comité de l'antibiogramme de la sociéte française de microbiologie Recommandations 2012. 4. Livermore DM, Andrews JM, Hawkey PM, Ho PL, Keness Y, Doi Y, Paterson D, Woodford N 2012 Are susceptibility tests enough, or should laboratories still seek ESBLs and carbapenemases directly? J Antimicrob Chemother 67:1569-1577. 5. Schwaber MJ, Navon-Venezia S, Kaye KS, Ben-Ami R, Schwartz D, Carmeli Y 2006 Clinical and economic impact of bacteremia with extended- spectrumbeta-lactamase-producing Enterobacteriaceae. Antimicrob Agents Chemother 50:1257-1262. 6. Drieux L, Brossier F, Sougakoff W, Jarlier V 2008 Phenotypic detection of extended-spectrum beta-lactamase production in Enterobacteriaceae: review and bench guide. Clin Microbiol Infect 14:90-103. 7. Garrec H, Drieux-Rouzet L, Golmard JL, Jarlier V, Robert J 2011 Comparison of nine phenotypic methods for detection of extended-spectrum beta-lactamase production by Enterobacteriaceae. J Clin Microbiol 49:1048-1057. 8. Gazin M, Paasch F, Goossens H, Malhotra-Kumar S 2012 Current trends in culture-based and molecular detection of extended-spectrum-beta-lactamaseharboring and carbapenem-resistant Enterobacteriaceae. J Clin Microbiol 50:1140-1146. 9. Nijhuis RH, van Zwet AA, Savelkoul PH, Roovers EA, Bosboom RW, Postma B, van Griethuysen AJ 2011 Distribution of extended-spectrum beta-lactamase genes using a commercial DNA micro-array system. J Hosp Infect 79:349-353. 10. Ellem J, Partridge SR, Iredell JR 2011 Efficient direct extended-spectrum betalactamase detection by multiplex real-time PCR: accurate assignment of phenotype by use of a limited set of genetic markers. J Clin Microbiol 49:3074-3077. 11. Hrabak J, Chudackova E, Walkova R 2013 Matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry for detection of antibiotic resistance mechanisms: from research to routine diagnosis. Clin Microbiol Rev 26:103-114. 14

337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 12. Schaumann R, Knoop N, Genzel GH, Losensky K, Rosenkranz C, Stingu CS, Schellenberger W, Rodloff AC, Eschrich K 2012 A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry. Med Sci Monit 18:MT71-MT77. 13. Sparbier K, Schubert S, Weller U, Boogen C, Kostrzewa M 2012 Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against beta-lactam antibiotics. J Clin Microbiol 50:927-937. 14. Ben Soltana M, Dallenne C, Birgy A, Compain F, Verdet C, Vimont S, Favier C, Juvin M, Arlet G 2012 Evaluation of a new chromogenic test (betalacta test) for rapid detection of third-generation cephalosporins nonsusceptible Enterobacteriaceae. Poster session 22nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID). 15. Laurent T, Huang TD, Bogaerts P, Glupczynski Y 2013 Evaluation of the betalacta test, a novel commercial chromogenic test for rapid detection of ceftazidime non-susceptible Pseudomonas aeruginosa isolates. J Clin Microbiol 51:1951-1954. 16. Hanaki H, Koide Y, Yamazaki H, Kubo R, Nakano T, Atsuda K, Sunakawa K 2007 Substrate specificity of HMRZ-86 for beta-lactamases, including extended-spectrum beta-lactamases (ESBLs). J Infect Chemother 13:390-395. 17. Livermore DM, Warner M, Mushtaq S 2007 Evaluation of the chromogenic Cicabeta-Test for detecting extended-spectrum, AmpC and metallo-betalactamases. J Antimicrob Chemother 60:1375-1379. 18. Cockerill FR, III, Wikler MA, Alder J, Dudley MN, Eliopoulos G, Ferraro MJ, Hardy DJ, Hecht DW, Hindler JA, Patel JB, Powell M, Swenson JM, Thomson BJ, Traczewski MM, Turnidge JD, Weinstein MP, Zimmer BL 2012 CLSI Performance standards for antimicrobial susceptibility testing; twenty-second informational supplement., 32 ed. 19. Dallenne C, Da Costa A, Decre D, Favier C, Arlet G 2010 Development of a set of multiplex PCR assays for the detection of genes encoding important betalactamases in Enterobacteriaceae. J Antimicrob Chemother 65:490-495. 20. Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C, Ould-Hocine Z, Barnaud G, Delisle F, Rossier A, Lambert T, Philippon A, Arlet G 2004 Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother 48:1249-1255. 21. Courvalin P, Leclercq R, Rice LB 2010 Antibiogram. ESKA. 22. Livermore DM 1995 beta-lactamases in laboratory and clinical resistance. Clin Microbiol Rev 8:557-584. 23. ECDC 2012 Annual Epidemiological Report. 15

378 379 380 381 382 383 384 385 386 387 388 24. Nordmann P, Gniadkowski M, Giske CG, Poirel L, Woodford N, Miriagou V 2012 Identification and screening of carbapenemase-producing Enterobacteriaceae. Clin Microbiol Infect 18:432-438. 25. Nordmann P, Dortet L, Poirel L 2012 Rapid detection of extended-spectrum-betalactamase-producing Enterobacteriaceae. J Clin Microbiol 50:3016-3022. 26. Cohen-Bacrie S, Ninove L, Nougairede A, Charrel R, Richet H, Minodier P, Badiaga S, Noel G, La Scola B, De Lamballerie X, Drancourt M, Raoult D 2011 Revolutionizing clinical microbiology laboratory organization in hospitals with in situ point-of-care. PLoS One 6:e22403. 16

Table 1. Characteristics of the strains included in the study; mechanisms of resistance to third-generation cephalosporins (3GC) and results of ßLACTA TM. Mechanisms of resistance to 3GC Species No. of strains No. of 3GC-R a strains (%.) ESBL Plasmidic AmpC betalactamase CTX-M SHV TEM DHA-1 CMY-2 AmpC stable overproduction Other Escherichia coli 1403 132 (9.4%) 105 4 5 1 6 0 11 a,b Klebsiella pneumoniae 328 84 c (25.6%) 66 7 4 6 0 0 2 a,b Enterobacter spp. Morganella spp. Serratia spp. Providencia spp. Citrobacter freundii 360 109 c (30.3%) 28 15 3 0 0 65 d 0 Proteus mirabilis 172 0 (0%) 0 0 0 0 0 0 0 Citrobacter koseri Klebsiella oxytoca 124 7 (5.6%) 1 1 2 0 0 0 3 e Total 2387 332 c (13.9%) 200 27 14 7 6 65 16 No of strains with ßLACTA TM + 273 265 c 200 27 14 4 1 14 8 a strains intermediate or resistant to ceftazidime or cefotaxime b not producing ESBL nor plasmidic AmpC c three strains (1 K. pneumoniae and 2 Enterobacter spp) produced two distinct beta-lactamases, each of them counted twice in the columns mechanisms of resistance d based on AST (see text for definition) e high level expression of chromosomal KOXY type enzyme

Table 2. Performances of the LACTA test for detecting resistance to third-generation cephalosporins; non interpretable results (n = 57, see text for definition) were excluded. LACTA Susceptibility testing % (95 CI) c 3GC-R a 3GC-S b Total Sensitivity Specificity All strains Positive 265 8 273 87.7 (83.0-92.5) 99.6 (99.3-100) Negative 37 2020 2057 Total 302 2028 2330 E. coli Positive 119 1 120 96.0 (91.6-100) 99.9 (99.7-100) Negative 5 1258 1263 Total 124 1259 1383 K. pneumoniae Positive 79 0 79 96.3 (91.2-100) 100 (100-100) Negative 3 240 243 Total 82 240 322 P. mirabilis Positive 0 1 1-99.4 (CI not applicable) Negative 0 171 171 Total 0 172 172 Species naturally producing inducible AmpC beta-lactamase Positive 60 1 61 67.4 (55.0-79.8) 99.6 (98.6-100) Negative 29 244 273 Total 89 245 334 K. oxytoca and C. koseri Positive 7 5 12 100 (100-100) 95.5 (90.7-100) Negative 0 107 107 Total 7 112 119 a strains intermediate or resistant to ceftazidime or cefotaxime b strains fully susceptible to ceftazidime and cefotaxime c CI, confidence interval

Table 3. Detailed characterization of strains with false-positive, false-negative, or non-interpretable results.* False positive results (n=8) E. coli 1 strain producing OXA-1 P. vulgaris 1 wild type strain K. oxytoca 5 strains with a high-level expression of chromosomal beta-lactamase P. mirabilis 1 strain producing CTX-M-15 susceptible to 3GC False negative results (n=37) E. coli 2 strains producing plasmidic AmpC beta-lactamase (CMY-2) 3 strains not producing ESBL nor plasmidic AmpC K. pneumoniae 3 strains producing plasmidic AmpC beta-lactamase (DHA-1) Species naturally producing inducible AmpC ** 29 strains overexpressing chromosomal AmpC beta-lactamase * Non-interpretable results among 3GC-R strains (n=30) Non-interpretable results among 3GC-S strains (n=27) E. coli 3 strains producing plasmidic AmpC beta-lactamase (CMY-2) K. pneumoniae 5 strains not producing ESBL nor plasmidic AmpC 1 strain with a high-level expression of chromosomal beta-lactamase 1 strain not producing ESBL nor plasmidic AmpC Species naturally producing inducible AmpC ** 20 strains overexpressing chromosomal AmpC beta-lactamase * E. coli 5 strains producing OXA-1 K. pneumoniae Species naturally producing inducible AmpC 7 strains not producing ESBL nor plasmidic AmpC 1 strain not producing ESBL nor plasmidic AmpC 1 strain with a high-level expression of chromosomal beta-lactamase 1 strain producing OXA-1 1 strain producing plasmidic AmpC beta-lactamase (DHA-1) 6 wild type strains K. oxytoca 4 wild type strains C. koseri 1 wild type strain * see text for definition ** Enterobacter spp., Serratia spp.