MICs Of Temocillin Research Articles

Impact of the phenotypic expression of temocillin resistance in Escherichia coli on temocillin efficacy in a murine peritonitis model.

Temocillin is a narrow spectrum β-lactam active against MDR Enterobacterales. Mechanisms of acquired resistance to temocillin are poorly understood. We analysed resistance mechanisms in clinical isolates of Escherichia coli and evaluated their impact on temocillin efficacy in vitro and in a murine peritonitis model. Two sets of isogenic clinical E. coli strains were studied: a susceptible isolate (MLTEM16S) and its resistant derivative, MLTEM16R (mutation in nmpC porin gene); and temocillin-resistant derivatives of E. coli CFT073: CFT-ΔnmpC (nmpC deletion), CFTbaeS-TP and CFTbaeS-AP (two different mutations in the baeS efflux-pump gene).Fitness cost, time-kill curves and phenotypic expression of resistance were determined. Temocillin efficacy was assessed in a murine peritonitis model. MICs of temocillin were 16 and 64 mg/L for MLTEM16S and MLTEM16R, respectively, and 8, 128, 256 and 256 mg/L for E. coli-CFT073, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP, respectively. No fitness cost of resistance was evidenced. All resistant strains showed heteroresistant profiles, except for CFTbaeS-AP, which displayed a homogeneous pattern. In vitro, temocillin was bactericidal against MLTEM16R, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP at 128, 256, 512 and 512 mg/L, respectively. In vivo, temocillin was as effective as cefotaxime against MLTEM16R, CFT-ΔnmpC and CFTbaeS-TP, but inefficient against CFTbaeS-AP (100% mortality). Heteroresistant NmpC porin alteration and active efflux modification do not influence temocillin efficacy despite high MIC values, unfavourable pharmacokinetic/pharmacodynamic conditions and the absence of fitness cost, whereas homogeneously expressed BaeS efflux pump alteration yielding similar MICs leads to temocillin inefficacy. MIC as sole predictor of temocillin efficacy should be used with caution.

Implications of two-component systems EnvZ/OmpR and BaeS/BaeR in in vitro temocillin resistance in Escherichia coli.

BaeS/BaeR is a two-component system of Escherichia coli that controls the expression of porins and efflux pumps. Its role in beta-lactam resistance is limited. To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. E. coli strain BW25113 and single-gene deletion mutants related to two-component systems were collected from the KEIO collection. Double-gen deletion mutants were generated. Temocillin-resistant mutant frequencies were determined at 32 mg/L. E. coli BW25113 mutants were selected by selective pressure from serial passages. Biological costs were analysed by growth curves. Genomes of the generated mutants were sequenced. The expression level of the mdtA, mdtB, mdtC, acrD and tolC in the ΔbaeS mutant was determined by RT-PCR (with/without temocillin exposure). The frequency of temocillin mutants ranged from 2.12 × 10-8 to 4.51 × 10-8 in single-porin mutants. No mutants were recovered from E. coli BW25113 (>10-9). Selection of temocillin-resistant variants by serial passage yielded mutants up to 128 mg/L. Mutations were found in the baeS gene. Temocillin MICs ranged from 4 to 32 mg/L (highest MICs for ΔbaeS and ΔompR). The efflux pumps mdtA, mdtB, mdtC and acrD pumps were overexpressed 3-10-fold in the presence of temocillin in ΔbaeS compared to control. Mutations in the sensor histidine kinase, baeS, may be involved in temocillin resistance through the expression of the efflux pumps mdtABC and acrD. In addition, the low mutation rate may be a good predictor of temocillin activity.

Population pharmacokinetics and dosing simulations of total and unbound temocillin in the plasma and CSF of neurocritically ill patients with external ventricular drain-related cerebral ventriculitis.

Cerebral ventriculitis might be caused by Gram-negative bacteria, including ESBL producers. Temocillin may be a useful treatment option in this scenario; however, no consistent data are available regarding its penetration into the CSF. To describe the population pharmacokinetics of temocillin in plasma and CSF and to determine the probability for different simulated dosing regimens to achieve pharmacokinetic/pharmacodynamic (PK/PD) targets in the CSF. Ten post-neurosurgical critically ill adult patients requiring continuous drainage of CSF were included in this monocentric, prospective, open-label, non-randomized study. They received 2 g loading dose temocillin over 30 min IV infusion, followed by a 6 g continuous infusion over 24 h. Total and unbound concentrations were measured in plasma (n = 88 and 86) and CSF (n = 88 and 88) samples and used to build a population PK model. Monte Carlo simulations were performed to estimate the PTA at 100% Css>MIC (steady state concentration above the MIC) in CSF. All patients were infected with Enterobacterales with temocillin MICs ≤8 mg/L. The median (min-max) temocillin penetration in CSF was 12.1% (4.3-25.5) at steady state. Temocillin unbound plasma pharmacokinetics were best described by a one-compartment model. PTA for the applied dosing regimen was >90% for bacteria with MIC ≤ 4 mg/L. The currently approved dose of 6 g by continuous infusion may be adequate for the treatment of ventriculitis by Enterobacterales with MIC ≤ 4 mg/L if considering 100% Css>MIC as the PK/PD target to reach. Higher maintenance doses could help covering higher MICs, but their safety would need to be assessed.

Biochemical characterization of OXA-244, an emerging OXA-48 variant with reduced β-lactam hydrolytic activity.

OXA-48-producing Enterobacterales have widely disseminated globally with an increasing number of variants identified. Among them, OXA-244 is increasingly reported, despite detection difficulties. To determine the steady-state kinetic parameters of OXA-244. The blaOXA-244 gene was amplified, cloned into plasmids p-TOPO and pET41b+, and transformed into Escherichia coli TOP10 for MIC determination and E. coli BL21 DE3 for purification. Steady-state kinetic parameters and IC50s of clavulanic acid, tazobactam and NaCl were determined using purified OXA-244. Molecular modelling was also performed. A reduction in MICs of temocillin and carbapenems was observed in E. coli expressing OXA-244 as compared with OXA-48. The kinetic parameters revealed a reduced carbapenemase activity of OXA-244 as compared with OXA-48, especially for imipenem, which was 10-fold lower. Similarly, catalytic efficiency (kcat/Km) was reduced by 4-fold and 20-fold for ampicillin and temocillin, respectively. Kinetic parameters for cephalosporins were, however, similar. Molecular modelling studies evidenced the key role of R214 in OXA-48, establishing salt bridges with D159 and with the carboxylate group of the R1 substituent of temocillin. These interactions are not possible with G214 in OXA-244, explaining the reduced affinity of temocillin for this enzyme. The R214G mutation in OXA-244 is also likely to induce changes in the active site's water network that would explain the decrease in the hydrolysis rate of carbapenems. Our data confirm that the R214G mutation (present in OXA-244) results in reduced carbapenem- and temocillin-hydrolysing activity, confirming the crucial role of residue 214 in the hydrolysis of these substrates by OXA-48-like β-lactamases.

OXA-484, an OXA-48-Type Carbapenem-Hydrolyzing Class D β-Lactamase From Escherichia coli.

OXA-48-like carbapenemases are among the most frequent carbapenemases in Gram-negative Enterobacterales worldwide with the highest prevalence in the Middle East, North Africa and Europe. Here, we investigated the so far uncharacterized carbapenemase OXA-484 from a clinical E. coli isolate belonging to the high-risk clone ST410 regarding antibiotic resistance pattern, horizontal gene transfer (HGT) and genetic support. OXA-484 differs by the amino acid substitution 214G compared to the most closely related variants OXA-181 (214R) and OXA-232 (214S). The blaOXA–484 was carried on a self-transmissible 51.5 kb IncX3 plasmid (pOXA-484) showing high sequence similarity with plasmids harboring blaOXA–181. Intraspecies and intergenus HGT of pOXA-484 to different recipients occurred at low frequencies of 1.4 × 10–7 to 2.1 × 10–6. OXA-484 increased MICs of temocillin and carbapenems similar to OXA-232 and OXA-244, but lower compared with OXA-48 and OXA-181. Hence, OXA-484 combines properties of OXA-181-like plasmid support and transferability as well as β-lactamase activity of OXA-232.

Screening of OXA-244 producers, a difficult-to-detect and emerging OXA-48 variant?

OXA-244, a single amino acid variant of OXA-48, demonstrates weaker hydrolytic activity towards carbapenems and temocillin compared with OXA-48. Of note, these antimicrobials are present in high concentrations in several carbapenemase-producing Enterobacterales (CPE) screening media. As a result, some screening media fail to grow OXA-244-producing isolates, while the prevalence of OXA-244 producers is constantly increasing in France. Here, we evaluate the performance of three commercially available CPE screening media [ChromID® CARBA SMART (bioMérieux), Brilliance™ CRE (Thermo Fisher) and mSuperCARBA™ (MAST Diagnostic)] for their ability to detect OXA-244 producers (n = 101). As OXA-244 producers may also express an ESBL, two additional ESBL screening media were tested (Brilliance™ ESBL and ChromID® BLSE). MICs of temocillin and imipenem were determined by broth microdilution. The clonality of OXA-244-producing Escherichia coli isolates (n = 97) was assessed by MLST. Overall, the sensitivity of the ChromID® CARBA SMART, Brilliance™ CRE and mSuperCARBA™ media were 14% (95% CI = 8.1%-22.5%), 54% (95% CI = 43.3%-63.4%) and 99% (95% CI = 93.8%-100%), respectively, for the detection of OXA-244 producers. Among the 101 OXA-244-producing isolates, 96% were E. coli and 77%-78% grew on ESBL screening media. MLST analysis identified five main STs among OXA-244-producing E. coli isolates: ST38 (n = 37), ST361 (n = 17), ST69 (n = 12), ST167 (n = 11) and ST10 (n = 8). Our results demonstrated that the mSuperCARBA™ medium is very efficient in the detection of OXA-244 producers, unlike the ChromID® CARBA SMART medium. The high prevalence of ESBLs among OXA-244 producers allowed detection of 77%-78% of them using ESBL-specific screening media.

Temocillin breakpoints in pyelonephritis: evaluation in a murine model due to ESBL-producing Escherichia coli clinical isolates.

Due to a spectrum restricted to Enterobacteriaceae and stability against ESBL and AmpC enzymes, temocillin is of major interest for the treatment of pyelonephritis. But there are still uncertainties about the optimal regimen and clinical breakpoints. To study in a murine model of pyelonephritis the activity of temocillin against Escherichia coli isolates with different MICs in order to evaluate clinical breakpoints. Four clinical uropathogenic E. coli isolates with temocillin MICs of 8 mg/L (Ec8), 16 mg/L (Ec16), 32 mg/L (Ec32) and 64 mg/L (Ec64) were evaluated. Antibiotic 24 h T>MIC achieved in humans was reproduced in mice with either intravenous temocillin (2 g q12h or 2 g q8h) or intravenous imipenem (1 g q8h). Efficacy was assessed by bacterial count in kidneys. Compared with controls, temocillin at 2 g q12h was highly efficient against Ec8 (-3.32 log10 cfu/g and negative cultures in 93% of mice; P < 0.001); imipenem gave similar results. Temocillin at 2 g q12h also induced high reduction of bacterial count against Ec16 (-2.92 log10 cfu/g; P < 0.001), albeit cultures were negative in only 48% of mice. In contrast, no significant effect was observed in mice infected by Ec32 (-0.01 log10 cfu/g; P = 0.981) or Ec64 (-0.55 log10 cfu/g; P = 0.523). Even temocillin at 2 g q8h failed to control Ec32 infection (-1.55 log10 cfu/g; P = 0.197). This model suggests a clinical breakpoint up to 16 mg/L for non-severe pyelonephritis treated with temocillin at 2 g q12h, a value consistent with the few previous available data.

Evaluation of temocillin for phenotypic carbapenemase screening of Escherichia coli and Salmonella enterica isolates in relation to the presence of genes encoding ESBLs and carbapenemase production.

The expression of enzymes of the OXA-48 carbapenemase group is difficult to detect by phenotypic methods owing to frequent low levels of carbapenem resistance and negative results with some screening methods. Temocillin has been shown to be a good option for phenotypic screening as it is hydrolysed by the OXA-48-group enzymes, whereas ESBLs, AmpC and some other carbapenemases have a lower hydrolytic effect on this antimicrobial. However, no epidemiological cut-off for temocillin is available. To evaluate temocillin MICs in relation to the presence or absence of genes encoding ESBLs and carbapenemases in Escherichia coli and Salmonella enterica. In this study, 111 E. coli and 102 S. enterica isolates, including WT and well-characterized ESBL-, AmpC- or carbapenemase-producing isolates, were tested by three independent laboratories. MICs were determined according to the CLSI guidelines by agar dilution with the test range from 0.5 to 512 mg/L temocillin and WGS was performed and analysed with ResFinder. Some overlap was detected between temocillin MICs for WT and ESBL- or AmpC-producing isolates. However, isolates carrying genes encoding carbapenemases showed a broader range of MICs for both E. coli and S. enterica. Higher MICs were observed for the OXA-48 group, VIM and some NDM-producing isolates, whereas isolates harbouring KPC enzymes showed low MICs. The results indicate that temocillin MICs enable phenotypic distinction between strains producing OXA-48-group enzymes and both WT susceptible and ESBL/AmpC-carrying isolates, whereas the distinction from other carbapenemases likely requires genotypic testing.

Activity of fosfomycin alone or combined with temocillin in vitro and in a murine model of peritonitis due to KPC-3- or OXA-48-producing Escherichia coli.

Alternative therapeutic regimens are urgently needed against carbapenemase-producing Enterobacteriaceae. Fosfomycin often remains active against KPC and OXA-48 producers, but emergence of resistance is a major limitation. Our aim was to determine whether the association of temocillin with fosfomycin might be useful to treat KPC- or OXA-48-producing Escherichia coli infections. Isogenic derivatives of E. coli CFT073 with blaKPC-3- or blaOXA-48-harbouring plasmids (named CFT073-KPC-3 and CFT073-OXA-48, respectively) were used. The addition of temocillin to fosfomycin was tested using the chequerboard method and time-kill curves as well as in a fatal peritonitis murine model. Mice were treated for 24 h with fosfomycin alone or in combination with temocillin. Bacterial loads, before and after treatment, were determined in the peritoneal fluid and fosfomycin-resistant mutants were detected. Temocillin MICs were 8, 32 and 256 mg/L for CFT073 (WT), CFT073-KPC-3 and CFT073-OXA-48, respectively. Fosfomycin MIC was 0.5 mg/L for all strains. The chequerboard experiments demonstrated synergy for all three strains. In time-kill curves, combining temocillin with fosfomycin was synergistic, bactericidal and prevented emergence of resistance for CFT073-pTOPO and CFT073-KPC-3, but not CFT073-OXA-48. In vivo, for the three strains, bacterial counts were lower in peritoneal fluid with the combination compared with fosfomycin alone (P < 0.001) and inhibited growth of resistant mutants in all cases. The combination of fosfomycin and temocillin demonstrated a benefit in vitro and in vivo against E. coli strains producing KPC-3 or OXA-48-type carbapenemases. This combination prevented the emergence of fosfomycin resistance and proved to be more bactericidal than fosfomycin alone.

Temocillin against Enterobacteriaceae isolates from community-acquired urinary tract infections: low rate of resistance and good accuracy of routine susceptibility testing methods.

Temocillin is an old 'revived' antibiotic that may play an important role in the treatment of febrile urinary tract infection (UTI). Data regarding its activity against current Enterobacteriaceae isolates as well as the performance of routine susceptibility testing methods are, however, scarce. To determine the MICs of temocillin for Enterobacteriaceae strains reflecting the current epidemiology and to analyse the accuracy of three commercial methods. Enterobacteriaceae isolates causing community-acquired UTI were prospectively collected from September 2015 to January 2017 in two French centres. Temocillin MIC was determined by agar dilution (AD) as the reference method and then compared with: (i) susceptibility testing by disc diffusion; (ii) MIC determination by Etest; and (iii) MIC estimation by the Vitek 2 automated system. A total of 762 Enterobacteriaceae were analysed comprising 658 (86.4%) Escherichia coli and 37 (4.9%) ESBL-producing isolates. Susceptibility rate assessed by AD was 99.6% according to the 8 mg/L clinical breakpoint and was significantly lower against the ESBL-producing isolates than the non-ESBL-producing isolates (94.6% versus 99.9%, P < 0.01). The MIC50 and MIC90 for the total set were 3 and 6 mg/L, respectively. According to the 8 mg/L clinical breakpoint, the major error rate was <1% for disc diffusion and Etest, and significantly higher for Vitek 2 (4.3%, P < 0.01), but still low. No very major error was noticed. Temocillin showed a high level of activity against Enterobacteriaceae from community-acquired UTI and good to excellent reliability of routine methods for susceptibility testing in such a setting.

Mechanisms of intrinsic resistance and acquired susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients to temocillin, a revived antibiotic

The β-lactam antibiotic temocillin (6-α-methoxy-ticarcillin) shows stability to most extended spectrum β-lactamases, but is considered inactive against Pseudomonas aeruginosa. Mutations in the MexAB-OprM efflux system, naturally occurring in cystic fibrosis (CF) isolates, have been previously shown to reverse this intrinsic resistance. In the present study, we measured temocillin activity in a large collection (n = 333) of P. aeruginosa CF isolates. 29% of the isolates had MICs ≤ 16 mg/L (proposed clinical breakpoint for temocillin). Mutations were observed in mexA or mexB in isolates for which temocillin MIC was ≤512 mg/L (nucleotide insertions or deletions, premature termination, tandem repeat, nonstop, and missense mutations). A correlation was observed between temocillin MICs and efflux rate of N-phenyl-1-naphthylamine (MexAB-OprM fluorescent substrate) and extracellular exopolysaccharide abundance (contributing to a mucoid phenotype). OpdK or OpdF anion-specific porins expression decreased temocillin MIC by ~1 two-fold dilution only. Contrarily to the common assumption that temocillin is inactive on P. aeruginosa, we show here clinically-exploitable MICs on a non-negligible proportion of CF isolates, explained by a wide diversity of mutations in mexA and/or mexB. In a broader context, this work contributes to increase our understanding of MexAB-OprM functionality and help delineating how antibiotics interact with MexA and MexB.

Activity of temocillin in a lethal murine model of infection of intra-abdominal origin due to KPC-producing Escherichia coli.

Temocillin is a 6-α-methoxy derivative of ticarcillin that shows in vitro activity against Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC). Our objective was to assess in vivo temocillin activity against KPC-producing Escherichia coli. Isogenic derivatives of the WT E. coli CFT073 producing KPC-2, KPC-3 or OXA-48 were constructed. An experimental murine model of intra-abdominal infection with sepsis was used. Mice were treated subcutaneously with temocillin 200 mg/kg every 2 h for 24 h, reproducing the duration of time that the free serum concentration of temocillin exceeded the MIC in humans with a regimen of 2 g every 12 h or 2 g every 8 h. Blood, peritoneal fluid (PF) and spleen were collected; 24 h survival and sterility rates were assessed. Temocillin MICs were 8, 16, 32, and 256 mg/L for the susceptible strain and KPC-2-, KPC-3-, and OXA-48-producing strains, respectively. In mice treated with temocillin, significant bacterial reduction was obtained in PF, blood, and spleen for the susceptible strainand KPC-2- and KPC-3-producing strains (P < 0.001) but not for the OXA-48-producing strain. Sterility rates in PF were 53%, 10%, 0% and 0% (P < 0.001) and sterility rates in blood were 77%, 40%, 3% and 0% (P < 0.001), while survival rates were 97%, 97%, 57%, 0% (P < 0.001) for mice infected with the susceptible strainand KPC-2-, KPC-3- and OXA-48-producing strains, respectively. In a lethal-infection model with bacteraemia from intra-abdominal origin, temocillin retained significant activity in PF, blood and spleen and prevented death in mice by effectively working against KPC-producing E. coli with temocillin MICs ≤16 mg/L.

In vitro activity of temocillin against multidrug-resistant clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp., and evaluation of high-level temocillin resistance as a diagnostic marker for OXA-48 carbapenemase

Sir, Temocillin is a narrow-spectrum penicillin active primarily against Enterobacteriaceae and resistant to hydrolysis by penicillinases, extended-spectrum b-lactamases (ESBLs), and AmpC enzymes. It has been shown also to retain some activity in vitro against Enterobacteriaceae with KPC-type carbapenemases, although not against bacteria with OXA-48-like non-metalloenzymes or the metalloenzymes (including IMP, NDM and VIM types). The emergence and spread of bacteria with acquired carbapenemases raises public health concerns, and there is a clear need for reliable diagnostic tests in routine bacteriology laboratories. Simple phenotypic tests with b-lactam/b-lactamase inhibitor combinations can aid the identification of isolates with KPC-type or metallo-carbapenemases, but cannot detect OXA-48 carbapenemases, for which there is currently no good inhibitor. Molecular tests could overcome this shortcoming but are not an option in many laboratories. The Carba NP acido/colorimetric phenotypic test detects carbapenem hydrolysis per se, but its sensitivity is reportedly lower for OXA-48 producers than for isolates with other carbapenemases. High-level temocillin resistance is a characteristic of many OXA-48 producers and has been included in algorithms to aid their recognition. – 8 We investigated the in vitro activity of temocillin against 2280 clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp. submitted to Public Health England’s Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit for the investigation of unusual resistance between January 2012 and April 2013. They included 1029 producers of KPC, OXA-48 or IMP, NDM and VIM carbapenemases. We also evaluated the diagnostic potential of high-level temocillin resistance (MIC ≥128 mg/L) to predict the production of an OXA-48 carbapenemase. Temocillin (Eumedica, Brussels, Belgium) was tested in the range 1–128 mg/L using agar dilution methodology; MICs were interpreted using BSAC breakpoints (susceptibility: systemic infections, MICs≤8 mg/L; urinary infections, MICs≤32 mg/L). Isolates were also tested against AMRHAI’s standard Gram-negative antibiotic panel, which includes ertapenem, imipenem (tested+EDTA to indicate likely metallo-carbapenemase producers) and meropenem. Carbapenemase production was sought using in-house PCRs and/or a commercial microarray (Check-MDR CT102; Check-Points, Wageningen, The Netherlands). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of temocillin MIC ≥128 mg/L as an indicator of OXA-48 carbapenemase production were determined using an online tool (http://www. medcalc.org/calc/diagnostic_test.php). Temocillin MIC distributions for the 2280 isolates are shown in Table 1. These were submissions to a reference laboratory so were biased towards multiresistance, with proportions of ‘unusual’ isolates far higher than would be observed in most diagnostic laboratories. Allowing for the fact that a larger proportion of Klebsiella spp. isolates produced carbapenemases (760/1133; 67.1%) than did isolates of E. coli (206/701; 29.4%) or Enterobacter spp. (63/446; 14.1%), no major differences were observed in the MIC distributions of temocillin for the three genera. Overall, 920 (40.4%) isolates were susceptible to temocillin at the systemic breakpoint (MICs ≤8 mg/L); those considered resistant included 329/669 (49.2%) KPC producers, 671/1251 (53.6%) isolates without carbapenemases, and all 360 (100%) isolates with other types of carbapenemase (OXA-48, IMP, NDM or VIM). At the higher, urinary breakpoint, 1758 (77.1%) isolates were susceptible to temocillin (MICs ≤32 mg/L), including 628 (93.4%) KPC producers and 1098 (87.8%) isolates without carbapenemases. However, 328 (91.4%) isolates with other carbapenemases (OXA-48, IMP, NDM or VIM) remained resistant with MICs .32 mg/L. Temocillin MICs ≥128 mg/L were observed for 355 (15.6%) isolates overall, most (292; 82.3%) of which had a carbapenemase. However, 63 isolates (17.7%; 30 E. coli, 22 Klebsiella spp. and 11 Enterobacter spp.) lacked these mechanisms and the nature of their high-level temocillin resistance warrants further investigation. Many of them had complex b-lactam antibiograms—for example, 57 were also non-susceptible to ertapenem (MICs .0.5 mg/L)—consistent with ESBL/AmpC production combined with impermeability (data not shown). High-level temocillin resistance (MICs ≥128 mg/L) was recorded for 98/108 (90.7%) OXA-48 producers compared with 257/2172 (11.8%) isolates lacking OXA-48 or with 72/1920 (3.8%) isolates lacking a metallo-carbapenemase. Based on these proportions, the detection of high-level resistance was not a sufficiently robust marker when used as the sole criterion to predict the presence of an OXA-48-like carbapenemase; the PPV of the test was only 27.6% (Table 2). However, if combined with an absence of imipenem/EDTA synergy (i.e. when there was no phenotypic evidence of metallo-carbapenemase production), the PPV of high-level temocillin resistance predicting OXA-48

Activity of Temocillin against KPC-Producing Klebsiella pneumoniae and Escherichia coli

Temocillin, a 6-α-methoxy derivative of ticarcillin, is currently approved for treatment of infections due to members of the Enterobacteriaceae in Belgium and the United Kingdom. It is stable against hydrolysis by most β-lactamases, including extended-spectrum β-lactamases (ESBLs) and AmpC-type β-lactamases, with studies reporting MICs at which 90% of bacteria are inhibited (MIC90s) between 16 and 32 μg/ml (3, 4, 8). Temocillin is thus drawing attention as a potential alternative to carbapenems in treatment of infections caused by the Enterobactericeae producing these broad-spectrum β-lactamases. Carbapenem-resistant Klebsiella pneumoniae producing KPC-type β-lactamase has emerged in recent years and caused hospital outbreaks of serious infections in the United States and other parts of the world (7). Furthermore, KPC-type β-lactamase is increasingly identified in other species of the Enterobacteriaceae as well, including Escherichia coli. One concerning recent phenomenon is the occurrence of urinary tract infections due to KPC-producing organisms at nursing homes (10). Currently, the limited treatment options for infections due to KPC-producing organisms include colistin and tigecycline. Concern over nephrotoxicity due to colistin limits its use outside closely monitored settings, whereas tigecycline does not achieve a therapeutic concentration in urine (2). Furthermore, emergence of resistance to these agents has recently been recorded for the Enterobacteriaceae (5, 6). The present study was conducted to evaluate the in vitro activities of temocillin against clinical isolates of K. pneumoniae and E. coli producing KPC-type β-lactamase. A total of 33 KPC-producing clinical isolates (30 K. pneumoniae isolates and 3 E. coli isolates) were used. KPC production was confirmed by an ertapenem resistance phenotype, a positive modified Hodge test, and positive PCR for the KPC structural gene. The isolates were collected from hospitals in three states in the United States. The MIC of temocillin was determined by the standard agar dilution method (1). Temocillin was provided by Eumedica (Brussels, Belgium). In addition, MICs of temocillin against an E. coli isogenic clone producing KPC-3 were tested to determine the direct effect of KPC production on the temocillin MIC. E. coli ATCC 25922 was used as the control strain. Table ​Table11 summarizes the results. For K. pneumoniae, the MICs ranged between 16 μg/ml and 64 μg/ml (MIC at which 50% of bacteria were inhibited = 32 μg/ml; MIC90 = 32 μg/ml). The E. coli clinical isolates had MICs between 8 and 16 μg/ml. E. coli DH10B both with and without the cloning vector pBCSK− (Stratagene, La Jolla, CA) encoding blaKPC-3 had an MIC of 8 μg/ml. An inoculum effect was not observed at 105 CFU, whereas a mild inoculum effect averaging within a twofold MIC difference was seen with K. pneumoniae when 106 CFU was inoculated (Table ​(Table1).1). This result was in line with those of a previous study documenting a modest inoculum effect of temocillin for non-KPC-producing isolates (9). The frequencies of mutants of representative clinical isolates that grew at their MICs and at 2× MICs were calculated to be approximately 1 × 10−10 and 0 for K. pneumoniae and 3 × 10−10 and 1 × 10−10 for E. coli, respectively. TABLE 1. Susceptibilities of KPC-producing K. pneumoniae and E. coli isolates to temocillin Currently, the British Society for Antimicrobial Chemotherapy (BSAC) is the only organization that defines temocillin MIC breakpoints for the Enterobacteriaceae. The BSAC defines temocillin susceptibilities at ≤8 and ≤32 μg/ml in systemic and urinary tract infections, respectively (http://www.bsac.org.uk/). One gram of temocillin is known to achieve a peak serum concentration of approximately 160 μg/ml, with serum binding of 85% and a half-life of 4 to 5 h (9). The urinary concentration after a 500-mg dose is approximately 500 μg/ml (9). These pharmacokinetic properties of temocillin make it a potential alternative treatment option for mild to moderate urinary tract infections caused by KPC-producing members of the Enterobacteriaceae.

Activity of temocillin and other penicillins against beta-lactamase-inducible and -stably derepressed enterobacteria.

MICs of temocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin and ampicillin were determined for mutant series of Enterobacter cloacae, Citrobacter freundii, Proteus vulgaris, Morganella morganii and Serratia marcescens with inducible, stably derepressed or basal expression of chromosomal Class I beta-lactamases. Ampicillin was inactive (MIC greater than 256 mg/l) both against beta-lactamase-inducible organisms (except C. freundii) and their stably derepressed mutants, whereas basal mutants were sensitive (MIC 2-8 mg/l). Carbenicillin, ticarcillin, mezlocillin, piperacillin (and ampicillin for C. freundii) were active against both inducible and basal organisms (MIC less than 16 mg/l), but inactive against the derepressed mutants (MICs usually greater than 64 mg/l). Temocillin inhibited all the organisms, including the stably derepressed mutants, at 16 mg/l. Derepressed S. marcescens, M. morganii and Pr. vulgaris were as susceptible as the inducible strains and basal mutants. MICs of temocillin for derepressed mutants of E. cloacae and C. freundii were 4-16 fold above those for their inducible parent strains, but remained within the clinical range.