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JAC Advance Access originally published online on February 24, 2006
Journal of Antimicrobial Chemotherapy 2006 57(4):771-774; doi:10.1093/jac/dkl046
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vitro activity of temocillin against extended spectrum ß-lactamase-producing Escherichia coli

Hector Rodriguez-Villalobos*, Vincent Malaviolle, Joëlle Frankard, Ricardo de Mendonça, Claire Nonhoff and Marc J. Struelens

Microbiology Department, Erasme Hospital–Université Libre de Bruxelles, Brussels, Belgium

* Corresponding author. Tel: +32-2555-4518; Fax: +32-2555-3110; E-mail: hrodrigu{at}ulb.ac.be

Received 29 August 2005; returned 27 October 2005; revised 2 February 2006; accepted 2 February 2006


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Objectives: Temocillin is a semi-synthetic 6-{alpha}-methoxy derivative of ticarcillin. It is highly stable to most bacterial ß-lactamases. However, data concerning its activity against extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae are limited. We have analysed the in vitro activity of temocillin against clinical isolates of ESBL-producing Escherichia coli over the past 4 years in a university hospital in Brussels, Belgium.

Methods: Strains were screened for ESBL production using the double-disc synergy test. The MIC of 12 antimicrobial agents was determined for ESBL-producing E. coli isolates (n = 162) using the agar dilution method. ESBLs were characterized by isoelectric focusing; multiplex PCR for bla genes of the SHV, TEM and CTX-M families; and DNA sequencing.

Results: ESBL-producing E. coli isolates harboured CTX-M+TEM (35%), TEM alone (44%), CTX-M alone (6%), CTX-M+SHV (2%) and other ESBL combinations (10%). The proportion of temocillin-susceptible isolates was 92%, with MIC50 and MIC90 values of 8 and 32 mg/L, respectively. Co-resistance to ciprofloxacin and co-trimoxazole in ESBL-producing E. coli was frequent (39%). The proportion of isolates not susceptible to aminoglycosides was 55, 37 and 4% for tobramycin, gentamicin and amikacin, respectively. The proportion of isolates not susceptible to ceftazidime, cefotaxime, cefepime and piperacillin/tazobactam was 70, 52, 37 and 11%, respectively. No resistance to meropenem was observed. The proportion of strains exhibiting resistance to temocillin by year was stable over the study period.

Conclusions: These data indicate good in vitro activity of temocillin against multiresistant ESBL-producing E. coli. Prospective clinical studies are necessary to examine temocillin's potential role in the treatment of non-complicated infections caused by ESBL-producing E. coli.

Keywords: ESBLs , E. coli , antimicrobial susceptibility


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Temocillin is a semi-synthetic penicillin derivative of ticarcillin, supplied as the disodium salt for parenteral administration [6-ß-(2-carboxy-2-thien-3-yl-acetamido)-6{alpha}-methoxypenicillanic acid sodium salt]. The methoxy group in the 6-{alpha} position confers loss of activity against Gram-positive cocci and anaerobic Gram-negative bacilli but excellent activity against the Enterobacteriaceae family, Haemophilus influenzae and Moraxella catarrhalis. It has no activity against Pseudomonas aeruginosa or Acinetobacter spp., but Pseudomonas acidovorans and Burkholderia cepacia are normally susceptible. Temocillin came on to the market in Belgium in 1988 and is one of the drugs recommended for parenteral treatment of acute pyelonephritis (BAPCOC guidelines: www.health.fgov.be/vesalius). It was approved recently in the USA for treatment of B. cepacia lung infection in cystic fibrosis. A recent survey in Belgium showed susceptibility rates of 80–100% among clinical isolates of Enterobacteriaceae.1 Recently, the rates of resistance to many ß-lactam drugs have increased among Enterobacteriaceae, but temocillin has been an exception in maintaining excellent activity against many resistant isolates. This conserved activity is related to the 6-{alpha}-methoxy group, which confers on this drug stability against a variety of ß-lactamases, including AmpC and extended-spectrum ß-lactamases (ESBLs). However, there are limited data in the literature about its activity against ESBL-producing strains, which prevents recommendation of its use in the treatment of patients infected by such strains.

The aim of this study was to determine the in vitro activity of temocillin against clinical isolates of ESBL-producing E. coli collected over a 4 year period in a university hospital in Brussels, Belgium.


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Bacterial strains

During the period from 1 January 2000 to 31 December 2003, 162 ESBL-producing E. coli isolates from clinical and epidemiological surveillance were collected from patients hospitalized in our institution and stored at –80°C. Only one strain per patient was included.

Detection of ESBL production

Screening for ESBL production was routinely carried out using the double-disc diffusion test method with NEOSENSITABS tablets (ROSCO, Denmark) to detect synergy between oxyimino ß-lactam discs and clavulanate in amoxicillin/clavulanate discs. Substrates included ceftriaxone, ceftazidime, cefepime and co-amoxiclav. Antibiotic discs were separated by a distance of 30 mm. The presence of ESBLs was inferred when the cephalosporin inhibition zone was expanded by the clavulanate (synergy). Three combination discs (CDs), CD04 (cefpirome 30 µg/clavulanic acid 7.5 µg), CD02 (ceftazidime 30 µg/clavulanic acid 10 µg) and CD03 (cefotaxime 30 µg/clavulanic acid 10 µg) (Oxoid, UK), were used for confirmation of ESBL production. A difference in zone size of ≥5 mm was used as an indicator of ESBL production.

ESBL characterization

ESBLs were characterized by multiplex PCR for bla genes of SHV, TEM and CTX-M families in all isolates, and by isoelectric focusing and DNA sequencing in selected strains.

Isoelectric focusing was performed on crude sonic extracts from bacterial growth to exponential phase in Luria–Bertani medium using the Phastsystem apparatus (Pharmacia AB, Uppsala, Sweden). Isoelectric points (pIs) were determined by comparing pI values of ß-lactamases with known pIs (pIs 5.4, 5.5, 5.6, 5.9, 6.3, 6.5, 7, 7.6, 7.8, 8.1 and 8.2) after staining of gels with 500 µg/mL nitrocefin (Oxoid).

Detection of blaTEM, blaSHV and blaCTX-M gene families was performed with multiplex PCR using three sets of specific primers designed in-house (Table 1). PCR mixtures (25 µL) contained 0.2 µM each primer, 1 µL of crude DNA extract from one or two bacterial colonies, 0.25 mM each dNTP, 1x buffer, 1.5 mM MgCl2 and 2.5 U of Taq polymerase (AmpliTaq, Applied Biosystems). PCR cycling conditions were initial denaturation (94°C for 5 min); 30 cycles of denaturation (94°C for 30 s), annealing (58°C for 1 min) and polymerization (72°C for 1 min); and an additional polymerization step (72°C for 7 min). PCR products were analysed on a 1.0% agarose gel. Identification of SHV, TEM and CTX-M subgroups was performed using DNA sequencing of full-length ESBL gene PCR products (ABI 3100, Perkin Elmer). Sequence homology was determined using BLASTX (EMBL and SwissProt databases).


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  		Table 1.. Nucleotide sequences of primers used in ESBL detection and sequencing

 
Susceptibility testing

MICs were determined using the agar dilution method according to CLSI guidelines for temocillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, ceftazidime, cefotaxime, cefoxitin, cefepime, meropenem, gentamicin, tobramycin, amikacin, trimethoprim/sulfamethoxazole and ciprofloxacin using interpretative criteria published by the CLSI.2 Susceptibility to temocillin was determined according to breakpoints provided by Fuchs et al.3: susceptible if MIC ≤16 mg/L and resistant if MIC ≥32 mg/L. The E. coli strain ATCC 25922 was used as a control.


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Bacterial isolates included surveillance cultures, rectal swabs obtained biweekly from patients admitted to intensive care units (38%) and clinical isolates from urinary specimens (37%), respiratory tract specimens (9.3%), wounds (8.6%), abdominal specimens (2.5%), blood (1.9%) and samples from other body sites (1.2%). Samples were collected from 82 female and 80 male patients admitted in our institution, with a mean age (range) of 66 (18–94) years and 61 (0–94) years, respectively.

ESBL-producing E. coli isolates harboured CTX-M+TEM (35%), TEM alone (44%), CTX-M alone (6%), CTX-M+SHV (2%) and other ESBL combinations (10%). DNA sequencing identified three groups of CTX-M enzymes: CTX-M group 1 (CTX-M-1, CTX-M-15 and CTX-M-22) in 76% of isolates, CTX-M-2 in 14% and CTX-M-9 in 10%. All SHV enzymes were SHV-12. ESBLs of the TEM family included TEM-1 (in isolates with a combination of TEM with other ESBL enzymes) and TEM-17, TEM-20, TEM-24, TEM-30 and TEM-52 in the group harbouring TEM alone. Two strains presented ESBL phenotypic activity but were shown using PCR to be negative for TEM, SHV and CTX-M genes.

The MIC distributions of antibiotics tested are shown in Table 2. Meropenem showed the best activity, with MIC100 ≤0.06 mg/L. The MIC50 and MIC90 (95% confidence interval) of temocillin were 5.14 (4.5–5.8) and 13.6 (11.3–16.3) mg/L, respectively. Together with meropenem and amikacin, temocillin showed the best activity, with 92% of isolates being susceptible. Co-resistance to ciprofloxacin and co-trimoxazole was frequent (39%). CTX-M-positive strains showed a higher percentage of resistance to cefotaxime and cefepime (86 and 72%, respectively) than CTX-M-negative strains (28 and 10%, respectively) (P < 0.001).


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  		Table 2.. MIC distribution of ESBL-producing E. coli for 12 antimicrobial drugs

 
The distribution of temocillin MICs was not influenced by the ESBL produced, even in isolates producing a combination of ESBL enzymes (Figure 1). Twelve strains showed temocillin MICs >32 mg/L. Of these strains, 10 harboured TEM-1 in combination with CTX-M-15 and one strain harboured TEM-17. The last strain showed positive ESBL results with both screening and CD tests, but only TEM-1 was detected by PCR and the sequencing analysis. Other strains that produced CTX-M-15 had lower MICs of temocillin (2–16 mg/L). The proportion of temocillin resistance in E. coli assessed by year remained stable.


Figure 1
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  		Figure 1.. Distribution of temocillin MICs for ESBL-producing E. coli by ESBL family or combination.

 

   Discussion
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Twenty years after the first identification of ESBL enzymes in Germany, the emergence of ESBL-producing Enterobacteriaceae has become a widespread problem. More recently, the emergence of clinical isolates of ESBL-producing E. coli that produce CTX-M enzymes was related to hospital and community outbreaks in several European countries.4–6 These enzymes typically confer a high level of resistance to cefotaxime, ceftriaxone and cefepime but are less capable of degrading ceftazidime. The CTX-M genes, which are associated with integrons, have become widespread in other Enterobacteriaceae species and represent a challenge for the treatment of infections caused by these bacteria both in the community and within hospitals.7 Since 2000, the proportion of ESBL-producing E. coli clinical isolates has increased in our institution, from 21 (0.92%) in 2000 to 33 (1.25%) in 2001, 48 (1.85%) in 2002, 64 (2.34%) in 2003 and 87 (3.40%) in 2004 (P < 0.001). Particularly notable was the increasing proportion of isolates carrying blaCTX-M genes.8 Among ESBL-producing Enterobacteriaceae, multiresistance is a common phenomenon that severely limits the options for effective antibacterial treatment. Usually, the only active antimicrobial agents are the carbapenems.

Although the efficacy of carbapenems against ESBL-producing strains of Enterobacteriaceae has been widely documented, carbapenem resistance may emerge, either by porin loss or by acquisition of plasmid-mediated carbapenemases such as IMP and VIM enzymes. This risk justifies efforts to limit carbapenem use. In addition, carbapenem MICs for metallo-ß-lactamase-producing E. coli isolates do not usually exceed the resistance breakpoint. Thus, detection of such isolates may be missed in routine clinical laboratory practice, leading eventually to the failure of a carbapenem-based therapy. Temocillin has high activity against most Enterobacteriaceae except for Serratia marcescens. It has no activity against P. aeruginosa, Acinetobacter spp. and Campylobacter spp. This drug has an excellent stability against the majority of ß-lactamases, including cephalosporinases, exhibits narrow MIC and MBC ranges, and shows bactericidal activity at concentrations close to MIC values and a lack of cross-resistance to third-generation cephalosporins.9–11 Temocillin could be considered for the treatment of infections caused by ESBL-producing strains based on studies demonstrating its activity against ESBL-producing E. coli, its bactericidal activity and its minimal inoculum effect.10,12,13 However, limited data are available regarding the clinical efficacy of this drug against multiresistant strains with a high level of ß-lactamase production. A previous Belgian study showed excellent activity of temocillin against Enterobacteriaceae recovered from inpatients in five general hospitals in Belgium, with no resistance detected in E. coli and <6% resistance among inducible Enterobacteriaceae strains. In our study, at a single tertiary care centre in Brussels, resistance to temocillin was uncommon (<10%) and stable over the study period in ESBL-producing E. coli. Although the majority of temocillin-resistant strains were CTX-M-15 producers, other isolates harbouring this enzyme were temocillin susceptible, suggesting that additional resistance mechanisms such as porin modifications were required for temocillin resistance. Further studies are required to examine this hypothesis. Meropenem, amikacin, temocillin and piperacillin/tazobactam were the most active antimicrobial agents in this study. This high activity of piperacillin/tazobactam could be related to the high proportion of strains that produced CTX-M enzymes. However, use of piperacillin/tazobactam for treating infection caused by ESBL-producing E. coli remains controversial due to a major inoculum effect.

In conclusion, this study, which examined the largest series to date, confirmed the in vitro activity of temocillin against >90% of multiresistant ESBL-producing E. coli isolates from patients admitted to a university hospital. Our data suggest that temocillin could be a valuable option in the treatment of infections caused by ESBL-producing E. coli. Prospective clinical studies are warranted to confirm its therapeutic efficacy.


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None to declare.


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1. Vanhoof R, Berin C, Carpentier M et al. Comparative in vitro activity of temocillin and other antimicrobial agents against Enterobacteriaceae isolated from patients admitted to five Belgian hospitals. Acta Clin Belg 2001; 56: 354–9.[Web of Science][Medline]

2. National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing: Approved Standard M100-S11. NCCLS, Wayne, PA, USA, 2001.

3. Fuchs PC, Barry AL, Thornsberry C et al. Interpretive criteria for temocillin disk diffusion susceptibility testing. Eur J Clin Microbiol 1985; 4: 30–3.[CrossRef][Web of Science][Medline]

4. Rodriguez-Bano J, Navarro MD, Romero L et al. Epidemiology and clinical features of infections caused by extended-spectrum ß-lactamase-producing Escherichia coli in nonhospitalized patients. J Clin Microbiol 2004; 42: 1089–94.[Abstract/Free Full Text]

5. Woodford N, Ward ME, Kaufmann ME et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum ß-lactamases in the UK. J Antimicrob Chemother 2004; 54: 735–43.[Abstract/Free Full Text]

6. Bonnet R. Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004; 48: 1–14.[Free Full Text]

7. Livermore DM, Hawkey PM. CTX-M: changing the face of ESBLs in the UK. J Antimicrob Chemother 2005; 56: 451–4.[Abstract/Free Full Text]

8. Rodriguez-Villalobos H, Malaviolle V, Frankard J et al. Emergence of CTX-M extended spectrum ß-lactamase-producing Escherichia coli in Belgium. EuroSurveillance Weekly 2005; 10: http://www.eurosurveillance.org.

9. Chanal M, Sirot J, Cluzel M et al. In vitro study of the bacteriostatic and bactericidal activity of temocillin (BRL 17421). Pathol Biol (Paris) 1983; 31: 467–70.[Medline]

10. Van Landuyt HW, Boelaert J, Piot P et al. In vitro activity of temocillin against clinical isolates. Drugs 1985; 29 Suppl 5: 1–8.[Web of Science][Medline]

11. Verbist L. Temocillin sensitivity of Enterobacteriaceae species resistant to cefotaxime. Drugs 1985; 29 Suppl 5: 38–42.

12. Van Landuyt HW, Pyckavet M, Lambert A et al. In vitro activity of temocillin (BRL 17421), a novel ß-lactam antibiotic. Antimicrob Agents Chemother 1982; 22: 535–40.[Abstract/Free Full Text]

13. Williams JD, Chen HY. The place of temocillin in the treatment of hospital infections. Drugs 1985; 29 Suppl 5: 234–9.


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