Trimethoprim/sulfamethoxazole resistance in Burkholderia pseudomallei

Sir, 
 
With the recent publication of the MERTH study [1], trimethoprim/sulfamethoxazole (SXT) monotherapy will be used increasingly in the treatment of melioidosis during the eradication phase, and possibly as the only treatment for some mild infections. It is therefore important to know the prevalence of SXT resistance in Burkholderia pseudomallei. This is difficult to test in vitro, with disc diffusion testing overestimating resistance, and indistinct endpoints in all methods [2]. Even using the Etest method to estimate the minimum inhibitory concentration (MIC), SXT resistance rates as high as 24% in a year have occasionally been reported from Thailand [3]. Recent data from Northern Australia published in this journal [4] are consistent with our own experience in Southeast Asia and suggest that true resistance to SXT in B. pseudomallei is actually very rare. 
 
As our own experience indicated a substantially lower prevalence of SXT resistance than the literature suggests, we reviewed routine data from our diagnostic laboratories in Laos (February 2003 to October 2012) and Cambodia (February 2006 to December 2012). Etest (bioMerieux, Basingstoke, UK) was performed according to the manufacturer's instructions on all isolates in Vientiane (Laos) and on all isolates that appeared non-susceptible by disc diffusion testing [Clinical and Laboratory Standards Institute (CLSI) M02-A11 method; zone diameter < 16 mm] in Siem Reap (Cambodia), as previously recommended [3]. The sources of the strains tested are shown in Table 1. 
 
 
 
Table 1 
 
Source of isolates. 
 
 
 
Of 769 sequential isolates, 99.2% (615/620) from Laos and 100% (149/149) from Cambodia were classified as susceptible to SXT according to CLSI criteria (trimethoprim MIC ≤ 2 mg/L). Three isolates had a trimethoprim MIC of 3 mg/L and two isolates had a trimethoprim MIC of 4 mg/L. The range of MICs is shown in Fig. 1. 
 
 
 
Fig. 1 
 
Distribution of trimethoprim/sulfamethoxazole (co-trimoxazole) Etest minimum inhibitory concentrations (MICs) of clinical isolates of Burkholderia pseudomallei from (a) Laos and (b) Cambodia. 
 
 
 
This confirms that primary resistance of B. pseudomallei to SXT is extremely uncommon and should rarely be a contraindication to SXT monotherapy. These results from Vientiane and Siem Reap closely mirror those of Crowe et al. in Darwin [4]. Although the CLSI currently only recommends broth microdilution testing for B. pseudomallei [5], many years of experience in melioidosis-endemic areas suggests that disc diffusion testing is reliable for all agents except SXT, for which Etest gives acceptable results. We think that the misleading data in the literature are due to the difficulty of interpreting endpoints in testing susceptibility of B. pseudomallei to SXT. We tried to follow the manufacturer's instructions and read the Etest at 80% inhibition, but this is a somewhat subjective endpoint. However, the really important thing that remains to be established is whether SXT MICs can predict the outcome of SXT monotherapy in melioidosis. 
 
Funding: The Laos-Oxford-Mahosot Hospital-Wellcome Trust Research Unit and the Cambodia-Oxford Medical Research Unit are funded by the Wellcome Trust of Great Britain. The authors thank all of the laboratory staff in the microbiology laboratories in Cambodia (Angkor Hospital for Children, Siem Reap) and Laos (Mahosot Hospital, Vientiane) who undertook the testing for their assistance. 
 
Competing interests: None declared. 
 
Ethical approval: Not required.

Funding: This work was supported by the Faculty of Pharmaceutical Sciences, Prince of Songkla University (Songkhla, Thailand). The study sponsors had no involvement in the study design, collection, analysis or interpretation of data, writing of the manuscript or the decision to submit the manuscript for publication.
Competing interests: None declared.

Sir,
With the recent publication of the MERTH study [1], trimethoprim/sulfamethoxazole (SXT) monotherapy will be used increasingly in the treatment of melioidosis during the eradication phase, and possibly as the only treatment for some mild infections. It is therefore important to know the prevalence of SXT resistance in Burkholderia pseudomallei. This is difficult to test in vitro, with disc diffusion testing overestimating resistance, and indistinct endpoints in all methods [2]. Even using the Etest method to estimate the minimum inhibitory concentration (MIC), SXT resistance rates as high as 24% in a year have occasionally been reported from Thailand [3]. Recent data from Northern Australia published in this journal [4] are consistent with our own experience in Southeast Asia and suggest that true resistance to SXT in B. pseudomallei is actually very rare. As our own experience indicated a substantially lower prevalence of SXT resistance than the literature suggests, we reviewed routine data from our diagnostic laboratories in Laos (February 2003 to October 2012) and Cambodia (February 2006 to December 2012). Etest (bioMérieux, Basingstoke, UK) was performed according to the manufacturer's instructions on all isolates in Vientiane (Laos) and on all isolates that appeared non-susceptible by disc diffusion testing [Clinical and Laboratory Standards Institute (CLSI) M02-A11 method; zone diameter < 16 mm] in Siem Reap (Cambodia), as previously recommended [3]. The sources of the strains tested are shown in Table 1.
Of 769 sequential isolates, 99.2% (615/620) from Laos and 100% (149/149) from Cambodia were classified as susceptible to SXT according to CLSI criteria (trimethoprim MIC ≤ 2 mg/L). Three isolates had a trimethoprim MIC of 3 mg/L and two isolates had a trimethoprim MIC of 4 mg/L. The range of MICs is shown in Fig. 1. This confirms that primary resistance of B. pseudomallei to SXT is extremely uncommon and should rarely be a contraindication to SXT monotherapy. These results from Vientiane and Siem Reap closely mirror those of Crowe et al. in Darwin [4]. Although the CLSI currently only recommends broth microdilution testing for B. pseudomallei [5], many years of experience in melioidosis-endemic areas suggests that disc diffusion testing is reliable for all agents except SXT, for which Etest gives acceptable results. We think that the misleading data in the literature are due to the difficulty of interpreting endpoints in testing susceptibility of B. pseudomallei to SXT. We tried to follow the manufacturer's instructions and read the Etest at 80% inhibition, but this is a somewhat subjective endpoint. However, the really important thing that remains to be established is whether SXT MICs can predict the outcome of SXT monotherapy in melioidosis. Funding

Carbapenem-resistant Enterobacter gergoviae harbouring bla KPC-2 in Brazil
Sir, Worldwide, Klebsiella pneumoniae carbapenemase (KPC) detection has been extensively reported every year, and the detection of new species capable of producing this enzyme is of great concern. KPC production is currently the main mechanism of resistance to carbapenems in Enterobacteriaceae strains [1]. In Brazil, reports of new species carrying the bla KPC gene have been made in different regions [2]. However, there are rare reports of KPC-producing Enterobacter gergoviae strains; as far as we are aware, there are only two reports, both in the USA [3,4].
During routine bacteriological diagnosis in a public teaching hospital in Recife (Brazil), a bacterial isolate identified as Enterobacter spp. (named EG2) was selected. This isolate was derived from blood of a patient hospitalised in the intensive care unit (ICU). The patient was hospitalised in February 2008 with hepatic failure. During their hospital stay, the patient received piperacillin/tazobactam (TZP) [500 mg every 6 h (q6 h)] empirically for 2 days. After that, therapy was changed to imipenem (250 mg q6 h). Ten days later the patient died. On the same day, blood samples showed a carbapenem-resistant Enterobacter spp. isolate. Confirmation of the isolate identity by 16S ribosomal DNA sequencing revealed an E. gergoviae isolate. This isolate was used for further analyses.
Minimum inhibitory concentrations (MICs) were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) 2013 guidelines (M100-S23). Molecular analyses were performed through sequencing after specific PCR for bla KPC , for the most common ␤-lactamase genes (bla CTX-M , bla TEM and bla SHV ) and the class 1 integron variable region. Furthermore, the genetic environment of the bla KPC-2 gene was analysed by specific PCR for the region between ISKpn7 and bla KPC , according to Cuzon et al. [1].
Plasmid DNA was obtained by a standard protocol and was used to transform Escherichia coli DH5␣ chemocompetent cells. Transformant cells were selected on Mueller-Hinton agar containing 2 g/mL imipenem and 50 g/mL ticarcillin. Plasmid typing was performed by PCR-based replicon typing (PBRT) as previously described by Carattoli et al. [5].
Results of susceptibility assays showed that isolate EG2 was multidrug-resistant (Table 1). Isolate EG2 harboured the bla KPC-2 , bla CTX-M-15 , bla SHV-5 and bla TEM-1 genes (Table 1). Moreover, the dfrA22 gene, which confers resistance to trimethoprim, was present in the variable region of a class 1 integron. Plasmid analysis showed that EG2 had 133, 65, 25 and 2 kb plasmids. The presence of bla KPC-2 , bla CTX-M-15 and bla SHV-5 genes was confirmed by PCR in transformant recipient cells, named T-EG2. Analysis of the plasmid profile from donor and recipient cells showed that they shared only a