JAC Advance Access published online on July 12, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm256
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Rapid detection of extended-spectrum ß-lactamase-producing Gram-negative bacilli in blood cultures
Department of Microbiology, City Hospital, Dudley Road, Birmingham B18 7QH, UK
* Corresponding author. Tel: +44-121-507-4228; Fax: +44-121-551-7763; E-mail: nigel.brenwald{at}swbh.nhs.uk
Received 21 February 2007; returned 25 March 2007; revised 14 June 2007; accepted 21 June 2007
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Abstract |
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Objectives: Conventional detection of extended-spectrum ß-lactamase (ESBL) producers in blood cultures usually requires overnight incubation. This could delay the prescribing of appropriate therapy. We evaluated whether the chromogenic cephalosporin HMRZ-86, which is hydrolysed by ESBLs, could be used for the rapid detection of ESBL producers directly in blood culture broths.
Methods: The HMRZ-86 test was first applied to broth cultures of isolates producing known ß-lactamases. A colour change indicating hydrolysis, which was inhibited by clavulanic acid, was taken as an indication of ESBL production. A similar method was used for testing blood culture supernatants and broth subcultures of blood cultures.
Results: The HMRZ-86 test detected all the ESBL producers among 83 clinical isolates and control strains. Only one false positive was seen. The usefulness of HMRZ-86 for the direct testing of blood culture broths was limited by the presence of lysed blood. However, by using a 2 h broth subculture of the blood culture broths, the HMRZ-86 test was able to detect all those blood cultures containing an ESBL producer. No false positive or negative tests occurred according to the results of our standard phenotypic tests.
Conclusions: The HMRZ-86 test is a simple and rapid method that can be used for detecting ESBL producers in blood cultures.
Key Words: HMRZ-86 , bacteraemia , chromogenic cephalosporin
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Introduction |
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In recent years, there has been an increase in infections caused by extended-spectrum ß-lactamase (ESBL)-producing strains of Gram-negative bacilli. These infections are associated with significantly higher morbidity and mortality.1,2 Several studies have associated a delay in appropriate antimicrobial treatment with an increased mortality in bloodstream infections caused by ESBL-producing Escherichia coli and Klebsiella pneumoniae.3,4 Therefore, there is a need for the rapid detection of ESBL producers, from blood cultures.
Current methods for detecting ESBL-producing blood culture isolates are based on determining the susceptibility of the isolate to third-generation cephalosporins such as cefpodoxime. This requires overnight growth and means that up to 24 h can elapse before ESBL production is detected. This may lead to a delay in the initiation of appropriate antibiotic therapy.
HMRZ-86 is a new chromogenic cephalosporin (Kanto Chemical Co Inc., Tokyo, Japan), which is hydrolysed by ESBLs and metallo-ß-lactamases (MBLs), but not by common non-ESBL enzymes such as TEM-1 or SHV-1.5,6 Hydrolysis occurs rapidly and is indicated by a simple colour change from yellow to red. HMRZ-86 has been used to detect ESBLs directly from bacterial colonies.6 However, using this approach to detect ESBL producers in blood cultures would not be substantially quicker than conventional methods; the results of direct susceptibility tests would normally be available at the same time as growth on agar subculture plates. Therefore, we investigated whether HMRZ-86 could be used to detect ESBL producers directly in blood culture broths, before an agar subculture of the isolate was available.
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Materials and methods |
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Bacterial strains
Eighty-three bacterial strains were used to evaluate the performance of HMRZ-86. The strains included 63 recent blood culture isolates (16 ESBL-positive E. coli, 23 ESBL-negative E. coli, 18 ESBL-positive K. pneumoniae and 6 ESBL-negative K. pneumoniae) and 20 control strains producing a variety of known ß-lactamases. The control strains included one each of the following ESBL producers: E. coli CTX-M-15, SHV-2, SHV-5, TEM-3, TEM-6, TEM-10, TEM-101; Enterobacter cloacae SHV-12; K. pneumoniae CTX-M-12, CTX-M-13, CTX-M-26. One each of the following non-ESBL producers were also tested: E. coli plasmid-mediated AmpC ß-lactamase, chromosomal AmpC ß-lactamase, SHV-1, TEM-1; E. cloacae inducible AmpC ß-lactamase, derepressed AmpC ß-lactamase; K. pneumoniae plasmid-mediated AmpC ß-lactamase; Klebsiella oxytoca K1 hyperproducer; Pseudomonas aeruginosa IMP-1 metallo-ß-lactamase. E. coli strain NCTC 10418 was used as a negative control.
A prospective study was carried out on blood cultures submitted to the Microbiology Department, City Hospital, Birmingham, UK, between June and December 2006. Blood cultures were processed on a BACTEC 9240 blood culture system (Becton–Dickinson, Oxford, UK) and submitted to the laboratory as a two-bottle set (Plus Aerobic/F and Lytic/10 Anaerobic/F) or single paediatric bottle (Peds Plus/F). Cultures that signalled for growth were examined microscopically by Gram stain and subcultured onto agar media. Isolates were identified by standard methods. All E. coli, K. pneumoniae, Proteus mirabilis and Salmonella sp. were screened for ESBL production using a cefpodoxime disc susceptibility method. Cefpodoxime-resistant isolates were confirmed as ESBL producers using a combined disc method (cefpodoxime 10 µg ± 1 µg clavulanic acid).7
Detection of ESBLs in broth cultures using HMRZ-86
Approximately 500 µL of blood culture broth was removed aseptically from those blood cultures in which Gram-negative bacilli had been seen. Two drops were inoculated into 3 mL of tryptone soya broth (TSB), which was then incubated without shaking for 2 h at 37°C in air. The remaining blood culture broth was centrifuged at 6000 g for 2 min and the supernatant used for testing. The detection of ESBLs was carried out by mixing equal volumes (5 µL) of the TSB culture or blood culture supernatant and HMRZ-86 reagent (supplied ready diluted by manufacturer) in a microtube. Each reaction was set up in duplicate. Clavulanic acid was added to one tube to a final concentration of 4 mg/L. A volume of water equalling that of the clavulanic acid was added to the remaining tube. The tubes were incubated at 37°C and examined at 30 min intervals for the hydrolysis of HMRZ-86 (indicated by a yellow to red colour change). The test was considered positive for an ESBL if hydrolysis occurred, which was inhibited by clavulanic acid. A negative reaction in both tubes was taken as being negative for ESBL production.
The same method was used for detecting ESBL producers growing on solid medium, except that the isolate was first grown in TSB for 2 h at 37°C in air before being tested in the same way as described for blood cultures.
All processing of blood cultures was undertaken in a category 3 cabinet.
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Results |
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Bacterial isolates
Of the 83 isolates tested, 47 were positive by the HMRZ-86 test. These included all 45 known ESBL producers, a K1 ß-lactamase-hyperproducing K. oxytoca and an IMP-1-producing P. aeruginosa (the hydrolysis was not inhibited by clavulanic acid). No false negative results were seen. None of the AmpC ß-lactamase producers gave a positive result, except for the SHV-12-producing E. cloacae.
A total of 127 blood culture bottles from 71 patients were studied. In all cases the blood culture bottles had signalled for growth and Gram-negative bacilli had been seen microscopically. Forty-three bottles were subsequently shown to be growing an ESBL-producing E. coli or Klebsiella sp. resistant to cefpodoxime and ESBL confirmatory test positive.
Detection of ESBL producers directly from blood culture broths
The direct testing of the anaerobic blood culture broths with HMRZ-86 was not possible as the medium lysed the blood, which interfered with the interpretation of the test.
Of the 62 aerobic and 4 paediatric blood culture broths tested, 12 aerobic broths could not be processed directly because the blood was lysed. Of the remaining 54 aerobic and paediatric bottles, 20 were found to contain an ESBL producer using conventional tests. Nineteen of these aerobic broths were HMRZ-86 test positive; only one E. coli strain gave a false negative result. All 19 HMRZ-86 tests were positive after 30 min of incubation. When the HRMZ-86 test was incubated for longer than 1 h, eight of the aerobic blood cultures that were negative for ESBLs by conventional methods gave a very weak positive result with HMRZ-86 that was unaffected by clavulanic acid.
Detection of ESBL producers in a 2 h subculture of blood culture broths
Of the 127 bottles tested, 43 were positive by the HMRZ-86 test using the 2 h broth subculture. All 43 bottles were shown by conventional methods to harbour an ESBL producer (E. coli n = 39, Klebsiella sp. n = 4). While the majority of the HMRZ-86 tests (67.4%) were positive after 30 min of incubation of broth and reagent, 23.3%, 7.0% and 2.3% took 60, 90 and 120 min, respectively, to become positive. No false negative or positive results occurred with the HMRZ-86 test, when compared with the conventional methods for detecting ESBLs. It was noted that for eight of the HMRZ-86 positive broths, the clavulanic acid containing tube became weakly positive after prolonged incubation (>2 h). Table 1 shows the identity and ESBL status of the blood culture isolates and results of the HMRZ-86 test.
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Discussion |
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Our preliminary study using broth cultures of clinical isolates and control strains showed that the HMRZ-86 test successfully detected all of the ESBL producers with no false negative reactions. Only one false positive reaction occurred with the strain of K. oxytoca (K1 ß-lactamase hyperproducer). The number of different ESBL and non-ESBL producers tested was limited. Therefore, additional work needs to be carried out with other types of ß-lactamases to fully explore the potential of HMRZ-86.
When the HMRZ-86 test was applied to the direct testing of blood cultures only one false negative occurred; 19 of the 20 blood culture broths containing an ESBL producer were HMRZ-86 test positive. However, the usefulness of direct testing was limited as only 42.5% of the blood cultures included in our study were suitable for direct testing. The remaining blood cultures contained lysed blood, which masked the colour change that occurred following HMRZ-86 hydrolysis. In order to overcome the problem of lysed blood interfering with the test, an initial broth subculture of the blood culture medium was introduced. This had the effect of diluting out the lysed blood, while producing sufficient bacterial growth for the HMRZ-86 test. When the broth subculture step was used, the HMRZ-86 test correctly identified all the blood cultures that were growing an ESBL producer. No false positive or false negative tests occurred if the HMRZ-86 test was read at 2 h. Although the use of a broth subculture increased the time required for testing it is possible that the 2 h incubation period could be reduced if a shaken culture was used.
In summary, our preliminary study showed that HMRZ-86 has the potential to be very useful in the early detection of ESBL producers in blood culture broths. However, further work needs to be carried out to define the limitations of the test. The HMRZ-86 test was very simple to perform and could easily be adopted in routine laboratories. Even with the initial broth subculture step, all ESBL producers could be detected within 2.5–4 h of the blood culture signalling for growth. The rapid detection of ESBL producers in blood cultures would improve the likelihood of early appropriate antibiotic therapy being given.
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None to declare.
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Acknowledgements |
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We thank Dr R. Kubo, Kanto Chemical Co Inc., Tokyo, Japan, for kindly supplying the HMRZ-86 used in the study, and Dr D. Brown, HPA Cambridge and Professor P. Hawkey, HPA Birmingham for supplying control strains.
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References |
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6 . Colodner R, Reznik B, Gal V, et al. Evaluation of a novel kit for the rapid detection of extended-spectrum ß-lactamases. Eur J Clin Microbiol Infect Dis (2006) 25:49–51.[CrossRef][Web of Science][Medline]
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