Identification and characterization of ceftriaxone resistance and extended-spectrum ß-lactamases in Malawian bacteraemic Enterobacteriaceae
1 Malawi-Liverpool-Wellcome Trust Laboratories, PO Box 30096, Blantyre, Malawi; 2 Department of Medical Microbiology, Royal Liverpool University Hospital, Liverpool L7 8XP, UK; 3 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK
* Corresponding author. Tel: +265-167-6444; Fax: +265-167-5774; E-mail: kgray{at}mlw.medcol.mw
Received 8 November 2005; returned 20 November 2005; revised 20 January 2006; accepted 24 January 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Objectives: To enumerate and characterize extended-spectrum ß-lactamases (ESBLs) amongst ceftriaxone-resistant coliforms in Blantyre, Malawi, where third-generation cephalosporin use is currently highly restricted.
Methods: Over the period April 2004–March 2005 all ceftriaxone-resistant isolates from blood cultures were examined for the presence of ESBLs. Isoelectric focusing was performed on enzyme extracts. PCR and DNA sequencing of amplicons were used to identify the underlying genetic determinants responsible for the ESBL phenotypes. Transferability of the ESBL phenotypes was tested by conjugation to a susceptible Escherichia coli J53.
Results: Enterobacteriaceae were isolated from 1191 blood cultures, of which 19 (1.6%) were ceftriaxone resistant. Ten isolates (0.7% of all isolates) demonstrated an ESBL phenotype but only eight were characterized as three isolates were from the same patient. Genotypes SHV-11 (n = 1), SHV-12 (n = 3), SHV-27 (n = 1), TEM-63 (n = 2) and CTX-M-15 (n = 1) were detected. Plasmid transfer of the ESBL resistance phenotype was successful for all the isolates.
Conclusions: In a clinical setting of minimal cephalosporin usage there is already a diversity of ESBL genotypes. Increased use of cephalosporins in this setting is likely to result in a rapid expansion of ESBLs and their prevalence will need to be carefully monitored.
Keywords: molecular epidemiology , Africa , ESBLs
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
The use of broad-spectrum penicillins and cephalosporins has been associated with the emergence of extended-spectrum ß-lactamases (ESBLs) in Enterobacteriaceae. These have been described worldwide and are a major cause of nosocomial infections associated with high mortality. ESBLs have been described in Africa1–7 and have considerable implications for the developing world, where there is limited access to newer antibacterial agents.
Third-generation cephalosporins are used rarely in Malawi, but their inclusion as agents of first choice in cases of community-acquired bacteraemic illness is being considered. Streptococcus pneumoniae and non-typhoidal salmonellae (NTS) are the commonest causes of bacteraemia at the Queen Elizabeth Central Hospital (QECH), a large tertiary referral hospital in Blantyre.8 Over recent years there has been a dramatic increase in the number of multidrug-resistant NTS and a problem of reduced susceptibility to penicillin among the pneumococci. Current policy is to treat patients with community-acquired sepsis with one or a combination of benzyl-penicillin, chloramphenicol and/or gentamicin, depending on the clinical presentation. Ceftriaxone has been intermittently available in recent years in Blantyre, first in 1997 for use in paediatric patients and in 2002 for adults participating in a clinical trial of meningitis treatment.
Following the death of a patient, attributable to a multiresistant Klebsiella pneumoniae infection, for which no therapeutic options were available, we decided to review the scale of the ESBL problem at the QECH and characterize the ESBLs produced by Enterobacteriaceae in clinically important isolates. This assessment will provide a baseline for future surveillance for ESBLs as cephalosporins become adopted as the agents of choice for community-acquired sepsis.
As part of our investigation we also chose to look for the plasmid-mediated quinolone resistance determinant qnrA, which has been associated with clavulanic acid-inhibited ESBLs in Enterobacteriaceae.9 Ciprofloxacin is the only alternative drug for treatment available for multiresistant NTS isolates in our setting and the presence of both resistance determinants would compromise therapy further.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Patients and bacterial strains
Blood cultures were performed by the Malawi-Liverpool-Wellcome Trust Laboratories for admissions to the paediatric and adult medical services in the QECH. Samples were processed using a commercial blood culturing system (BacT Alert, BioMerieux, Lyons, France). Significant isolates were identified using standard procedures and all Enterobacteriaceae were identified to the species level using the API 20E system (BioMerieux).
Susceptibility testing
Disc diffusion susceptibility was performed in accordance with British Society for Antimicrobial Chemotherapy guidelines.10 Antimicrobials tested in the routine laboratory include ciprofloxacin, co-trimoxazole, ampicillin, gentamicin, chloramphenicol and ceftriaxone. Isolates reported as ceftriaxone resistant were further examined for ESBL production and susceptibility to ceftazidime, cefoxitin and imipenem. MICs were determined using the Etest method (AB Biodisk, Solna, Sweden). ESBL detection was performed using the double disc diffusion method with cefotaxime and ceftazidime discs placed edge to edge 15 mm from a disc containing amoxicillin/clavulanic acid.
Conjugation experiments
Broth matings were carried out with the clinical isolates as donors and Escherichia coli J53 (sodium azide resistant) at a bacterial cell ratio of 1:10. After 24 h, the mating mixture was centrifuged at 13 000 g for 5 min and the supernatant removed (to remove any cell free ß-lactamase). The mixture was resuspended in an equal volume of PBS and 30 µL was plated separately onto MacConkey agar (Oxoid Ltd, Basingstoke, UK) containing sodium azide (100 mg/L) supplemented with either cefotaxime (1 mg/L) or ceftazidime (4 mg/L). After overnight incubation, transconjugants were identified, E. coli was confirmed as the recipient and MICs were determined.
PCR screening and DNA sequencing
Total DNA for PCR was extracted by suspending bacteria in 5% (w/v) Chelex-100 resin slurry (Bio-Rad, Hemel Hempstead, UK) in injection grade water followed by boiling for 10 min. Samples were centrifuged (10 min at 13 400 g) and used immediately or stored at –20°C. Isolates with the ESBL phenotype were examined by PCR for the presence of the blaTEM,11 blaSHV,12 blaCTX-M13 and qnrA14 genes. Positive and negative controls were included for all the PCRs. Control DNA for the qnrA PCR was obtained from a previous study.15 PCR products were excised from 1% (w/v) agarose gels and purified using a commercial kit (QIAquick gel extraction kit, Qiagen, GmbH). Sequence determination of amplicons for both parents and transconjugants was performed on both strands using the respective PCR primers. Sequence analysis was performed using commercial software (Lasergene, DNAStar Inc., Madison, WI, USA), and sequences were aligned to the following GenBank accession numbers: blaSHV-11, AF187732 [GenBank] ; blaSHV-12, AY940490 [GenBank] ; blaSHV-27, AF293345 [GenBank] ; blaTEM-63, AF332513 [GenBank] ; blaCTX-M-15, AY044436 [GenBank] .
Analytical isoelectric focusing
ß-Lactamase extraction and isoelectric focusing (IEF) were performed as described previously16 on polyacrylamide gels containing ampholines in the pH range 3.5–9.5 (Amersham Pharmacia Biotech, Buckinghamshire, UK). Detection of cefotaxime hydrolysis by the separated proteins post-electrophoresis was achieved by overlaying the ampholine gel with agar containing cefotaxime (0.4 mg/L). The gel overlay method was used to attempt to define the differing contribution of these bands to the hydrolysis of cefotaxime as many bacteria produce both major and minor ß-lactamases. After incubation for 2 h at 37°C, the agar was flooded with a McFarland standard 0.5 suspension of E. coli (NCTC 10418), re-incubated overnight and examined for bacterial growth.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Patients and bacterial strains
From 1 April 2004 to 31 March 2005, 6487 blood cultures were drawn from adults, from which 649 (10%) isolates of Enterobacteriaceae were grown. Seven (1.1% of all Enterobacteriaceae) were ceftriaxone resistant, of which five had the ESBL phenotype. In each case these were community-acquired infections. One organism (H309) was isolated three times at sequential intervals from the same patient with cavitating pneumonia and thus was counted only once. In addition, there were 5964 paediatric blood cultures, from which 542 (9.1%) Enterobacteriaceae were isolated. Of the 542 isolates, 12 (2.2% of all Enterobacteriaceae) were reported to be ceftriaxone resistant, of which five had the ESBL phenotype.
Characteristics of the ESBL phenotypes are summarized in Table 1. All isolates were resistant to co-trimoxazole, amoxicillin, gentamicin, chloramphenicol and ceftazidime, as shown by disc testing. Only isolates H308 and H309 were also resistant to ciprofloxacin, but all were susceptible to imipenem. Isolates H305 and H306 (both Enterobacter cloacae) were resistant to cefoxitin. MICs of ceftazidime and cefotaxime with or without clavulanate, for both original isolates and their transconjugants, are shown in Table 1.
|
Conjugation experiments
Transfer of the ESBL resistance phenotype to E. coli J53 was successful for all the isolates.
IEF, PCR and DNA sequencing
IEF profiles of the isolates are shown in Table 1. At least two bands were detected for all isolates except H305. All isolates except H305 produced TEM-like enzymes, as evidenced by bands in the region pI 5.4–6.3. It was assumed that the band at pI 5.4, found in all isolates except H305, was most likely to represent TEM-1. This was confirmed for H309 by DNA sequencing of the amplicon from the TEM PCR. Isolates H304, H309, H310 and H311 also produced SHV-like enzymes, as evidenced by bands in the region pI 7.0–8.2. PCR screening confirmed the presence of blaSHV in isolates H304, H309, H310 and H311.
The genotypes determined by DNA sequencing are shown in Table 1. The blaSHV amplicon for H309 was not sequenced as no SHV enzymes have been reported with pIs between 7.1 and 7.5. The nucleotide sequences of both the TEM-63 genes had the same silent point mutation in the codon for glycine 218, which is encoded by GGG in the originally described TEM-63 but by GGA in isolates H306 and H307. (The amino acid residues are numbered according to the numbering system of Ambler et al.17) The plasmid-mediated quinolone resistance determinant qnrA was not detected in any of the isolates.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
This study suggests that ESBLs are currently found in only a small number of clinically relevant isolates in paediatric and adult patients of the QECH, Blantyre, Malawi. However, there is already a genetic diversity of ESBLs despite the highly restricted use of third-generation cephalosporins.
SHV-12 was the most commonly identified genotype. This was first described in 199718 and since then has been reported worldwide and more recently from Tunisia,4 Cameroon5 and Tanzania7 on the African continent. In our study it was isolated only from paediatric patients and was the commonest paediatric genotype, although it was carried by three different bacterial genera. The gene was shown to be transmissible in all cases and this suggests that plasmid-encoded SHV-12 may be circulating within this population. Further work is required, including studies of enteric flora, to identify risk factors for possession of this ESBL on the paediatric wards. It is interesting that there was an absence of bands detectable by IEF for the SHV-12 enzymes, and cefotaxime hydrolysis was not detected for any of these either. It may be that there were other contributors to the ESBL phenotype which were not detected or that there were low levels of these enzymes in the periplasmic space ensuring that a toxic threshold was achieved, but on protein extraction insufficient protein was released for IEF detection.
Of the other ESBL genotypes, SHV-27 has been reported previously only from Brazil19 and SHV-11 is also infrequently reported (although there is some debate as to whether SHV-11 is really an ESBL).18 CTX-M-15 has been reported worldwide, including from Cameroon6 and Tanzania7 in Africa. The patient from whom the multiresistant K. pneumoniae carrying CTX-M-15 was isolated died due to bacterial pneumonia, as effective antimicrobial chemotherapy was unavailable. Also, the CTX-M-15 gene was not detected by the nitrocefin reaction although cefotaxime hydrolysis was shown by the bioassay at a pI close to 9.0 in the expected region of CTX-M-15. We, like others,20,21 have previously observed the lack of nitrocefin hydrolysis on IEF, particularly with CTX-M-mediated ß-lactamases.
It is interesting that we detected TEM-63, which was transmissible from both the E. cloacae and E. coli isolates, from both the adult and the paediatric patients. This ESBL has previously been reported only from South Africa, where it was reported initially in ESBL-producing Klebsiella spp., Proteus spp., Enterobacter spp. and E. coli strains2,22 and more recently in Salmonella strains.3 This suggests that there has been a spread of the TEM-63 gene within southern Africa. Of greater concern is that in all probability it is only a matter of time before it spreads into the NTS in Malawi. NTS are particularly important causes of bloodstream infections in children and in HIV-infected adults,8,23 and the rise in multidrug resistance among these bacteria shows that therapeutic options at present are limited to quinolones and cephalosporins. Although two of our isolates demonstrated ciprofloxacin resistance in association with ESBLs, it is reassuring that we did not detect the plasmid-mediated quinolone resistance determinant qnrA in any of the isolates.
We did not specifically look for AmpC-type ß-lactamases in this study although isolates H305 and H306 were both resistant to cefoxitin. It is likely that the cephamycin resistance was porin mediated, as we detected no basic pI values of >9.0 on the IEF, and ESBLs were detected phenotypically in both the isolates by disc testing systems.
We have identified only small numbers of ESBLs, but it is likely that we have not fully recognized their prevalence. In our routine susceptibility testing we test only ampicillin and ceftriaxone (which is the only cephalosporin therapy available to any great extent in Malawi) and do not routinely test for ESBLs. As ceftazidime was initially recommended for ESBL screening (now cefpodoxime is being recommended), it is possible that we may have missed rare isolates in this collection that are resistant to ceftazidime but fully susceptible to ceftriaxone. Nevertheless, our results provide a baseline from which changes in the numbers and characteristics of ESBLs can be monitored in the coming years. If the use of ceftriaxone increases, as seems likely at the present time, then it is inevitable that ESBL prevalence will also increase. Unfortunately, the healthcare institutions of Malawi, as in much of the developing world, lack effective infection-control practice and infrastructure (e.g. lack of hand-washing facilities) and are permanently overcrowded. Each of these factors will serve to promote the dissemination of ESBLs. Furthermore, unlike the situation in the developed world, the financial resources to provide alternative agents such as carbapenems are lacking, and the option to tailor therapy based on antimicrobial resistance testing is unavailable in all except a handful of hospitals. Thus, it would be prudent to monitor carefully the evolving epidemiology of ESBLs with changing antibiotic practice and to act quickly on this information to control the prescription of such antibiotics if necessary.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
We thank the staff and patients of the Queen Elizabeth Central Hospital, Blantyre, for their contribution to this work and Professor Malcolm Molyneux, Director of the Malawi-Liverpool-Wellcome Trust Laboratories, for his support. The Wellcome Trust, UK, provided financial support for the work (grant numbers 058390 and 061230), with a contribution from the University of Liverpool.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
1. Pitout JD, Thomson KS, Hanson ND et al. ß-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa. Antimicrob Agents Chemother 1998; 42: 1350–4.
2.
Essack SY, Hall LM, Pillay DG et al. Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum ß-lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa. Antimicrob Agents Chemother 2001; 45: 88–95.
3.
Kruger T, Szabo D, Keddy KH et al. Infections with nontyphoidal Salmonella species producing TEM-63 or a novel TEM enzyme, TEM-131, in South Africa. Antimicrob Agents Chemother 2004; 48: 4263–70.
4. Ben-Hamouda T, Foulon T, Ben-Mahrez K. Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in a Tunisian neonatal ward. Microb Drug Resist 2004; 10: 132–8.[CrossRef][ISI][Medline]
5.
Gangoue-Pieboji J, Bedenic B, Koulla-Shiro S et al. Extended-spectrum-ß-lactamase-producing Enterobacteriaceae in Yaounde, Cameroon. J Clin Microbiol 2005; 43: 3273–7.
6.
Gangoue-Pieboji J, Miriagou V, Vourli S et al. Emergence of CTX-M-15-producing enterobacteria in Cameroon and characterization of a blaCTX-M-15-carrying element. Antimicrob Agents Chemother 2005; 49: 441–3.
7.
Blomberg B, Jureen R, Manji KP et al. High rate of fatal cases of pediatric septicemia caused by gram-negative bacteria with extended-spectrum ß-lactamases in Dar es Salaam, Tanzania. J Clin Microbiol 2005; 43: 745–9.
8. Gordon MA, Walsh AL, Chaponda M et al. Bacteraemia and mortality among adult medical admissions in Malawi—predominance of non-typhi salmonellae and Streptococcus pneumoniae. J Infect 2001; 42: 44–9.[CrossRef][ISI][Medline]
9.
Nordmann P, Poirel L. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother 2005; 56: 463–9.
10.
Andrews JM. BSAC standardized disc susceptibility testing method (version 4). J Antimicrob Chemother 2005; 56: 60–76.
11.
Sayeed S, Saunders JR, Edwards C et al. Expression of Aeromonas caviae bla genes in Escherichia coli. J Antimicrob Chemother 1996; 38: 435–41.
12. Nuesch-Inderbinen MT, Hachler H, Kayser FH. Detection of genes coding for extended-spectrum SHV ß-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test. Eur J Clin Microbiol Infect Dis 1996; 15: 398–402.[CrossRef][ISI][Medline]
13.
Gniadkowski M, Schneider I, Palucha A et al. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing ß-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob Agents Chemother 1998; 42: 827–32.
14.
Jacoby GA, Chow N, Waites KB. Prevalence of plasmid-mediated quinolone resistance. Antimicrob Agents Chemother 2003; 47: 559–62.
15.
Corkill JE, Anson JJ, Hart CA. High prevalence of the plasmid-mediated quinolone resistance determinant qnrA in multidrug-resistant Enterobacteriaceae from blood cultures in Liverpool, UK. J Antimicrob Chemother 2005; 56: 1115–17.
16. Corkill JE, Hart CA, McLennan AG et al. Characterization of a ß-lactamase produced by Pseudomonas paucimobilis. J Gen Microbiol 1991; 137: 1425–9.[Medline]
17. Ambler RP, Coulson AF, Frere JM et al. A standard numbering scheme for the class A ß-lactamases. Biochem J 1991; 276: 269–70.
18. Nuesch-Inderbinen MT, Kayser FH, Hachler H. Survey and molecular genetics of SHV ß-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12. Antimicrob Agents Chemother 1997; 41: 943–9.[Abstract]
19.
Corkill JE, Cuevas LE, Gurgel RQ et al. SHV-27, a novel cefotaxime-hydrolysing ß-lactamase, identified in Klebsiella pneumoniae isolates from a Brazilian hospital. J Antimicrob Chemother 2001; 47: 463–5.
20.
Corkill JE, Hart CA, Shears P. Plasmid mediated ceftazidime resistance associated with a ß-lactamase giving a slow nitrocefin reaction. J Antimicrob Chemother 1989; 24: 467–70.
21.
Kim J, Lim YM, Jeong YS et al. Occurrence of CTX-M-3, CTX-M-15, CTX-M-14, and CTX-M-9 extended-spectrum ß-lactamases in Enterobacteriaceae clinical isolates in Korea. Antimicrob Agents Chemother 2005; 49: 1572–5.
22. Hanson ND, Smith ME, Pitout JD. Enzymatic characterization of TEM-63, a TEM-type extended spectrum ß-lactamase expressed in three different genera of Enterobacteriaceae from South Africa. Diagn Microbiol Infect Dis 2001; 40: 199–201.[CrossRef][ISI][Medline]
23. Graham SM, Walsh AL, Molyneux EM et al. Clinical presentation of non-typhoidal Salmonella bacteraemia in Malawian children. Trans R Soc Trop Med Hyg 2000; 94: 310–4.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Kiratisin, A. Apisarnthanarak, C. Laesripa, and P. Saifon Molecular Characterization and Epidemiology of Extended-Spectrum- {beta}-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolates Causing Health Care-Associated Infection in Thailand, Where the CTX-M Family Is Endemic Antimicrob. Agents Chemother., August 1, 2008; 52(8): 2818 - 2824. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||