Effect of extended-spectrum ß-lactamases on the susceptibility of Haemophilus influenzae to cephalosporins

Stephen G. Tristram*

School of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250, Australia

Received 10 July 2002; returned 4 September 2002; revised 12 September 2002; accepted 25 September 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The extended-spectrum ß-lactamases (ESBLs) TEM-3, TEM-4 and TEM-5 were cloned into Haemophilus influenzae. These recombinant strains exhibited cefotaxime MICs of 0.5, 0.25 and 0.12 mg/L for TEM-3, -4 and -5, respectively, and the MIC of cefaclor was 4.0 mg/L. These MICs are higher than those of ß-lactamase-negative strains, or those producing simple wild-type TEM-1 ß-lactamase, but not high enough to be categorized as resistant according to the breakpoints of the NCCLS. The clones were also categorized as susceptible using NCCLS disc diffusion methodology and interpretive criteria. This study shows that current NCCLS susceptibility testing methods may have difficulty in detecting ESBLs if they were to occur in H. influenzae.

Keywords: extended-spectrum ß-lactamase, TEM ß-lactamase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The plasmid-mediated TEM-1 and TEM-2 ß-lactamases are the most common among Gram-negative bacteria, and have identical enzymic properties, even though TEM-2 has a Gln->Lys amino acid substitution at position 37 (numbered according to Sutcliffe1) due to a single base pair difference in the structural region of the gene.2 Another single base pair difference in the regulatory region of the blaTEM-2 gene results in a much stronger native promoter and the production of larger amounts of the enzyme.2 The blaTEM-1 and blaTEM-2 genes are known to be transposable, with the blaTEM-2 gene associated with transposon 1 (Tn1), and the blaTEM-1 gene associated with a number of transposons, including Tn2 and Tn3.3

It has been shown that the blaTEM-1 gene was probably transposed from plasmids in the Enterobacteriaceae on to cryptic plasmids already present in Haemophilus influenzae.4 Production of TEM-1 ß-lactamase is the most common mechanism for ampicillin resistance in H. influenzae, and has become increasingly common and widespread.57 The TEM-1 ß-lactamase does not produce cephalosporin resistance in H. influenzae, and although cephalosporin resistance does occur via other mechanisms, it is limited to earlier cephalosporins, such as cefaclor and cefuroxime.5,6,8,9 Resistance to the extended-spectrum cephalosporins, such as cefotaxime, has not yet been reported in H. influenzae.5,6,8,9

However, plasmid-mediated extended-spectrum ß-lactamases (ESBLs) and associated resistance to the third-generation cephalosporins have emerged in the Enterobacteriaceae as a result of mutations to the blaTEM-1 and blaTEM-2 genes.10,11 Therefore it might be possible for ESBLs to emerge in H. influenzae, either by similar mutations to the blaTEM-1 gene already in ß-lactamase-positive strains, or by transposition of modified blaTEM genes coding ESBLs from the Enterobacteriaceae.12,13

Problems with methodology and interpretive criteria resulted in problems with the detection of ESBLs in the Enterobacteriaceae,14 and the inherent problems associated with testing H. influenzae for susceptibility to ß-lactam antibiotics suggest that this may also be the case should ESBLs emerge in this organism.13,15,16

In order to determine the effect that ESBLs might have on the susceptibility to cephalosporins, three well-characterized ESBLs were cloned into H. influenzae. The ability of current NCCLS susceptibility testing methods and criteria to detect the resistance mechanism was determined by testing the recombinant strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and plasmids

Plasmids pGEM-3Z (Promega, Madison, WI, USA), pAT251, pAT266 and pAT268 (Institut Pasteur, Paris, France) were used as the sources of blaTEM-1, blaTEM-3, blaTEM-4 and blaTEM-5 genes, respectively.17,18 Plasmid pLS88 (kindly provided by P. J. Willson) is a 4.8 kb plasmid that replicates in both Escherichia coli and H. influenzae and carries a kanamycin resistance marker.19 This was used as a shuttle vector and host plasmid for various constructs produced during the study. Plasmids pLS88TEM-1, pLS88TEM-3, pLS88TEM-4 and pLS88TEM-5 were constructs produced during this study and consist of pLS88 with respective blaTEM genes inserted. E. coli JM109 (Promega) and H. influenzae Rd (kindly supplied by J. K. Setlow) were used as host strains.

Construction of pLS88 derivatives

The entire blaTEM-1, blaTEM-3, blaTEM-4 and blaTEM-5 genes, including promoter sequences, were PCR amplified from their respective plasmids and ligated into the pLS88 shuttle vector, as described by Balko et al.,20 and then used to transform E. coli JM109 competent cells (Promega) by standard methods. Transformants were selected by growth on Luria–Bertani agar containing 50 mg/L ampicillin and 30 mg/L kanamycin for the pLS88TEM-1 construct, and 1 mg/L cefotaxime and 30 mg/L kanamycin for the pLS88TEM-3, pLS88TEM-4 and pLS88TEM-5 constructs. H. influenzae Rd was subsequently transformed using the calcium chloride-induced artificial competence method with the various pLS88TEM constructs previously produced.21 The H. influenzae Rd transformants were selected on chocolate agar containing 30 mg/L kanamycin.

Validation of constructs and transformants

The presence of the appropriate pLS88TEM constructs was confirmed by plasmid extraction, EcoRI digestion and agarose gel electrophoresis, to demonstrate expected bands of 4.8 and 1.2 kb, corresponding to the pLS88 and TEM genes, respectively. The E. coli transformants were also tested for appropriate expression of the relevant ß-lactamase enzymes, using the double-disc synergy test described by Brun-Buisson et al.22 The TEM genes were sequenced using the ABI PRISM BigDye Terminator cycle sequencing kit on an ABI 377 automatic DNA sequencer (Applied Biosystems, Victoria, Australia). Sequencing was performed on the leading strand of double-stranded plasmid DNA, using the primers described by Chanal et al.23

The H. influenzae Rd host strain, and the subsequent transformants, were checked for production of functional ß-lactamase using nitrocefin touchsticks (Oxoid, Victoria, Australia).

Susceptibility testing of transformants

E. coli transformants were tested for susceptibility to ampicillin, cefotaxime and ceftazidime by macrobroth dilution, according to the recommended procedures of the NCCLS.24 Similarly, H. influenzae Rd transformants were tested for susceptibility to ampicillin, co-amoxiclav, cefotaxime and cefaclor by disc diffusion, and ampicillin, cefotaxime and cefaclor by broth dilution, according to the recommended procedures of the NCCLS.24,25


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results for the susceptibility tests of the various host strains and transformants are given in Tables 13.


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Table 1.  MICs for E. coli JM109 strains
 

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Table 3.  Disc diffusion results for H. influenzae Rd strains
 
The results of both the double-disc diffusion tests (data not shown) and broth dilution susceptibility tests performed on E. coli JM109 strains transformed with the various pLS88TEM constructs were consistent with the production of the appropriate TEM ß-lactamases. The identity of these ß-lactamases was also confirmed by the sequences of the respective blaTEM genes (data not shown).

The production of functional ß-lactamase in the H. influenzae transformants was confirmed by positive results with the nitrocefin hydrolysis test, which was negative in the non-transformed host strain.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The phenotypically expressed susceptibility of a particular bacterial strain to a ß-lactam antibiotic is brought about by a complex and dynamic combination of the permeability of the cell to the antibiotic, the presence, spectrum of activity and level of production of ß-lactamases, and the affinity of the antibiotic for the target site.26 In developing various pLS88TEM constructs in this study, it was important to consider some of these issues to ensure that any observed cephalosporin resistance in the transformants was as close to what might be expected should ESBLs emerge naturally in H. influenzae.

Plasmid copy number is known to affect both the amount of TEM ß-lactamase produced by some bacterial strains and the level of resistance expressed,27 so the influence of the copy number of the pLS88 constructs needed to considered. The similarity of ampicillin MICs for H. influenzae Rd transformed with pLS88TEM-1 to those seen for wild-type TEM-1 ß-lactamase-positive strains indicates that any differences in copy number of pLS88 derivatives and wild-type plasmids does not significantly alter the amount of ß-lactamase being produced or the level of resistance being expressed.

There are now over 100 TEM-derived ESBLs,28 and the rationale for selecting TEM-3, TEM-4 and TEM-5 for these initial cloning experiments was to use variants that might be likely to emerge in H. influenzae naturally, and also to use variants that have particular clinical relevance. The three ESBLs chosen were amongst the first TEM variants to emerge naturally in the Enterobacteriaceae,10 and are also some of the most frequently seen,29 which might represent a significant gene pool for transfer to H. influenzae. TEM-4 and TEM-5 have a Gln at position 37 and also have the mutation in the regulatory region to give the TEM-2 type promoter,10 both of which would be expected to occur should the ESBLs emerge from mutation of TEM-1 in situ in H. influenzae. Both TEM-3 and TEM-4 are primarily cefotaximases, which is relevant since cefotaxime is used widely in the treatment of invasive infections with H. influenzae.7 Cefaclor is used widely to treat respiratory infections, including those caused by H. influenzae,7 but little is known about the activity of various ESBLs on this antibiotic. TEM-5, which has greater activity against ceftazidime, was included in this study in order to determine whether ESBLs with greater activity to cefotaxime or ceftazidime would produce significantly different levels of resistance to cefaclor when expressed in H. influenzae.

The observed cephalosporin MICs were significantly lower for the ESBL-producing H. influenzae transformants compared with the respective E. coli transformants. This was expected since the outer membrane of the Enterobacteriaceae is known to be a significant barrier to the penetration of ß-lactams into the cell, and this slow access allows the periplasmically located ß-lactamase to hydrolyse the antibiotic efficiently before it reaches the target.30 In contrast, the outer membrane of H. influenzae provides very little resistance to the penetration of ß-lactams, and the hydrolytic capacity of the ß-lactamase is more easily overwhelmed, allowing unhydrolysed antibiotic to reach the target, and thus the ß-lactam MICs are comparatively lower.30

Although the various H. influenzae Rd strains expressing ESBLs used in this study all exhibited increased MICs of both cefotaxime and cefaclor, none of them would be classified as resistant on the basis of current NCCLS breakpoints.24 In addition, all strains also tested as susceptible by NCCLS disc diffusion testing.25 However, the fact that the MICs of various cephalosporins for these strains are lower than the current NCCLS breakpoints does not necessarily indicate that such resistance would not be clinically relevant.

First, many of the ESBLs in the Enterobacteriaceae fail to raise the MIC of some third-generation cephalosporins above the breakpoint, yet the presence of the enzymes is still considered to indicate clinical resistance.10,11 This is due, at least in some part, to the inoculum effect, where the level of resistance rises as the inoculum rises,10,11 and since the inoculum effect has also been shown to occur in TEM-1-producing strains of H. influenzae,20 a similar situation may occur with ESBL-producing strains. In addition, given that the breakpoints for H. influenzae and the third-generation cephalosporins were established in the absence of any resistant strains, their value in determining the clinical relevance of new resistance mechanisms is questionable, particularly in the light of experience with the emergence of ESBLs in the Enterobacteriaceae. On the other hand, resistant strains were available for the determination of the cefaclor breakpoints, but again the relevance of these breakpoints to the possible emergence of ESBLs in H. influenzae is uncertain as the resistance mechanism in existing resistant strains is due to altered penicillin-binding proteins and not the production of ß-lactamases.6 Furthermore, NCCLS breakpoints are established on a number of criteria, including the level of antimicrobial in the blood following usual dosage, and their suitability for infections in other tissues has been questioned.31 For example, the mean cefaclor concentration in lung secretions following a standard dose has been reported to be as low as 0.4 mg/L,32 yet the NCCLS breakpoint for sensitive strains is <=8.0 mg/L.24 The actual breakpoints for cefaclor have also been questioned for cases of acute otitis media caused by H. influenzae, because of high treatment failure rates for strains with cefaclor MICs as low as 1.0 mg/L.33

In summary, this study has shown that the production of ESBLs will reduce the susceptibility of H. influenzae to both cefotaxime and cefaclor, although the clinical significance of the reduced susceptibility is unclear. The study also shows that current NCCLS susceptibility testing methodologies would be unlikely to detect the emergence of ESBLs in H. influenzae. Further experimental work in this area is warranted, particularly the development of a sensitive screening test and subsequent sentinel surveillance programme to search for ESBLs in H. influenzae.

The generalization, ‘Where they have been found largely reflects where they have been looked for’,11 was apt for ESBLs in the Enterobacteriaceae, and may well prove to be equally apt for H. influenzae.


    Acknowledgements
 
pLS88 was kindly supplied by Dr Philip J. Willson (Veterinary Infectious Disease Organization, Saskatchewan, Canada), pAT251, pAT266 and pAT268 were kindly supplied by the Institut Pasteur (Paris, France) and the H. influenzae Rd was kindly supplied by Dr Jane K. Setlow (Brookhaven National Laboratory, Upton, New York). The expert technical advice of Dale Kunde is gratefully acknowledged. This work was supported by a grant from the Clifford Craig Medical Research Trust, Launceston, Tasmania.


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Table 2.  MICs for H. influenzae Rd strains
 

    Footnotes
 
* Tel: +61-3-63-243323; Fax: +61-3-63-243658; E-mail: Stephen.Tristram{at}utas.edu.au Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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33 . Dagan, R., Abramson, O., Leibovitz, E., Greenberg, D., Lang, R., Goshen, S. et al. (1997). Bacteriologic response to oral cephalosporins: are established susceptibility breakpoints appropriate in the case of acute otitis media? Journal of Infectious Diseases 178, 1253–9.