Time taken to travel to work by region of workplace: Great Britain, October to
3. Knowle: South-east Baptist Church 4 Windmill Hill: South-west Baptist Church.
4.4. Approach to sample groups:
4.5.2 Phase 2: Questionnaire
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central venous lines, arterial lines), total parenteral nutrition, recent surgery, haemodialysis, presence of decubitus ulcers, poor nutritional status, and heavy antibiotic use have been implicated as risk factor. Use of a variety of other antibiotic classes has been found to be associated with subsequent infections due to ESBL-producing organisms. Conversely, prior use of β-lactam/ β -lactamase inhibitor combinations, penicillins, or carbapenems seems not to be associated with frequent infections with ESBL-producing organisms.137 Other risk factors identified include poor functional status, liver disease, and the use of histamine2 receptor antagonists.139
2.6 DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASES
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ESBLs do not always increase MICs to levels characterized as resistant. These enzymes are usually less efficient at hydrolysis than their parent enzymes, and consequently their detection by currently used routine susceptibility tests is difficult.
Therefore, ESBL-producing Klebsiella spp. and E. coli may falsely appear to be susceptible to newer cephalosporins. Because current breakpoints forcephalosporin sensitivity are set for clinical efficacy, theyare too high to detect ESBL mutations;
therefore, there is clearly a requirement to detect the resistance mechanism itself ratherthan to rely on in vitro susceptibility testing.140
Methods of Detection
The detection methods can be divided into phenotypic and molecular methods.
Phenotypic methods:
The CLSI has published guidelines for ESBL detection in enterobacteriaceae specifically for E. coli, Klebsiella spp. and Proteus spp. In the UK, the Health Protection Agency (HPA) has also prepared guidelines. These guidelines include an initial screening with either 8 mg/L (CLSI) or 1 mg/L (HPA) of cefpodoxime, 1 mg/L each of cefotaxime, ceftazidime, ceftriaxone, or aztreonam, followed by confirmatory tests using both cefotaxime and ceftazidime in combination with clavulanate, or the E-test ESBL strips. Automated systems that use similar
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detection principles have proved to be popular in clinical laboratories, especially those in North America.141
Detection of ESBLs in E. coli and K. pneumoniae
There are two main approaches to detecting ESBLs in the clinical microbiology laboratory – screening tests and specific (or confirmatory) tests. The CLSI uses a screening test to detect ESBL producing organisms that is compliant with currently published standards. The screening test used by CLSI is based on detecting reduced susceptibility to any of the following indicator drugs: cefotaxime, ceftriaxone, ceftazidime, cefpodoxime, and aztreonam, either by disc diffusssion or standard broth microdilution method. Each Klebsiella pneumoniae, K. oxytoca, or Escherichia coli isolate should be considered a potential ESBL-producer if the test results are as follows:
Table 2.2 Zone of inhibition diameter and MIC limits for ESBL screening
Disk Diffusion MICs
cefpodoxime < 17 mm cefpodoxime > 8 µg/ml ceftazidime < 22 mm ceftazidime > 2 µg/ml
aztreonam < 27 mm aztreonam > 2 µg/ml cefotaxime < 27 mm cefotaxime > 2 µg/ml ceftriaxone < 25 mm ceftriaxone > 2 µg/ml
The sensitivity of screening for ESBLs in enteric organisms can vary depending on which antimicrobial agents are tested. The use of more than one of the five
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antimicrobial agents suggested for screening will improve the sensitivity of detection. Cefpodoxime and ceftazidime show the highest sensitivity for ESBL detection.142 Any isolate that is screen positive must be tested by a confirmatory test. The ESBL confirmatory test involves testing two indicator drugs, cefotaxime and ceftazidime, alone and in combination with clavulanate, either by disc diffusssion or standard broth microdilution method. If an ESBL is produced, clavulanate increases the size of the inhibition zone of at least one of the drugs by at least 5 mm, or in an MIC test it reduces the MIC by at least 3 doubling dilutions.
For all confirmed ESBL-producing strains, the test interpretation should be reported as resistant for all penicillins, cephalosporins (except the cephamycins, cefoxitin, and cefotetan), and aztreonam and, the infection control team should be notified accordingly. The CLSI quality control recommendations are that simultaneous testing with a non-ESBL-producing organism (Escherichia coli ATCC 25922) and an ESBL-producing organism (Klebsiella pneumoniae ATCC 700603) also be performed. 142
The phenotypic confirmatory tests are highly sensitive and specific compared to genotypic confirmatory tests. However, false positive confirmatory tests have been reported in Klebsiella pneumoniae or E.coli isolates which lack ESBLs but hyper-produce SHV-1. The coexistence of both ESBLs and plasmid-mediated AmpC-type β-lactamases in Klebsiella pneumoniae may result in false negative tests.
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AmpC-type β-lactamases resist inhibition by clavulanate and hence obscure the synergistic effect of clavulanate and cephalosporin against ESBL.126 The use of cefepime, a fourth-generation cephalosporin that is less rapidly inactivated by cephalosporinase than by ESBL, improves the detection of synergy with clavulanate when there is simultaneous stable hyperproduction of a cephalosporinase; alternatively, the cephalosporinase can be inactivated by performing phenotypic tests on a cloxacillin-containing agar. Some β-lactamases can hydrolyze both third-generation cephalosporins and carbapenems, such as the metallo-β-lactamases, which are not inhibited by clavulanate, but rather by EDTA.
The production of an ESBL masked by a metallo-β-lactamase can be detected by means of double inhibition by EDTA and clavulanate. Since extended-spectrum Ambler class D oxacillinases are weakly inhibited by clavulanate and not inhibited by EDTA, their detection is difficult in the routine laboratory.143
Other methods of ESBL detection, which are commercially available include:
Etest for ESBLs, Vitek ESBL cards, MicroScan panels, BD Phoenix Automated Microbiology System, Cephalosporin/clavulanate combination disks on Iso-Sensitest agar, Double-disk diffusion test, Agar supplemented with clavulanate, Disk replacement method, Three-dimensional test.139
Clinical laboratories from several countries, especially in Europe, have taken a more pro-active approach to the detection of ESBL-producing organisms. First-line
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screening tests in these countries are based on double disc synergy testing, combination discs and microdilution tests (with isolates grown in broth containing 1 mg/L of extended-spectrum cephalosporins). Second-line, confirmatory tests require determination of MIC by broth dilution of extended-spectrum cephalosporins with and without clavulanic acid or Etest ESBL confirmation tests.141
Molecular Methods:
There are a number of methods which can be used to characterize ESBLs. These methods, which have been used with varying degrees of success, include:
Iso-electric focusing, Beta-lactamase gene detection using specific DNA probes, PCR with gene specific oligonucleotide primers, Oligotyping, Gene Amplification and subsequent restriction enzyme analysis, PCR-single-strand conformation polymorphism
Ligase chain reaction, southern blot analysis, Nucleotide sequencing.144,145 PCR with gene specific oligonucleotide primers is the easiest and most common molecular methods, but will not discriminate among different variants of TEM or SHV.
Nucleotide sequencing remains the standard for determination of the specific ß-lactamase gene present in a strain. However, this too can give variable results
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depending on the method used.145 These techniques are available only in research centres and are beyond the scope of routine clinical microbiology laboratories.
Treatment of Infections Caused By Extended Spectrum Beta-Lactamases The third generation cephalosporins should not be used to treat infections with ESBL producing organisms because clinical outcome is poor even in the presence of apparent susceptibility.146 The drugs with the most reliable activity against ESBL producers include the fluoroquinolones, carbapenems (Imipenem, Meropenem or Eterpenem) and cephamycins such as cefoxitin. It should be noted, however, that resistance to the fluoroquinolones has been seen in some ESBL-producing Enterobacteriaceae and resistance to cefoxitin has emerged during therapy with this drug. This may leave only a carbapenem for therapy. There are no randomized controlled trials on therapy of infections with ESBL producing organisms.
The usual gold standard of evidence for determining optimal treatment of any type is the randomized controlled trial. Unfortunately, no trial exists which proves that one particular antibiotic is better than any other antibiotic for treatment of infections by ESBL producers. Therefore the decision as to which antibiotic to use for treatment of ESBL producers depends on either in-vitro susceptibility data, results from animal studies, data from observational studies of antibiotic use or sub-analyses of randomized controlled trials. Each of these four lines of evidence
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suggests that carbapenems are the treatment of choice for serious infections with ESBL producing organisms. This finding is consistent with the knowledge that carbapenems are stable to the hydrolytic effects of ESBLs.116 Possible alternatives include quinolones (such as ciprofloxacin) but the observed increase in quinolone resistance will limit their role in treatment options. Other possible alternatives are Tigecycline, colistin and Polymixin B. Nitrofurantoin and fosfomycin may be useful in uncomplicated urinary tract infections.147
Current literature supports the action of piperacillin–tazobactam against susceptible strains of ESBL-producing bacteria based on the structure–activity relationship between inhibitors and the ESBLs, as well as on recent clinical outcome studies,124 but there is paucity of clinical information on use of other β-lactam / β-β-lactamase inhibitor combinations for treatment of infections due to ESBL producing organisms. Aminoglycosides are not recommended as monotherapy for serious infections whether caused by ESBL producers or not.
Some isolates exhibit synergy between β-lactam antibiotics and aminoglycosides but this is unpredictable for any given strain and no clinical data exists favouring routine use of combination therapy for infections with ESBL-producing organisms.116 With all these uncertainties, the choice of definitive treatment is best guided by in-vitro sensitivity tests.
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2.7 PREVENTION AND CONTROL OF INFECTIONS BY ESBL