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In this article we will discuss about the Introduction and Categories of Antimicrobial Susceptibility Test.
Introduction to Antimicrobial Susceptibility Test:
One of the prime duty of clinical microbiology laboratory is to undertake antimicrobial susceptibility test(s) if an isolated bacteria is found to be pathogenic and whose antimicrobial susceptibility pattern cannot be predicted empirically. Aim of these tests is to determine the phenotypic property of resistance or sensitivity profile of the isolate to help clinician to prescribe suitable antimicrobial agent(s).
In addition to this routine practice this is done for research purposes, e.g. surveillance of antimicrobial agent susceptibility of bacteria causing diarrhoeal disease (V. cholerae, E.T.E.C.*, diarrhoea- genic serovars of Salmonella etc.)
Categories of Antimicrobial Susceptibility Test:
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(a) Sensitive:
Ordinary recommended dose achieve a systemic concentration which is > M.I.C.**of the antimicrobial agent tested against the isolate.
(b) Intermediate sensitive:
Isolate is amenable to treatment with a larger dose of the antimicrobial agent, so that concentration at the site of infection is above the breakpoint value to tackle with high M.I.C. value of the isolate.
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(c) Resistance:
M.I.C. value of the isolate is so high that the required concentration is unachievable at the site of infection.
Methods:
A. Tube dilution method
B. Agar dilution method
C. Disc diffusion method:
(a) Kirby Bauer Method
(b) Stake’s Method
(A) Tube dilution method:
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Principle:
Ability (i.e. resistance) or inability (i.e. sensitive) of the test bacterium to grow in tubes containing broth medium with serial dilution of a given antibiotic, is noted to determine M.I.C. value. If M.I.C. > breakpoint value, the test organism is considered resistant.
Method:
1. Medium:
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A number of tubes with nutrient broth are taken. Serial dilutions of antibiotic to be tested are to be prepared in broth. The concentrations are determined according to breakpoint*** value of the antibiotic. For an initial test some tubes are adjusted above the breakpoint value and others below. One tube is kept aside as control without antibiotic.
2. Inoculum:
One loopful of overnight broth culture of the isolate to be tested.
3. Incubation:
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37°C for overnight period is the usual method. If ‘time killing curve is desired, serial subculture is to be done at hourly or ½ hourly interval.
4. Reading:
(a) Growth in control tube validates the result.
(b) Tubes where there is growth or no growth are noted.
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(c) The highest concentration where growth occurs and lowest concentration where no growth occurs are noted.
Concentration in between these two values is the M.I.C. value of this antimicrobial agent for the isolate tested.
5. Interpretation:
Resistance — MIC value > Breakpoint value
Sensitive — MIC value < Breakpoint value
Advantages:
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1. An accurate method for MIC determination.
2. Mutants if grown in between tubes showing no growth could be easily recognised.
3. Compounds of high molecular weight and poorly diffusible in agar plates could be tested.
4. Whether the agent is bacteriostatic or bactericidal, could be determined by subculture from tubes showing no growth.
Disadvantages:
1. A cumbersome and laborious test method.
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2. Mutants grown in tube adjacent to the tube showing growth of the bacteria under test could not be recognised. This may lead to false interpretation of MIC value.
3. Unsuitable method for clinical microbiology laboratory, where large number of organisms are to be tested.
4. Fastidious organism could not be tested.
(B) Agar dilution method:
Principle:
Ability (i.e. resistance) or inability (i.e. sensitive) to grow, in Muller Hinton Agar plates containing different concentrations of a given antibiotic, is noted.
Method:
1. Medium:
M.H.A. plates are to be poured with different concentrations of antibiotic to be tested. Like tube dilution method, concentrations of antibiotics are adjusted at above and below the breakpoint value. One plate is incorporated without antibiotic as control.
2. Inoculum:
With loop of internal diameter 1 mm, from young broth culture adjusted to 0.5 Macfarland standard, inoculums are lifted and spot inoculation is done.
3. Incubation:
37°C for overnight period.
4. Reading:
i. Growth on the plate without antibiotic validates the result.
ii. Spots showing no growth or growth of up to 3 colonies, indicates susceptibility at that concentration.
iii. Spots showing growth of more than 3 colonies indicates resistance at that concentration.
5. Interpretation:
Resistance — M.I.C. value > Breakpoint value
Sensitive — M.I.C. value < Breakpoint value
Advantages:
1. A number of isolates could be tested in a single test.
2. M.I.C. value could be determined.
3. Any mutant or contaminants developed in inoculum, could be recognised immediately.
Disadvantages:
(1) Only one antimicrobial agent could be tested in a single set of test.
(C) Disc diffusion method:
(a) Kirby Bauer Method:
Principle:
Production of inhibitory zone of a recommended size as recommended in Kirby Bauer Chart in a lawn culture of an isolate around an antimicrobial agent impregnated filter paper disc indicates sensitivity.
Medium:
Nature of the medium affects the result of susceptibility test. A totally defined medium which support reproducible result is yet to be formulated with satisfaction.
Factors related to the media affecting the result:
i. Quality of agar — affects diffusibility of antimicrobials.
ii. Quality of protein hydro-lysate — Varies from batch to batch affecting the result.
iii. Final pH of the stored medium — affects the action of antimicrobial.
iv. Cationic (Mg++ and Ca++) concentration — affects the result.
v. Presence of thymidine and thymine at higher concentration antagonizes the action of antimicrobials (sulfonamides) which are inhibitors of folate metabolism.
vi. Hydration, unless optimum, might influence the diffusibility of antimicrobials.
Considering the above facts, it has been recommended by CLSI, the international agency for quality control of antimicrobial susceptibility test, to use ‘Mueller-Hinton Agar’ in Kirby-Bauer method for non-fastidious bacteria.
Media recommended for non fastidius bacteria:
(a) N. gonorrhoeae:
G.C. Agar (Difco) supplemented with 10% (final concentration) saponin lysed horse/sheep blood.
(b) Fastidious anaerobes:
Wilkin-Chalgren Agar (Oxoid) supplemented with 5% (final concentration) saponin lysed horse/sheep blood.
(c) Other fastidious bacteria:
Isosensitest agar (oxoid) supplemented with 5% saponin lysed horse/sheep blood and 10 mg/L nicotinamide adenine dinucleotide (NAD).
Inoculum:
Inoculum is to be prepared from pure culture. Its concentration affects the result. A heavy inoculum may cause reduction of zone size and indistinctness of margin of the zone. Optimum inoculum should produce, evenly distributed semi- confluent colony after overnight incubtion.
Preparation of inoculums:
Bacteria is picked up by a loop touching the tip of similar looking 5 colonies. A suspension is to be made in peptone water. The opacity is adjusted to Macfarland opacity tube 0.5. This will produce a concentration of 1.5 x 108/ml bacteria. Macfarland 0.5 capacity tube is prepared by mixing 0.5 ml concentrated H2SO4 with 99.5 ml of saturated solution of BaCl2.
Choice of disc:
Choice of disc depends on the following considerations:
1. Identity of the organism isolated
2. Prevalent resistance patterns and antibiotic prescription policy of the hospital.
3. Site of infection
4. Method used
Organisms isolated:
Antimicrobials to which the organism is intrinsically resistant are excluded from the test, e.g. Vancomycin in gram -ve isolates. Similarly, the same consideration may be applied for the antimicrobials, specifically developed for a particular bacteria but not others, e.g. ceftazidime for use against P. aeruginosa but not against staphylococcus.
Prevalent resistance patterns:
Routine testing is not warranted for an antimicrobial, against which resistance is quite prevalent across the isolates. If gram -ve rods commonly shows resistance to cephalexin, it could be omitted from test battery. On similar ground more potent agents need not be tested.
Site of infection:
1. Nitrofurantoin to be applied for urinary isolated, as it is not effective elsewhere.
2. Urinary isolates need high potency discs.
Method used:
Cefotaxime could be tested by tube dilution only, so should be omitted in disc diffusion method.
Method:
1. Preparation of Muller Hinton Agar plate — 4 mm thick solidified medium in 90 mm diameter petri plates. It is properly dried before use. Medium older than 7 days, even if properly preserved should not be used. Medium should not contain too much water or be too dry.
2. Inoculum size — young culture in liquid medium containing 1.5 x 108 bacteria, matching Macforland opacity tube 0.5 standard.
3. Inoculation on plate:
A sterile swab soaked in the inoculum is streaked evenly on all parts of agar surface. Inoculum is allowed to dry before application of disc.
4. Application disc:
Using flamed forceps, discs are lightly pressed on to the surface of the agar plates. A 90 mm diameter plate accommodates 5 discs at periphery and one at centre. It is desirable discs should be placed 20 mm apart.
5. Pre-incubation:
Next plates are kept for ½ hour in refrigerator at 8-10°C for pre-diffusion of antibiotic in medium.
6. Incubation:
Incubation is done at 35°-37°C for 18-24 hours in humidified air.
Interpretation:
Zone of inhibition is measured using caliper or template. Noted size is compared with Kirby-Bauer interpretative chart. Result is expressed as sensitive, intermediate sensitive or resistant as the interpretative chart dictates.
Quality assurance:
The result of Kirby Bauer method of disc diffusion test may be influenced by the following factors leading to variations of inhibitory zone size:
(i) Medium:
Composition, hydration status, thickness of the medium influence the zone size.
(ii) Disc:
Thickness and size of the disc affects its absorptive powder, consequently the concentration of antibiotic too.
(iii) Inoculum size:
More the size of the inoculum, less is the size of inhibitory zone and vice versa.
(iv) Incubation period and pre-incubation:
If pre-incubation time is shortened, zone size is reduced. Over incubation reduces the zone size too and development of mutant.
To overcome these factors, the following measures are undertaken in clinical microbiology laboratory:
(i) Medium:
Muller Hinton medium of 4 mm thickness and preferably used within 7 days of preparation.
(ii) Disc:
6 mm diameter discs are to be used punched out from Whattmann No.1 thickness filter paper disc for preparation of antibiotic impregnation. Concentrations of disc is determined according to CLSI guideline.
(iii) Inoculum size:
An young culture in liquid medium matching turbidity standard of Macfarland 0.5 (equivalent to 1.5 x 108 bacteria/ml) is to be used for preparation for a semi-confluent lawn.
(iv) Pre-incubation and incubation:
½ hr. pre-incubation followed by overnight incubation is the guideline.
(v) Check for the newly procured or prepared medium and discs:
Using reference strains, disc and medium is checked regularly for the recommended zone range according to CLSI guideline. If the experiment fails, the medium or disc batch is to be discarded.
(vi) External quality control — in collaboration with reference centre of repute.
(b) Stoke’s Method:
Stoke’s method of disc diffusion test for antimicrobial sensitivity test overcomes many of the questions raised regarding quality assurance, accuracy and reproducibility of result in Kirby Bauer’s Method.
In Stoke’s Method, inbuilt control measures are incorporated for quality assurance. Here in the same plate both control (E. coli NCTC 10418 for rapid growing gram -ve isolates, S. aureus NCTC 6571 for gram +ve non-fastidious cocci) and test strains are inculated as shown in the Fig. 20.1.
Method:
1. Inoculum preparation:
The same method as is described in Kirby Bauer’s Method is followed both for test and control strain.
2. Inoculation:
Inoculation done with swab on M.H. Agar plate to get semi-confluent colony as shown in Fig. 20.1. A narrow zone of uninoculated area should be kept to keep apart the control and test bacteria.
3. Placement of disc:
Antibiotic inpregnated discs of recommended concentration are placed over the uninoculated line in such a way, so that disc margins just covers a short area of both inoculated zones. In 90 mm diameter plate, it is recommended that 4 discs could be placed.
4. Incubation:
Pre-incubation at 10°C in refrigerator for V2 hour is to be followed by incubation at 35°-37° for overnight period.
5. Interpretation of result (Fig. 20.2):
Sensitive:
(1) Inhibitory zone of test is equal to that of control (A).
(2) Inhibitory zone is of test is greater than control (B).
(3) Inhibitory zone diameter of test is lesser than control but not more than 3 mm (C).
Resistent:
Inhibitory zone diameter of test is lesser than control by more than 3 mm (D).
Advantage:
Both tests and control bacteria are tested on same medium, using same disc and environment. As the control bacteria is sensitive to all the discs, a comparison of inhibitory zone diameter of test yields a quality assured report.
Disadvantage:
Only 4 antibiotics could be tested in a single plate. If 3 discs are applied on the line, the inhibitory zone diameter might overlap.
Some areas of difficulties in disc diffusion test:
(i) Test to detect Methicillin sensitivity— requires special medium (Columbia agar containing 5% v/v horse/sheep blood with NaCl 2%), 5 μg methicillin disc (freshly prepared) and incubation at 30°C. As methicillin is not stable for a long time, alternatively Oxacillin (2 μg) disc may be used. Recent reports shows Cefoxitin (30 μg) disc may be used as a surrogate marker on M.H. Agar media to detect methicillin resistance.
(ii) Tests to detect ‘Extended Spectrum β-Lactamase (ESBL)’ producing bacteria:
ESBLs, found in a wide range of Gram-negative rods, are plasmid mediated enzymes capable of hydrolyzing and inactivating a wide variety of β-lactams, including 3rd generation cephalosporin’s, penicillins and aztreonam, but sensitive to cephamycin (cefoxitin).
These enzymes are the result of mutations of TEM-1 and TEM-2 and SHV-1. Widespread use of 3rd generation cephalosporin’s and aztreonam is believed to be the major cause of mutation in these enzymes that has led to the emergence of ESBLs.
These enzymes mediate resistance to β-lactam antibiotics containing an oxyimino-group such as oxyimino-cephalosporins (e.g. ceftazidime, ceftriaxone, cefotaxime) as well as oxyiminomonobactam (aztreonam) but have no detectable activity against cephamycins and carbapenems.
Because of greatly extended substrate range, these enzymes were called extended spectrum β-lactamases. Generally, they are inhibited by β-lactamase-inhibitors such as clavulanate and tazobactam.
ESBL is very frequently detected in K. pneumoniae, K. oxytoca and E. coli and infrequently in Enterobacter spp, Salmonella spp, Morganella morganii, Proteus mirabilis, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter spp, Burkholderia cepacia and Alcaligenes fecalis.
Classical ESBLs have evolved from the widespread plasmid-encoded enzymes families TEM (> 130 type), SHV (> 50 type), cefotaxime (CTX-M) and oxacillin (OXA). New ones are being found every week.
Treatment failure rate is high and may exceed 90% when cephalosporins were used for serious infections caused by ESBL producing organisms, particularly when the MICs for used cephalosporins are elevated (e.g., 4 or 8 μg/ml) but are still within susceptible range.
Methods of detection:
Different ESBL types express variable levels of resistance to third generation cephalosporine. So, routine clinical microbiology laboratory employs ESBL detection methods which are sensitive enough to recognize the level of resistance that would be achieved by the situation given in vivo.
(A) Screening Test:
Initial screening, by disc diffusion test using cefpodoxime, cefotaxime, ceftriaxone, ceftazidime or aztreonam to note reduced susceptibility, if any, is done.
Next, phenotypic confirmatory test is recommended. As proposed by the CLSI, M100-S-16 document suggests the use of more than one of the five indicaor cephalosporins will improve the sensitivity of detection. But, if it is necessary to rely on a single screening substance, ceftazidime or cefpodoxime is suggested as the best choice.
The breakpoint zone diameter used in this study for ESBL, producers, as per CLSI guidelines (2007) is shown below in the table 20.1.
Note that the above criteria is different from Kirby-Buer interpretative inhibition zone criteria as depicted below (see table 20.2).
(B) Phenotypic test:
(a) Double Disc Synergy (DDS):
The Jarlier double disc approximation of double disk synergy (DDS) was the first detection test described in 1980’s. DDS is a disc diffusion test in which 30 mg antibiotic discs of ceftazidine, ceftriaxone, cefotaxime and aztreonam are placed on the plate, 30 mm (center to center) from the amoxicillin/clavulanate (20 μg/10 μg) disc.
A clear extension of the edge of the antibiotic’s inhibition zone towards the disk containing clavulanate is interpreted as synergy, indicating the presence of an ESBL (Fig. 20.3 & Fig. 20.4). The use of cefpodoxime as the expanded spectrum cephalosporin of choice has been suggested as evaluation of DDS has shown sensitivities and specificities ranged from 79% to 97% and 94% to 100% respectively in two reports.
False negative results have been observed with isolates harboring SHV-2, SHV-3 and TEM-12. In isolates which are suspicious for harboring ESBLs but are negative using the standard distance of 30 mm between disks, the test may be repeated with closer (e.g., 20 mm) or more distant (e.g. 40 mm) disks.
The test remains a reliable, convenient and inexpensive method of screening for ESBLs. However, the interpretation of the test is quite subjective. Sensitivity may be reduced when ESBL activity is very low leading to wide inhibition zones around the cephalosporin and aztreonam.
The disc on the left is cefotaxime (30 μg); the disc in the centre is coamoxyclav (20+10 μg); the disc on the right is ceftazidime (30 mg). Note the expansion of the zones around the cefotaxime and ceftazidime discs adjacent to the coamoxyclav. The organism is E. coli with a TEM-24 enzyme.
Principle:
It is based on the resistance that ESBLs confer to oxyimino-beta-lactams (cefotaxime, ceftriaxone and ceftazidime) and the ability of a beta lactam inhibitor, usually clavulanic acid, to block this resistance.
Materials required:
(1) Disks of ceftazidime (30 μg); cefotaxime (30 μg); ceftriaxone (30 μg).
(2) Disk of amoxycillin/clavulanic acid (20 μg/ 10 μg).
Procedure:
A 0.5 McFarland turbidity matched of test isolate was swabbed on Mueller-Hinton agar plate and 30 mg antibiotic disks of ceftazidime or ceftriaxone or cefotaxime were placed on the plate, 30 mm (center to center) from the amoxicillin/clavulanate (20 μg/10 μg) disk. The plate was incubated at 35°C for 18-24 hours.
Inference:
A clear extension of the edge of ceftazidime or ceftriaxone or cefotaxime inhibition zone towards the disk containing clavulanate is interpreted as synergy, indicating the presence of an ESBL.
(b) Combined disk method (Fig. 20.5):
Cephalosporin/clavulanate combination disks are used. The CLSI recommened use of cefotaxime (30 μg) or ceftazidime (30 μg) disks with or without clavulanate for phenotypic confirmation for the presence of ESBLs in Klebsiella and E.coli. The CLSI recommends that the disk test performed with semi confluent growth on Mueller-Hinton agar.
A difference of ≥ 5 mm between the zone diameters of either of the cephalosporin disks and their respective cephalosporin/clavulanate disk is considered to be phenotypic confirmation of ESBL production. The use of both antibiotic disks is advisable since the use of ceftazidime alone has resulted in the inability to detect CTX-M-producing organisms.
Principle:
It is an inhibitor potentiated disk diffusion method where the combination of cephalosporin and clavulanic acid shows an increase inhibitory zone diameter by ≥ 5 mm or 50% than that of cephalosporin alone.
Materials required:
(a) Disk of ceftazidime, Ca (30 μg).
(b) Disk of ceftazidime + clavulanic, Cac (30 μg/ 10 μg).
Procedure:
A disk of ceftazidime (30 μg) alone and a disk of ceftazidime + clavulanic acid (30 μg/10 μg) were used for this test. Both the disks were placed independently, 30 mm apart, on a lawn culture of 0.5 McFarland opacicity of the test isolate on Mueller Hinton Agar (MHA) plate and incubated for 18-24 hours at 35°C.
Inference:
An increase of ≥ 5 mm or 50% in zone inhibition diameter around the ceftazidime/clavulanic acid in comparison to ceftazidime alone confirmed ESBL production.
(c) MIC reduction test:
Phenotypic confirmatory testing can also be performed by broth micro-dilution assays using ceftazidime (0.25 to 128 mg/ml), ceftazidime plus clavulanic acid (0.25/4,128/4), cefotaxime (0.25 mg to 64 mg/ml) and cefotaxime plus clavulanic acid (0.25/4 to 64/4).
The use of both antibiotics is recommended. The test is done using standard methods. Phenotypic confirmation is considered as > 3-two-fold-serial-dilution decrease in MIC of either cephalosporin in the presence of clavulanic acid to its MIC when used alone.
Materials required:
(a) Antimicrobial powder (ceftriaxone, ceftriaxone-tazobactam, ceftazidime and ceftazidimetazobactam).
(b) Sterile distilled water.
(c) Nutrient agar test tubes (each of 15 ml).
(d) Micro-pipettes (50 μl, 100 μl, 500 μl, 1000 μl).
Procedure:
1. Stock solution preparation of ceftazidime:
i. Weight 54.4 mg of ceftazidime and dissolve in 2 ml of sterile D.W.
ii. To achieve a concentration of 27200 μg/1000 μl.
2. Working solution preparation and pouring of plates:
Make 2 sets of such plates. In the second set before pouring plate add Clavulanic acid to each tube achieve a final concentration of 4 μg/ml.
Thus in 1st set conc. of Ceftazidime range from 2- 128 μg/ml; in the 2nd set the conc. of ceftazidime/ clavulanic acid range from 2/4 to 128/4 μg/ml.
After the plates were set, they were further dried in an incubator at 35C for 20-30 minutes. The plates were divided by a market pen on the lids for the desired number of isolates. In a 90 mm plate, a total of 25 isolates could be inoculated.
The test strains were spot inoculated with a wire loop calibrated to deliver 0.001 ml and the final inoculum on the agar surface should be approximately 10 colony forming unit per spot. A control nutrient agar plate without antibiotics was inoculated with the test strains. The test and the control plates were incubated at 35 C for 18-24 hours.
Inference:
The antibiotic concentration of the plate showing ≥ 99% inhibition is taken as the MIC for the organism. Hazy growth and one or two colonies were ignored and considered as mutants. Growth of more than three colonies were taken as resistant.
A minimum of ≥ 3 fold reduction in MIC of these strains when tested in combination of ceftriaxone-tazobactam or ceftazidime-tazobactam as compared to MIC for ceftriaxone or ceftazidime alone, confirmed that the strains were ESBL producer.
(d) Etest ESBL strip (Fig. 20.6):
The Etest ESBL strip is a two-sided strip in which clavulanate is added to one side of a dual oxyiminobeta lactam gradient looking for a reduction in the MIC of cephalosporin’s of more than three fold in the presence of clavulanate.
The availability of cefotaxime as well as ceftazidime strips improves the ability to detect ESBL types which preferentially hydrolyse cefotaxime such as CTX-M-types enzymes. This method is useful for both screening and phenotypic confirmation of ESBL production. Sometime due to weak enzyme production, indeterminate results may be obtained.
The reported sensitivity as a phenotypic confirmatory test for ESBL is 87 to 100% and specificity is 95 to 100%. The test is limited by its indeterminate results, difficulties in recognizing subtle zone deformities and cost.
(e) The automated ESBL microbial susceptibility test system:
The automated antimicrobial susceptibility test systems (Vitek, MicroScan and BD phoenix) also produce ESBL test.
1. The Vitek ESBL test utilizes cefotaxime and ceftazidime alone and in combination. A predetermined reduction in the growth of the cefotaxime or ceftazidime wells containing clavulanate, compared with the level of growth in the well with cephalosporin alone indicates a positive test. Sensitivity and specificity of the method exceed 90%.
False-negative results have been observed in Klebsiella pneumoniae isolates producing both an ESBL and AmpC-type beta lactamase. Klebsiella oxytoca strains hyper-producing the K1 β-lactamase will usually be recorded as positive on the Vitek ESBL test.
2. The Micro Scan panels which contain combinations of ceftazidime or cefotaxime plus β-lactamase inhibitors have appeared highly reliable.
3. The Phoenix ESBL test uses growth response to cefpodoxime, ceftazidime, ceftriaxone and cefotaxime with or without clavulanate to detect the production of ESBLs. The results are usually available within six hours.
The Phoenix ESBL method detects ESBL production in greater than 90% of strains genotypically confirmed to produce ESBL. The method correctly detects ESBL production by Klebsiella, E. coli, Enterobacter, Proteus and Citrobacter spp.
Molecular Methods:
For identification of specific ESBL expressed by a clinical isolate, the following molecular detection methods can be applied:
(a) Iso-electric focusing:
Gives a presumptive identification since many of them possess identical isoelectric points.
(b) Specific DNA probes:
Labour intensive. Applicable for TEM and SHV enzymes.
(c) PCR with oligonucleotide primers oligotyping:
For PCR with oligonucleotide primers, several molecular methods that will aid in the detection and differentiation of ESBLs without sequencing have been suggested. However, PCR will not discriminate among different variants of TEM or SHV.
These probes are less sensitive for the detection of mutations which are responsible for the extended substrate range. In some cases these mutations lead to the appearance or disappearance of restriction sites.
(d) PCR followed by restriction fragment length polymorphism analysis (RFLP):
Amplification of the relevant part of the gene by PCR followed by restriction enzyme analysis can thus indicate the presence or absence of specific TEM or SHV derived ESBLs.
(e) PCR-single-strand conformation polymorphism (PCR-SSCP) has also been applied to the study of ESBLs with satisfactory results. This method has been used to detect a single base mutation at specific location within the beta-lactamase gene. The combination of PCR-SSCP with PCR- restriction fragment length polymorphism (PCR- RFLP) allows the identification of newer SHV variants.
(f) Ligase chain reaction:
The ligase chain reaction (LCR) is used for the identification of SHV genes. LCR allows the discrimination of DNA sequences that differ by a single base pair.
These techniques are available only in research centres and are beyond the scope of routine clinical microbiology laboratories.
Regarding the reporting of ESBL producing isolates, the microbiology laboratory report should state that the strain produced ESBL and should be considered resistant to all penicillins, cephalosporin’s and aztreonam.
(iii) Tests to detect ‘AmpC-β-lactamase’ producing bacteria:
(a) AmpC Disk Test (Figs. 20.7A&B) :
AmpC class β-lactamase is a cephalosporinase, substrate range of which includes third generation of cephalosporins (as E.S.B.L) including cephamycin and poorly inhibited by clavulanic acid.
AmpC-β- lactamase are encoaded by genes located both in plasmid and chromosome. It is demonstrated in K. pneumoniae, E. coli, Salmonella spp. Proteus mirabilis, Citrobacter freundii, Enterobacter spp, Acinetobacter spp and Pseudomonas spp.
Principle:
A known sensitive strain shows blunting or indentation of flattening of the inhibitory zone around the Cefoxitin (30 mg) disc in response to placement of a sterile blank filter paper (6 mm in diameter, Whatman filter paper No.2) inoculated with several colonies of AmpC-β-lactamase.
Materials required:
(a) E.coli ATCC 25922
(b) Cefoxitin disk (30 μg)
(c) Peptone water
(d) Muller Hinton Agar plate
(e) Micro-pipette
(f) Sterile blank filter paper disc (6 mm in diameter, Whatman filter paper No.2)
Procedure:
A lawn culture of E. coli ATCC 25922 was prepared on MHA plate. Sterile disks (6 mm) were moistened with sterile saline (20 μl) and inoculated with several colonies of test organism. The inoculated disc was then placed beside a cefoxitin disk (almost touching) on the inoculated plate. The plates were incubated overnight at 35°C.
Inference:
A positive test appeared as a flattening or indentation of the cefoxitin inhibition zone in the vicinity of the test disc. A negative test had an undistorted zone.
(b) Modified three dimensional test (Fig. 20.8):
Principle:
A known sensitive strain shows blunting or indentation or flattening of the inhibitory zone around the Cefoxitin (30 mg) disc in response to addition of culture filtrate of AmpC β-lactamase producing strains.
Materials required:
(a) E. coli ATCC 25922
(b) Cefoxitin disk, (30 μg)
(c) Peptone water
(d) Mueller Hinton Agar plate
(e) Micro-pipette (50 μl)
(f) No. 15 surgical scapel blade with handle
Procedure:
The suspected isolate of AmpC β-lactamase producer from MHA plate was suspended in peptone water and incubated overnight at 35°C. The growth in the peptone water was then centrifuged at 3000 rpm for 15-20 minutes and the supernatant was used as a crude enzyme extract for the test.
Lawn culture of E. coli ATCC 25922 was prepared on MHA plates and cefoxitin (30 μg) disk were placed on the plates. Linear slits (3 cm) were cut using sterile surgical blade, 3 mm away from cefoxitin disk.
At the other end of the slit a small circular well was made and the enzyme extract was loaded. A total of 30 μl of extract was loaded in the well at a 10 μl increment. The plates were kept upright for 5 to 10 minutes until the liquid dried and were incubated at 35°C for 18-24 hours.
Inference:
Enhanced growth of the surface organism at the point where the slit inserted the zone of inhibition of cefoxitin was considered a positive three-dimensional test and was interpreted as evidence for the presence of AmpC β-lactamases.
Three different results were recorded. Isolates showing clear distortion of the zone of inhibition were considered as AmpC producers. Isolates showing minimal distortion were taken as intermediate strains and isolates with no distortion as negative strains.
Detection of ESBL in presence of AmpC β-lactamases:
For the detection of the co-existence phenotype of both ESBL and AmpC, a double disk synergy test (DDST) using a cefepime disk (30 μg) separated by a distant of 20 mm (center to center) from a amoxycillin/clavulanate disk (20 μg/10 μg) was performed.
After incubating at 35°C for 18-24 hours, an increase in zone of inhibition of cefepime towards the disk of amoxycillin-clavulanate was considered positive for ESBL production.
(iv) Tests to detect ‘Metallo-β-lactamase’:
Metallo-beta-lactamase pose a perplexing problem. These enzymes are capable of hydrolysing β-lactams from all chemical classes except the mono- lactams and the beta-lactamases inhibitors as clavulanic acid or tazobactam are ineffective against it.
The IMP-1 metalloenzyme was initially confined to Japanese isolates and was thought to be a major threat when it appeared on a transposable element in 1991. Now it is being reported all over the world including our country.
Metallo-beta-lactamases have been found in a limited number of species, such as Bacteroides fragilis, Klebsiella pneumonia, Pseudomonas aeruginosa and other members of gram-negative non-fermenters that frequently cause nosocomial outbreaks, and selection pressure for this enzyme is increasing.
Method (IMP-EDTA combination disk diffusion) (Figs. 209 and 20.10):
The inoculum was prepared emulsifying 5-6 colonies of the suspected isolate in Mueller Hinton broth and turbidity adjusted to 0.5 McFarland opacity standard.
Two 10 μg Imipenem discs were placed on the Mueller Hinton (MH) media inoculated with test organism. To one of the Imipenem discs, 10-25 mm apart a blank filter study disc was placed to which 5 μL 0.5 M EDTA solution was added. The combinations used in DDST was I (10 μg) + EDTA (5 μL of 0.5 M. solution).
Interpretation:
After 24 hours of incubation at 35°C, the zone of inhibition around Imipenem and Imipenem + EDTA discs were compared. If the zone of inhibition of Imipenem + EDTA discs compared to Imipenem alone is greater than 7 mm, then the test organism can be considered as MBL producing.
The zone of inhibition around Imipenem disc if expands towards EDTA disc, compared to the other Imipenem disc, placed on the far side, could also be interpreted as a positive result.
