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Introduction of Beta  Lactamases

Beta Lactamases is the commonest cause of bacterial resistance to β – lactam antibiotics. These enzymes are produced by some bacteria and confer resistance to β -lactam antibacterials. These antibacterials are called so because of the four atom β – lactam ring in their molecular structure (with the ring mimicking two amino acids in the pentapeptide cross-link of the peptidoglycan bacterial cell wall).  β -lactamases enzyme breaks this ring open, deactivating the molecule’s antibacterial properties. Many Gram-negative bacteria possess naturally occurring, chromosomally mediated β -lactamases that are thought to help the bacteria compete with other β – lactam- producing bacteria or to remove β – lactam like molecules that bacteria may use as natural regulators of cell wall synthesis. The ability of a β – Lactamase to confer resistance depends on its location, kinetics, quantity, and physicochemical conditions. At least 400 different types of β -lactamase, originating from clinical isolates, have been described. The first plasmid-mediated β- lactamase in gram-negative bacteria was discovered in Greece in the 1960s. It was named TEM after the patient from whom it was isolated (Temoniera). Subsequently, a closely related enzyme was discovered and named TEM – 2. These two enzymes are the most common plasmid-mediated β- lactamases in gram-negative bacteria, including Enterobacteriaceae, P. aeruginosa, H. influenzae, and N. gonorrhea. TEM 1 and TEM 2 hydrolyze penicillins and narrow-spectrum cephalosporins. However, they are not effective against higher generation cephalosporins with an oxyimino side chain, such as Cefotaxime, ceftazidime, ceftriaxone, or Cefepime. Consequently, when these antibiotics were first introduced, they were effective against a broad group of otherwise resistant bacteria. A related but less common enzyme was termed SHV because sulfhydryl reagents had a variable effect on substrate specificity.

Types of beta  Lactamases

β-Lactamase can be classified into four molecular classes according to the Ambler scheme.

1. A (Penicillinases, including ESBLs)
2. B (Metallo β- lactamase, MBLs
3. C (AmpC enzymes)
4. D (Oxacillinase)

Detection methods beta Lactamases

For detection of ESBL

Introduction 

ESBL stands for Extended-spectrum β- lactamase. In modern medical practice, newer antimicrobials drugs have been used extensively resulting in the emergence and rapid dissemination of resistant bacterial strains. Since one of the mechanisms of bacterial resistance to β-lactam antibiotics is the production of β- lactamase enzyme that breaks down the structural β- lactam ring of penicillin and its synthetic derivatives. The property of stability to many bacterial β- lactamase was increased with the later generations of cephalosporins. However, the persistent exposure of bacterial strains to a multitude of β- lactams have induced a dynamic and continuous production and mutation of lactamase in many bacteria, expanding their activity even against the third and fourth-generation cephalosporins. These new β- lactamases are called ESBLs which were first reported in Germany.

ESBL detection method

Screening method

Antibiotics      Zone of Inhibition (ZOI)

Ceftazidime(30µg)  ≤ 22 mm

Cefotaxime (CTX ) ≤ 27 mm

Cefpodoxime (CPD) ≤ 17 mm

Confirmatory method

Put ceftazidime (30 µg) and combination of ceftazidime (30 µg) and clavulanic acid ( 10  µg) disks with distance of 20 mm from disk to disk on Muller-Hinton agar as shown above picture.

After overnight incubation, ZOI of ceftazidime clavulanic acid (CAC) should be equal to and greater than 5 mm that of ceftazidime.

The above isolate is showing the ESBL producer.

Other methods of detection-

Double disk Synergy Test (DDST): 

The disk of third-generation cephalosporin placed at 15 mm distance from amoxicillin-clavulanic acid. Enhanced inhibition indicates ESBL.

Micro Dilution Test:  Growth in a broth containing 1 µg/ml third-generation cephalosporins indicates ESBL.

MIC Broth Dilution: 

MIC of third-generation cephalosporin alone or combined with CA. A decrease in the MIC of the combination of 3 twofold dilutions indicates ESBL.

E- Test (MIC ESBL Strip): 

Two-sided strip containing CAZ on one side and CAZ- CA on the other. The ratio of MIC of the combination to that of CAZ alone > 8 or the presence of a phantom zone (or both) indicates ESBL.

Automated Instruments (Vitek): Measures MICs and compares the growth of bacteria in presence of CAZ vs. CAZ-CA.

Molecular (DNA probes, PCR, RFLP): Targets specific nucleotide sequences to detect different variants of TEM and SHV genes.

Details of ESBL 

ESBLs are β- lactamases containing serine at the active site and belong to Ambler’s molecular class -A (majority of them). They hydrolyze extended-spectrum cephalosporins with an oxyimino side chain. These cephalosporins include Cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino- monobactam aztreonam. ESBLs confer resistance to expanded spectrum, aztreonam, and related oxyimino β- lactams; but they are not specific to cephamycins (e.g. Cefoxitin and cefotetan) or carbapenems (e.g. Meropenem or imipenem). Typically they derive from genes for TEM 1, TEM 2, or SHV 1 by a mutation that alter the amino acid configuration around the active site of this β- lactamases. This extends the spectrum of β- lactam antibiotics susceptible to hydrolysis by these enzymes. They are also generally susceptible to β- lactamase inhibitors, such as clavulanate, sulbactam, and tazobactam, which consequently can be combined with a β-lactam substrate to test for the presence of this resistance mechanism. The majority of ESBLs contain a serine at the active site. These enzymes are most commonly produced by Klebsiella spp.  and E. coli but may also occur in other gram-negative bacteria, including Enterobacter, Salmonella, Proteus, Citrobacter, Morganella morganii, Serratia marcescens, Shigella dysenteriae, P. aeruginosa, Burkholderia cepacia, Capnocytophaga ochracea.

TEM β- lactamases (Class A): Although TEM type β- lactamase is most often found in E. coli and K. pneumoniae, they are also found in other species of gram-negative bacteria with increasing frequency. The amino acid substitutions responsible for the ESBL phenotype cluster around the active site of the enzyme and change its configuration, allowing access to oxyimino β – lactam substrates. Opening the active site to β lactam substrate also typically enhances the susceptibility of the enzyme to β- lactamase inhibitors such as clavulanic acid. Single amino acid substitution at positions 104,164, 238, and 240 produce the ESBL phenotype, but ESBL with the broadest spectrum usually have more than a single amino acid substitution. Based upon different combinations of changes, currently, 140 TEM-type enzymes have been described.

SHV β- lactamases (Class A): These have amino acid changes around the active site, most commonly at positions 238 or 238 and 240. More than 60 SHV varieties are known. They are the predominant ESBL type in Europe and the United States and are found worldwide.

CTX – M – β lactamases (Class A): These enzymes were named for their greater activity against Cefotaxime than other oxyimino β- lactam substrates (e.g. ceftazidime, ceftriaxone, or cefepime). More than 40 CTX M enzymes are currently known. Despite their name, a few are more active on ceftazidime than Cefotaxime. They have mainly been found in strains of Salmonella enterica serovar Typhimurium and E. coli, but have also been described in other species of Enterobacteriaceae and are the predominant ESBL type in parts of South America. CTX- M- 14, CTX- M -3, and CTX-M -2 are the most widespread.

OXA β- lactamases (Class D): The OXA- type β -lactamases confer resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and Cloxacillin and the fact that they are poorly inhibited by clavulanic acid. Amino acid substitutions in OXA enzymes can also give the ESBL phenotype. While most ESBLs have been found in E. coli, K. pneumonia,e, and other Enterobacteriaceae, the OXA- type ESBLs have been found mainly in P. aeruginosa. Some confer resistance predominantly to ceftazidime, but OXA -17 confer greater resistance to Cefotaxime and Cefepime than it does resistance to ceftazidime.

Other β- lactamases: Other plasmid-mediated ESBLs such as PER, VEB, GES, and IBC β- lactamases have been described but are uncommon and have been found mainly in P. aeruginosa and at a limited number of geographic sites.

Treatment option

ESBL producing isolates typically show greater than average resistance to other agents including aminoglycosides and fluoroquinolones. These relationships were illustrated in a review of 85 episodes of bacteremia due to ESBL production from 12 hospitals in seven countries. All isolates were susceptible to imipenem or meropenem, while 71% were resistant to gentamycin, 47% to piperacillin-tazobactam, and 20% to ciprofloxacin. When an oxyimino- β – lactam is used to treat severe infections caused by ESBL- producing K. pneumoniae, treatment failure is likely even if the organism tests susceptible to the antibiotic in vitro. In a review of 28 patients with ESBL – producing K. pneumoniae with reported susceptibility to cephalosporins, 15 failed to respond to cephalosporin therapy. A high degree of associated resistance to gentamycin, co-trimoxazole, and quinolones was found in ESBL producers. The majority of ESBL producers were detected among patients. Currently, carbapenems are generally regarded as the preferred agent for the treatment of infections due to ESBL- producing organisms. Carbapenems are resistant to ESBL- mediated hydrolysis and exhibit excellent in vitro activity against strains of Enterobacteriaceae expressing ESBLs.

Clinical outcome due to ESBL

ESBL producing bacteria are typically associated with MDR. The antibacterial choice is often complicated by multi-drug resistance. Thus, infection due to ESBL producing bacteria can result in avoidable failure of treatment and increased cost in patients who have received inappropriate antibiotic treatment. Colonization and infection with these bacteria have also been associated with indiscriminate use of antibiotics, prolonged hospitalization, increasing numbers of immune-compromised patients, and medical progress resulting in increased use of invasive procedures and devices. Once an ESBL producing strain is detected the laboratory should report it as ‘resistant” to all penicillins, cephalosporins, and aztreonam, even if they test as susceptible. So, updated knowledge of the susceptibility pattern of bacteria is important for the proper selection and use of antimicrobial drugs and for the development of an appropriate prescribing policy. The first prevalence study of ESBL producing bacterial isolates in Nepal by Pokhrel et al in 2005 showed that > 20% isolates were positive for ESBL. In this context, it is quite necessary to present the current scenario of ESBL production in our setting. Problems in identification arise because ESBLs are heterogeneous. OXA- type ESBLs e.g. are poorly inhibited by clavulanate. Some ESBLs are best detected with ceftazidime and others with cefotaxime (such as most CTX- M enzymes). Consequently, susceptibility to several oxyimino β –lactams must be tested. In the combination disk method, E. coli ATCC 25922 is used as ESBL negative control, and K. pneumoniae ATCC 700603 is used as ESBL positive references strains (132). Because different ESBLs hydrolyze β – lactams at different rates, several different agents must be examined to examine their presence.

Worldwide pattern of ESBL

 The prevalence of ESBLs among clinical isolates varies from country to country and from institution to institution.

This difference may be due to geographical variations, local antibiotic prescribing habits, etc. Although the prevalence of ESBLs is not known, it is clearly increasing, and in many parts of the world, 10-40 % of strains of E. coli and K. pneumoniae express ESBLs.

In the United States, the occurrence of ESBL production in Enterobacteriaceae ranges from 0 to 25%, depending on the institution, with the national average being around 3%. In Korea and Indonesia, the distribution of ESBL in E. coli is 5% and 23.3% respectively which is higher when compared to North America or Europe, but similar to that of South America. In Japan, the percentage of β- lactam resistance due to ESBL production in E. coli and K. pneumoniae remains very low. In a recent study of 196 institutions across the country, < 0.1% of E. coli and 0.3% of K. pneumoniae strains possessed and ESBL. Elsewhere in Asia, the percentage of ESBL production in E. coli and K. pneumoniae varies like 8.5% in Taiwan93 and 12% in Hong Kong, The first prevalence study of ESBL producing bacterial isolates in Nepal by Pokhrel et al showed that > 20% of isolates were positive for ESBL. In this context, it is quite necessary to present the current scenario of ESBL production in our setting.

For detection of MBL

MBL stands for Metallo β- lactamase and according to Ambler, the scheme comes under class B. This class of β -lactamases is characterized by the ability to hydrolyze carbapenems and by its resistance to the commercially available β- lactamase inhibitors (clavulanic acid and tazobactam) but susceptibility to inhibition by metal ion chelators. The substrate spectrum is quite broad; in addition to the carbapenems, most of these enzymes hydrolyze cephalosporins and penicillins but lack the ability to hydrolyze aztreonam.

Mechanism of action of MBL

The mechanism of hydrolysis is dependent on the interaction of the β-   lactams with zinc ions in the active site of the enzyme, resulting in the distinctive trait of their inhibition by EDTA, a chelator of zinc ion and other divalent cations. The first MBLs detected and studied were chromosomal enzymes present in environmental and opportunistic pathogenic bacteria such as Bacillus cereus, Aeromonas spp., and S. maltophilia. MBLs mediate resistance to β- lactams by cleaving the amide bond of the β-lactam ring. They possess a distinct set of amino acids that define the finite architecture of the active site which coordinated the zinc ions. The zinc ions in turn usually coordinate two water molecules necessary for hydrolysis. The principal zinc-binding motif is histidine- X–histidine –X aspartic acid. The proposed mechanism of hydrolysis suggests that the active site orients and polarizes the β- lactam bond to facilitate nucleophilic attack by zinc-bound water/ hydroxides. The MBL mechanism of hydrolysis is complex and varies from one MBL to another. Unlike serine β -lactamases, MBLs possess a wide plastic active site groove and accordingly can accommodate most β- lactam substrates, facilitating their very broad spectrum of activity. They are also impervious to the impending effects of serine inhibitors such as Clavulanic acid and sulbactam that are often treated as poor substrates. Interestingly, none of the MBLs hydrolyze aztreonam particularly well, and it has been speculated that it could be considered a therapeutic MBL inhibitor (see the section on inhibitors). However, in studies of animals with pneumonia caused by P. aeruginosa producing VIM- 2, the infection could not be eradicated with aztreonam even when the animals were given high drug doses. MBLs are currently divided into three subclasses based on a combination of structural features, zinc affinities for the two binding sites, and hydrolysis characteristics. MBLs like all β-lactamases can be divided into those that are normally chromosomally mediated and those that are encoded by transferable genes.

MBL Families

The most common MBL families include the IMP, VIM, GIM, SIM, and SPM enzymes which are located within a variety of integron structures, where they have been incorporated as gene cassettes. When these integrons become associated with plasmids or transposons, transfer between bacteria is readily facilitated.

IMP: The first indication of mobile MBLs (IMP 1) was with the discovery of P. aeruginosa strain GN 17203 in Japan in 1988. IMP enzymes spread slowly to other countries in the Far East, were reported from Europe in 1997, and have been found in Canada and Brazil.

VIM: VIM -1 (‘Verona integron – encoded MBL’) was first isolated in Verona, Italy, in 1997. Currently, it consists of 14 members which have a wide geographic distribution in Europe, South America, and the Far East and have also been found in the United States. VIM enzymes mostly occur in P. aeruginosa, also P. putida, and very rarely, Enterobacteriaceae.

SPM: SPM -1 (‘Sao Paulo MBL’) was first isolated in the P. aeruginosa strain in Sao Paulo, Brazil.

GIM: GIM-1 (‘German imipenemase’) was isolated in Germany in 2002. It has approximately 30% homology to VIM, 43% to IMPs, and 29% to SPM.

SIM: The latest family, i.e., SIM-1 (‘Seoul imipenemase’) was isolated in Korea. Since their initial discoveries, SPM, GIM, and SIM MBLs have not spread beyond their countries of origin. However, VIM and IMP continue to be detected worldwide, with an overall trend of these two MBLs moving beyond P. aeruginosa and into the Enterobacteriaceae.

Problems regarding Inhibitors of MBLs: Different combinations have been studied with inhibitors directed against MBLs. However, there are many obstacles to circumvent.

1. MBLs possess subtle but significant variations in their active site architecture so that designing a single inhibitor efficacious against even the transferable MBLs will be problematic
2. Unlike Clavulanic acid, which interacts directly with class- A enzymes and forms a stable covalent intermediate, MBLs do not form highly populated metastable reaction intermediate
3. Clavulanic acid has no homologous mammalian target, i.e. it possesses relatively low toxicity. Unfortunately, MBLs have active site motifs similar to those for mammalian enzymes that are highly likely to be quintessential for cellular functions.

MBL producing bacterial isolates have been responsible for several nosocomial outbreaks in tertiary centers in different parts of the world, illustrating the need for proper infection control practices (108).In some countries, P. aeruginosa possessing MBLs constitute nearly 20% of all nosocomial isolates. These isolates have been responsible for serious infections such as septicemia and Pneumonia (109) and have been associated with failure of therapy with carbapenems.

Detection of MBLs

The catalytic mechanisms of MBLs and non MBLs are different.  Therefore, different strategies are needed for the detection of pathogens harboring these enzymes in any attempt aimed at their control and eradication. Accurate identification of MBLs will therefore rely often on the availability of specific, sensitive, and simple assays able to differentiate MBLs from other Carbapenemases (146) unfortunately; there is no recommended phenotypic method available from CLSI for their detection. However, several phenotypic methods are practiced. All these methods are based on the ability of metal chelators, such as ethylene diamine tetraacetic acid (EDTA) and thiol-based compounds, to inhibit the activity of MBLs (147). These tests include

1. Double disk synergy tests (DDST) using EDTA with imipenem (IPM) or ceftazidime (CAZ)
2. Hodge test, a combined disk test using EDTA with CAZ or IMP.
3. MBL E- test (a threefold or greater decrease in the imipenem MIC in the presence of EDTA)
4. Micro dilution method using EDTA and phenanthroline with IMP
5. Isoelectric focusing
6. Polymerase chain reaction

The non-molecular “gold standard” is well established in research laboratories where bacterial crude cell extracts are examined for their ability to hydrolyze carbapenems and whether this hydrolysis is EDTA sensitive. These data indicate that the enzyme is being produced regardless of its genotype. However, this technique utilizes specialized spectrophotometric equipment that precludes its implementation in a routine diagnostic laboratory. Most of the current phenotypic methods described for the detection of MBLs, especially the DDST and the Hodge test, are difficult and subjective to interpret. This test can also be technically demanding and time- consuming. Besides, with these methods, MBL carrying organisms can appear susceptible to carbapenems using current CLSI or British Society for Antimicrobial Chemotherapy breakpoints. In a study over 30% of MBL carrying isolates were found to be susceptible to imipenem. Similarly, Yan et al reported that an outbreak of K. pneumoniae isolates carrying bla IMP- 8 and showed that 88% were susceptible to carbapenems. In this context, a method described by Peleg et al, 2006, seems to be effective for the phenotypic detection of carbapenem- susceptible as well as resistant MBL-producing gram-negative bacilli. They utilized several unique methodologies in an attempt to maximize the detection of such challenging organisms. They used a lower concentration of EDTA so that its bactericidal effect was minimized and they also used a lower cutoff for the increase in zone diameter with imipenem-EDTA as opposed to imipenem alone.

In brief

MBL detection method

Screening method

Antibiotics      Susceptibility

Imipenem    –  Resistance

Meropenem – Resistance

Ceftazidime – Resistance

Confirmatory method

Put imipenem (10 µg) and combination of imipenem (10 µg) plus EDTA ( 10  µl-concentration 292µg/ml i.e. 0.1 M) disks with a distance of 25 mm from disk to disk on Muller-Hinton agar as shown above picture. After overnight incubation, ZOI of imipenem plus EDTA  should be equal to and greater than 5 mm that of imipenem. The above isolate is showing the MBL producer.

For detection of AmpC

Introduction  and clinical significance of AmpC

AmpC β-lactamases are enzymes of gram-negative bacteria like many Enterobacteriaceae and a few other bacteria. It is clinically important cephalosporinases encoded on the chromosomes of these bacteria where they mediate resistance to cephalothin,  cefoxitin, cefazolin, most penicillins, and β-lactamase inhibitor or β-lactam combinations. The increasing antibiotic resistance is a notable example of how bacteria can procure, maintain and express new genetic information that can confer resistance to one or several antibiotics. Detection of bacteria producing these enzymes can be difficult because their presence does not always produce a resistant phenotype on conventional disc diffusion or automated antibiotics susceptibility testing methods. The false susceptibility to AmpC β-lactamases laboratory report is potentially fatal. These enzymes show tremendous variation in geographic distribution and therefore, their accurate detection and characterization are important for epidemiological as well as clinical, laboratory, and infection control points of view.

Test for AmpC Production in Gram-Negative Isolates

Screening for AmpC β-lactamase production was performed by the Cefoxitin disk test. Isolates that yielded a zone diameter less than 18 mm (Positive in screening) were further subjected to confirmatory testing. These positive Screen isolates were swabbed onto an MHA plate. Cefoxitin (30ug) and Cefotaxime (30ug) disks were placed at a 20 mm distance between the disks. Blunting of the zone of inhibition around the Cephotaxime disk at the side of the Cefoxitin disk after overnight incubation at 37°C was considered positive for AmpC production by the isolates.

 In brief,

Ampc detection method (Phenotypic)

Screening method

The zone of inhibition (ZOI) of Cefoxitin (30 µg)  should be less than 18 mm.

Confirmatory method

Put cefoxitin (30 µg) and a combination of cefoxitin and phenylboronic acid(PBA) 10  µg disks with a distance of 20 mm from disk to disk on Muller-Hinton agar as shown above picture.

After overnight incubation, the ZOI of the combined disk( cefoxitin+PBA) should be equal to and greater than 5 mm that of cefoxitin.

The organism (Gram-negative bacteria ) is an AmpC producer as shown above image.

For detection of KPC

Introduction of Klebsiella pneumoniae carbapenemase (KPC)

Klebsiella pneumoniae carbapenemase (KPC) is an enzyme produced by bacteria, Klebsiella pneumoniae that makes organism multidrug resistance. This enzyme falls in Ambler class A beta-lactamases.

Types of beta  Lactamases

β-Lactamase can be classified into four molecular classes according to the Ambler scheme.

1. A (Penicillinases, including ESBLs)
2. B (Metallo β- lactamase, MBLs
3. C (AmpC enzymes)
4. D (Oxacillinase)

Importance of Klebsiella pneumoniae carbapenemase (KPC) detection

The importance of KPC detection is due to the following reasons-

1. The emergence of KPCs producing bacteria has become a significant global public health challenge while the optimal treatment remains undefined.
2. This is particularly relevant with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) or other types of carbapenemase-producing Enterobacteriaceae (CPE) infections because commonly used regimens for empiric antimicrobial treatment do not normally cover multidrug-resistant (MDR)  pathogens.
3. KPC-KP has become one of the most important contemporary pathogens, especially in endemic areas.
4. To provide practical suggestions for physicians dealing with the management of KPC-KP infections in critically ill patients, based on expert opinions.
5. KPC-producing organisms can confer resistance to multiple different antimicrobial classes, including all available β-lactams, fluoroquinolones, and aminoglycosides. As such, infections due to KPCs are associated with high therapeutic failure and mortality rates of at least 50%.
6. The limited number of agents available for the treatment of KPCs presents a tremendous challenge to clinicians.
7. Klebsiella is resistant to multiple antibiotics. This is thought to be a plasmid-mediated property. Longer hospital stays and the performance of invasive procedures are risk factors for the acquisition of these strains.
8. Many isolates are a single sequence type, ST258, and susceptibility is limited to gentamicin, tigecycline, and colistin.

KPC detection method

Screening method

• Antibiotics      Susceptibility
• Imipenem    –  Resistance
• Meropenem – Resistance

Confirmatory method

Test Requirements 

• Muller-Hinton agar(MHA)
• Antimicrobial disks ( meropenem)
• phenylboronic acid
• EDTA
• Bunsen burner
• Inoculating loop
• Suspected isolate ( bacterial growth)
• Sterile cotton swab sticks
• Densitometer or O.5 McFarland Std.
• Inoculum suspension media (5 ml Tryptone Soya Broth)

Test procedure

• Inoculum Preparation: Use only pure cultures. Confirm by Gram-staining before starting susceptibility test. Transfer 4-5 similar colonies with a wire, needle, or loop to 5 ml Tryptone Soya. Broth and incubate at 35-37°C
  for 2-8 hours until light to moderate turbidity develops. Compare the inoculum turbidity with that of standard 0.5 McFarland. Dilute the inoculum or incubate further as necessary to attain comparative turbidity. Alternatively, the inoculum can be standardized by another appropriate optical method (0.08 – 0.13 OD turbid suspension at 625 nm).
• Swab the entire agar surface of the plate with standardized inoculum soaked onto the cotton swab.
• Put two meropenem (10 µg) disks with a distance of 20 mm from disk to disk on Muller-Hinton agar as shown above picture.
• Add phenylboronic acid  ( 10  µl-concentration 300µg/ml) over a disk and EDTA on another disk.
• Incubate the plate overnight at 37°C.

Observation

Observe for the zone of inhibition (ZOI).

Result Interpretation of KPC-KP

•  ZOI of meropenem plus phenylboronic acid  is equal and greater than 5 mm that of meropenem plus EDTA:  KPC producer
•  ZOI of meropenem plus phenylboronic acid  is less than 5 mm that of meropenem plus EDTA: Non- KPC producer strain
• Result: KPC producer as shown above image.

Betalactamase detection|ESBL| MBL| KPC |AmpC |Oxacillinase|MRSA|D Zone test positive| iMLSB strain as shown below-

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