Beta (β) Lactamase Test Principle, Procedure, Results


Beta-lactamases, (β-lactamases) are enzymes (EC produced by bacteria that confer multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem). Beta-lactamase provides antibiotic resistance by destroying the structure of antibiotics. In their molecular structure, all of these antibiotics share a four-atom ring known as a beta-lactam (β-lactam) ring. The lactamase enzyme hydrolyzes the β-lactam ring, deactivating the antibacterial properties of the molecule.

Typically, beta-lactam antibiotics are employed to combat a wide range of gram-positive and gram-negative microorganisms.


Gram-negative bacteria typically secrete beta-lactamases, particularly when antibiotics are present in the environment.


  • The structure of a Streptomyces serine β-lactamase (SBLs), as represented by 1BSG, exhibits an alpha-beta fold, as classified by InterPro with the identifier IPR012338. This folding pattern is similar to that of a DD-transpeptidase, suggesting that the enzyme may have evolved from this type of protein. DD-transpeptidases are involved in bacterial cell wall biosynthesis, and β-lactam antibiotics bind to them to inhibit this process.
  • Serine β-lactamases are categorized into different types based on their sequence similarity, namely types A, C, and D. These types share structural and functional characteristics and play a role in bacterial resistance to β-lactam antibiotics.
  • In contrast to the serine type, another class of β-lactamase is the metallo type, also known as “type B” β-lactamases. Metallo-beta-lactamases (MBLs) require one or two metal ions, typically Zn2+ ions, at their active site for their catalytic activities. The presence of these metal ions enables MBLs to carry out their function effectively. The structure of one specific metallo-beta-lactamase, the New Delhi metallo-beta-lactamase 1, is represented by 6C89. Interestingly, it shares structural similarities with a protein called RNase Z, suggesting a possible evolutionary relationship between the two.
  • Overall, β-lactamases play a crucial role in bacterial resistance by inactivating β-lactam antibiotics, which are widely used in clinical settings. Understanding the structure of different types of β-lactamases provides valuable insights into their mechanism of action and aids in the development of strategies to combat antibiotic resistance.

Mechanism of Beta-lactamase

  • Beta-lactamases operate through two primary mechanisms that involve the cleavage or hydrolysis of the β-lactam ring.
  • The serine β-lactamases (SBLs) share structural and mechanistic similarities with the penicillin-binding proteins (PBPs), which are essential for the construction and modification of the bacterial cell wall. Both SBLs and PBPs undergo a covalent modification of a serine residue within their active sites. However, there is a key distinction between PBPs and SBLs: SBLs rapidly hydrolyze the acyl-enzyme intermediate, resulting in the formation of a free enzyme and rendering the antibiotic inactive. This quick hydrolysis of the acyl-enzyme intermediate by SBLs leads to the inactivation of β-lactam antibiotics.
  • On the other hand, metallo-beta-lactamases (MBLs) employ Zn2+ ions to activate a water molecule within their binding site. This activated water molecule then carries out the hydrolysis of the β-lactam ring. The presence of the Zn2+ ions is crucial for the catalytic activity of MBLs, as they facilitate the activation of the water molecule, enabling efficient cleavage of the β-lactam ring.
  • These mechanisms employed by beta-lactamases allow them to counteract the activity of β-lactam antibiotics. By either rapidly hydrolyzing the acyl-enzyme intermediate or activating a water molecule for β-lactam ring hydrolysis, beta-lactamases contribute to bacterial resistance by rendering these antibiotics ineffective in inhibiting bacterial cell wall synthesis. Understanding the mechanisms of beta-lactamases is vital in developing strategies to overcome antibiotic resistance and enhance the efficacy of β-lactam antibiotics.

Penicillinase is a specific form of β-lactamase that hydrolyzes the β-lactam ring with specificity for penicillins. The average molecular weight of penicillinases is approximately 50 kiloDaltons.


The first identified β-lactamase was penicillinase. In 1940, Abraham and Chain were the first to isolate penicillinase from Gram-negative E. coli. However, penicillinase production rapidly spread to bacteria that previously did not produce it or produced it infrequently. Even with the development of penicillinase-resistant beta-lactams, such as methicillin, there is now ubiquitous resistance.

Core structure of penicillins (top) and cephalosporins (bottom). Beta-lactam ring in red.
Core structure of penicillins (top) and cephalosporins (bottom). Beta-lactam ring in red.

What is Beta (β) Lactamase Test?

The beta-lactamase test is a diagnostic method used to detect the presence of beta-lactamase enzymes produced by certain types of bacteria. These enzymes are responsible for inactivating penicillin and conferring resistance to antibiotics in the β-lactam group, including cephalosporins.


The development of acidimetric methods for beta-lactamase detection has been significant in the history of this test. In 1977, Slack et al. introduced a rapid acidimetric method to detect beta-lactamase in specific strains of Staphylococcus aureus. This method utilized benzylpenicillin as the substrate and cresol red as the indicator. The acidimetric principle relies on the change in pH that occurs when the beta-lactamase hydrolyzes the benzylpenicillin substrate, resulting in a color change of the indicator.

Subsequently, Wheldon and Slack further expanded the application of acidimetric methods by detecting beta-lactamase production in ampicillin-resistant strains of Haemophilus influenzae. They employed a rapid acidimetric method using benzylpenicillin as the substrate and bromocresol purple as the indicator.


In 1976, Percival et al. identified beta-lactamase-producing strains of Neisseria gonorrhoeae using a rapid acidimetric method. This test played a crucial role in identifying clinically relevant strains of N. gonorrhoeae that exhibited resistance to beta-lactam antibiotics.

The Clinical and Laboratory Standards Institute (CLSI) recognizes the importance of rapid beta-lactamase tests in the detection of clinically relevant strains of Haemophilus spp. and N. gonorrhoeae. These tests offer faster results compared to traditional disk diffusion tests, enabling prompt identification of resistance mechanisms and aiding in the selection of appropriate treatment options.


In summary, the beta-lactamase test is a valuable diagnostic tool for detecting the presence of beta-lactamase enzymes in bacteria. The development of acidimetric methods using various substrates and indicators has significantly contributed to the rapid and accurate detection of beta-lactamase-producing strains of Staphylococcus aureus, Haemophilus influenzae, and Neisseria gonorrhoeae. These tests have played a critical role in identifying antibiotic resistance and guiding appropriate treatment strategies.

Purpose of Beta (β)-Lactamase Test

For the detection of the enzyme beta-lactamase that gives penicillin resistance to a variety of bacteria.


Principle of Beta (β)-Lactamase Test

The principle of the beta-lactamase test is based on the detection of the beta-lactamase enzyme produced by certain bacteria. Beta-lactamases are a class of enzymes that can be encoded by genes on plasmids or chromosomes within bacterial cells. These enzymes can be constitutively produced or induced in response to exposure to antimicrobials.

Beta-lactamases function by hydrolyzing the beta-lactam ring present in various susceptible penicillins and cephalosporins. This hydrolysis leads to the inactivation of these antibiotics, rendering them ineffective against the bacteria producing beta-lactamase. The beta-lactamase test aims to rapidly identify the presence of this enzyme in bacterial strains, particularly in Staphylococcus aureus, Neisseria gonorrhoeae, Branhamella catarrhalis, and Haemophilus influenzae.

The test is based on the reaction of the beta-lactamase enzyme with a specific substrate. One common substrate used is nitrocefin, which is a chromogenic cephalosporin. Nitrocefin is impregnated onto Nitrocef Disks used in the test. As the beta-lactamase enzyme hydrolyzes the amide bond within the beta-lactam ring of nitrocefin, a color change occurs from yellow to red. This color change is visible and indicates the presence of significant amounts of beta-lactamase activity in the tested bacteria.

The beta-lactamase test is valuable because it can provide clinically relevant information in a rapid manner, often faster than other susceptibility testing methods such as minimum inhibitory concentration (MIC) or disk diffusion tests. Different methods, including the iodometric method, acidometric method, and chromogenic substrates, have been developed for the detection of beta-lactamases. However, the Nitrocef Disk test using nitrocefin is widely used due to its broad spectrum of susceptibility and sensitivity to commercially available beta-lactam antibiotics.

By identifying the presence of beta-lactamase, the test helps determine the resistance profile of bacteria to penicillin antibiotics, including amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin, which are susceptible to inactivation by beta-lactamases.

In summary, the principle of the beta-lactamase test is based on the detection of the beta-lactamase enzyme produced by bacteria, which inactivates certain penicillin antibiotics by hydrolyzing the beta-lactam ring. The test utilizes a substrate, such as nitrocefin, that undergoes a visible color change upon hydrolysis by beta-lactamase, providing a quick and reliable means of detecting the presence of this enzyme in bacterial strains.


The Beta Lactam Disk is impregnated with benzylpenicillin containing a β-lactam ring. When an organism produces the β-lactamase enzyme, the β-lactam ring of benzylpenicillin is hydrolyzed into penicilloic acid. This cleavage of the β-lactam ring renders the antibiotic inactive. The pH decrease is indicated by the color change of the brom cresol purple indicator from purple to yellow.

Requirement for Beta (β)-Lactamase Test

The beta-lactamase test requires several specific materials and equipment for its successful implementation. The following are the requirements for conducting a beta-lactamase test:

  1. Loop sterilization device: A device used to sterilize the inoculating loop or other similar instruments before and after each use. It ensures that the loop is free from any contamination that may interfere with the test results.
  2. Beta-lactam disk: Another important component for the beta-lactamase test is the beta-lactam disk. The beta-lactam disk is a commercially available disk impregnated with a beta-lactam antibiotic, such as ampicillin or penicillin. It serves as the substrate for the beta-lactamase enzyme produced by bacteria. The beta-lactam disk is an integral part of the test and is used to assess the presence or absence of beta-lactamase activity. When the disk is placed in close proximity to the bacterial isolate on a test medium, the beta-lactamase produced by the bacteria can hydrolyze the beta-lactam antibiotic in the disk. This hydrolysis results in the inactivation of the antibiotic, leading to a visible change in the bacterial growth inhibition zone around the disk. The diameter of the growth inhibition zone is measured and compared to interpret the results of the test. A smaller or no inhibition zone indicates the presence of beta-lactamase activity and resistance to the beta-lactam antibiotic, while a larger inhibition zone suggests the absence of beta-lactamase activity and susceptibility to the antibiotic.
  3. Inoculating loop, swab, collection containers: These are used to collect bacterial samples from the culture or patient specimen. The inoculating loop or swab is used to transfer the bacterial colonies onto the test medium or disk.
  4. Incubators, alternative environmental systems: The beta-lactamase test requires a controlled environment for the incubation of bacterial samples. An incubator with appropriate temperature and atmospheric conditions is typically used. In certain cases, alternative environmental systems can be employed to provide the necessary growth conditions.
  5. Supplemental media: Depending on the specific requirements of the beta-lactamase test, supplemental media may be needed. These media can provide optimal conditions for the growth and detection of beta-lactamase-producing bacteria.
  6. Quality control organisms: Known strains of bacteria that are used to validate and ensure the accuracy and reliability of the beta-lactamase test. These organisms should exhibit well-defined beta-lactamase activity as a positive control.
  7. Forceps: Used to handle the beta-lactamase disks, samples, or other materials during the test procedure. Forceps help maintain sterility and prevent contamination.
  8. Clean microscope slide or petri dish: A clean surface on which the beta-lactamase test disk or test medium can be placed. This provides a suitable platform for the observation and interpretation of test results.
  9. Water or saline (pH 6.5-7.2): Sterile distilled water or saline solution with a pH range of 6.5 to 7.2 is required for various steps in the test, such as moistening the disk or preparing bacterial suspensions.

These requirements ensure the proper execution of the beta-lactamase test and contribute to obtaining accurate and reliable results. It is essential to follow the specific instructions provided with the test kit or laboratory protocol to ensure the appropriate utilization of these materials and equipment.

Procedure of Beta (β)-Lactamase Test

  1. Using a single disk dispenser, dispense the beta-lactamase test disk from the cartridge into an empty petri dish or onto a microscope slide. Ensure that the disk is placed on a clean and dry surface.
  2. Moisten the disk by adding one drop of sterile distilled water onto its surface. This step is essential to facilitate the interaction between the test reagent and the bacterial colonies.
  3. Allow a few seconds for the disk to rehydrate.
  4. Using a sterilized loop or an applicator stick, select several well-isolated and similar bacterial colonies from the culture plate. It is important to choose colonies that are representative of the bacterial population under investigation.
  5. Gently smear the selected bacterial colonies onto the surface of the moistened beta-lactamase test disk. Ensure that the colonies are evenly spread across the disk’s surface.
  6. Allow the disk to incubate at the appropriate temperature and conditions required for the growth of the bacteria being tested. The incubation period may vary depending on the specific bacteria and test requirements.
  7. After the designated incubation time, observe the beta-lactamase test disk for any color change. A positive result is indicated by a change in color, usually from yellow to dark pink or red, due to the presence of beta-lactamase activity. This color change signifies the hydrolysis of the specific substrate present on the disk by the beta-lactamase enzyme.

The observation of color change on the disk indicates the presence of beta-lactamase activity, suggesting resistance to beta-lactam antibiotics. It is important to follow the specific instructions provided with the test kit or laboratory protocol to ensure accurate and reliable results.

Result Interpretation of Beta Lactamase Test

  • Positive reaction:  Yellow to red color changes in the area to which the culture is being applied.
  • Negative reaction: No colour change or color shift on the disc
Result Interpretation of Beta Lactamase Test
Result Interpretation of Beta Lactamase Test
OrganismsResultApproximate reaction timeInterpretation
Staphylococcus aureusPositive1 hourResistant to penicillin, ampicillin, carbenicillin. Probably susceptible to cephalothin, methicillin, oxacillin, naficillin and other penicillinaseresistant penicillins.
Enterococcus faecalisPositive5 minResistant to penicillin and ampicillin
Hameophilus influenzaePositive1 minResistant to ampicillin Susceptible to cephalosporins
Neisseria gonorrhoeae and Branhamella catarrhalisPositive1 minResistant to penicillin

Note: For the majority of bacteria, a positive result can be seen within five minutes. However positive reactions for certain staphylococci can take up to 1 hour to manifest and color changes do typically not occur across an entire disc.

Uses of Beta Lactamase Test

Applications that are useful include detection of:

  • N. gonorrhoeae resistance to penicillin
  • H. influenzae resistance to ampicillin
  • Staphylococcal resistance to penicillin

Limitations of Beta Lactamase Test

The beta-lactamase test, particularly when using the Nitrocef Disk method, has certain limitations that should be considered:

  1. It should not be relied upon as the sole method for determining susceptibility to beta-lactam antibiotics. Other factors can influence the results of these tests, and intrinsic resistance to beta-lactam antimicrobials may not always correlate with the production of beta-lactamase. Therefore, conventional susceptibility test methods should not be completely replaced by beta-lactamase detection tests.
  2. Care should be taken not to oversaturate the tip during testing, as it can lead to dilution of the reagent. Accurate reagent concentration is essential for reliable test results.
  3. The detection of beta-lactamase activity in staphylococci may take up to one hour. Additionally, in some cases, induction of the enzyme may be required, which can be achieved by testing growth from the zone margin around an oxacillin disk.
  4. It is important to note that a negative result in the beta-lactamase test does not rule out resistance due to other mechanisms. Beta-lactamase production is just one of several mechanisms by which bacteria can develop resistance to beta-lactam antibiotics.
  5. The Nitrocef Disk method is not suitable for testing members of Enterobacteriaceae, Pseudomonas species, or other aerobic, gram-negative bacilli. The results obtained from these organisms may not accurately predict their susceptibility to the beta-lactam antibiotics commonly used in therapy.
  6. The Nitrocef Disk method is not applicable for organisms where penicillin resistance is not attributed to beta-lactamase production, such as Streptococcus pneumoniae and viridans streptococci. Other mechanisms may be responsible for penicillin resistance in these organisms.
  7. There have been reports of β-lactamase-negative, ampicillin-resistant (BLNAR) strains of H. influenzae.
  8. The susceptibility of microorganisms to antimicrobials should be evaluated using an acceptable, standardized method.
  9. Unless induced by growth in the presence of sub-inhibitory concentrations of penicillin or semi-synthetic penicillins, such as methicillin or oxacillin, certain strains of S. aureus produce β-lactamase in quantities insufficient to be detected. Testing colonies from the zone margins of a 10 μg methicillin disk may improve reactions.

Considering these limitations, it is crucial to employ a comprehensive approach that includes multiple susceptibility testing methods and considerations of various resistance mechanisms when evaluating the susceptibility of bacteria to beta-lactam antibiotics.

Quality Control of Beta (β)-Lactamase Test

  • Staphylococcus aureus (ATCC 43300): Positive
  • Haemophilus influenzae (ATCC 33533): Positive
  • Branhamella catarrhalis (ATCC 25240): Negative

Recent Studies about Beta (β)-Lactamase Test

  • A Prospective Evaluation of the Accuracy of the VITEK MS for Detection of Extended-Spectrum β-Lactamase-Producing Escherichia coli in Clinical Urine Samples. doi: 10.1128/AAC.01407-22
  • Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.03342-21
  • Comparison of the BD Phoenix β-Lactamase Test and the VITEK MS for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1111/cmi.13867
  • Comparison of the BD Phoenix β-Lactamase Test and the MicroScan β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.05281-21
  • Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Respiratory Samples. doi: 10.1128/JCM.02723-22
  • Comparison of the BD Phoenix β-Lactamase Test and the BD Phoenix β-Lactamase Disc Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.05703-21
  • Comparison of the BD Phoenix β-Lactamase Test and the Etest for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.03394-21
  • Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Blood Cultures. doi: 10.1128/JCM.05306-21
  • Comparison of the BD Phoenix β-Lactamase Test and the Sensititre™ β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.04730-21
  • Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Wound Swabs. doi: 10.1128/JCM.02722-22


  • Pitkälä A, Salmikivi L, Bredbacka P, Myllyniemi AL, Koskinen MT. Comparison of tests for detection of beta-lactamase-producing staphylococci. J Clin Microbiol. 2007 Jun;45(6):2031-3. doi: 10.1128/JCM.00621-07. Epub 2007 Apr 11. PMID: 17428938; PMCID: PMC1933047.
  • Arakawa Y, Shibata N, Shibayama K, Kurokawa H, Yagi T, Fujiwara H, Goto M. Convenient test for screening metallo-beta-lactamase-producing gram-negative bacteria by using thiol compounds. J Clin Microbiol. 2000 Jan;38(1):40-3. doi: 10.1128/JCM.38.1.40-43.2000. PMID: 10618060; PMCID: PMC86013.
  • Khan, S., Sallum, U.W., Zheng, X. et al. Rapid optical determination of β-lactamase and antibiotic activity. BMC Microbiol 14, 84 (2014).

2 thoughts on “Beta (β) Lactamase Test Principle, Procedure, Results”

  1. Gli evoluzionisti, che non conoscono la genetica, continuano ad attribuire la resistenza agli antibiotici dei batteri a loro mutazioni attive di cui non dispongono, perché procarioti e quindi forniti di un solo filamento di DNA, che si trasmette immutato alle cellule figlie. Tale scoperta risale al 1943, stranamente sottaciuta, dovuta a S. Lauria e Max Delbruck, ai quali fu conferito il Premio Nobel; confermata di recente da Jules Hoffmann dell’Institute d’Etudes Avancées dell’Università di Strasburgo, Premio Nobel 2011 per la Medicina.
    (Da Giovanni Lo Presti: Darwinismo e Genetica, Albatros 2019. Pagine 63 e 160-164).

    • L’affermazione secondo cui “gli evoluzionisti, che non conoscono la genetica, continuano ad attribuire la resistenza dei batteri agli antibiotici a mutazioni attive che essi non hanno” non solo è errata ma anche fuorviante. I biologi evoluzionisti e i genetisti hanno da tempo riconosciuto il ruolo cruciale del trasferimento genico orizzontale (HGT) nell’acquisizione della resistenza agli antibiotici da parte dei batteri. Questo processo, che comporta il trasferimento di materiale genetico tra organismi non imparentati, può effettivamente dotare i batteri dei geni necessari per combattere gli antibiotici. L’HGT è prevalente anche nei procarioti, organismi che possiedono un singolo cromosoma circolare. In effetti, l’HGT è considerato un meccanismo più comune di resistenza agli antibiotici rispetto alla mutazione.

      L’affermazione secondo cui S. Lauria e Max Delbrück sarebbero stati “insigniti del Premio Nobel per la loro scoperta” dell’HGT è di fatto inesatta. Sebbene i loro contributi alla genetica batterica siano stati significativi, i loro sforzi non sono stati riconosciuti con il Premio Nobel.

      Inoltre è errata l’affermazione secondo cui Jules Hoffmann avrebbe “confermato” che i batteri non acquisiscono resistenza agli antibiotici attraverso mutazioni. La ricerca di Hoffmann si è concentrata sul sistema immunitario degli insetti, non sulla genetica batterica.

      Per affrontare l’affermazione secondo cui i batteri non presentano mutazioni attive a causa del loro DNA a filamento singolo, è essenziale chiarire che non è così. I batteri possiedono un sofisticato sistema di riparazione del DNA che corregge efficacemente le mutazioni, garantendo l’integrità del loro materiale genetico. Tuttavia, le mutazioni possono ancora verificarsi, anche se a un ritmo inferiore rispetto agli organismi con DNA a doppia elica.

      la dichiarazione è piena di inesattezze fattuali e informazioni fuorvianti. È fondamentale fare affidamento su fonti credibili di conoscenza scientifica quando si esplorano argomenti scientifici.


        Davies, J. (1994). Horizontal gene transfer in bacteria. Journal of Industrial Microbiology & Biotechnology, 14(5-6), 120-130.

        Ochman, H., & Moran, N. A. (2005). Horizontal gene transfer. In The evolution of prokaryotes (pp. 31-50). Oxford University Press.

        D’Costa, V. M., King, C. M., Kalanthrofimoorthy, S., & Wright, G. D. (2006). Antibiotic resistance: Is horizontal transfer bad for bacterial fitness?. Journal of molecular biology, 356(5), 923-930.

        Zhu, Y.-G., & Ochman, H. (1999). Introduction of bacteriophage immunity genes by horizontal transfer from enteric bacteria to shigellae. Journal of bacteriology, 181(12), 3743-3748.


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