Metallo-beta-lactamases (MBLs) are carbapenem-hydrolyzing beta-lactamases which belong to molecular Class B of Ambler beta-lactamase classification, and which have the ability to hydrolyze and confer resistance to carbapenems (imipenem, meropenem, ertapenem) and other beta-lactam antibiotics. Class B carbapenemases (i.e. the metallo-beta-lactamases) are found in Enterobacteriaceae, Acinetobacter species and Pseudomonas aeruginosa isolates. metallo beta lactamases, which are a type of carbapenemases, are an emerging public health problem among clinically important Gram-negative organisms including P. aeruginosa, A. baumannii and the Enterobacteriaceae.
]The carbapenems are very potent antimicrobial agents used for the treatment of serious Gram-negative bacterial infections including those that are ESBL-mediated; and because of the broad spectrum activity and stability of the carbapenems to most beta-lactamase enzymes, the carbapenems have been widely used under restricted conditions in most hospitals worldwide as the first-line treatment for severe Gram-negative infections. The metallo beta lactamases are known to confer variable range of high resistance to all beta-lactam antibiotics except the monobactams and their presence in clinically important Gram-negative bacteria have put the use of the carbapenems under threat.
The metallo beta lactamases belong to a group of beta-lactamases which requires divalent cations (e.g. zinc ions) as cofactors for their enzyme activity, and they share four main characteristics as follows:
- Activity against carbapenem antibiotics.
- No clear hydrolysis of monobactams.
- Inhibition by chelating agents such as ethylene diamine tetraacetic acid (EDTA) and dipicolinic acid.
- Requirement of zinc ions (Zn2+) for enzyme activity.
Genetically, the metallo beta lactamases are either plasmid-mediated or chromosomally-mediated, and those that are plasmid-mediated (or encoded by transferable genes or elements such as integrons and transposons) are found in more resistant bacteria such as P. aeruginosa, A. baumannii, and the Enterobacteriaceae while those that are chromosomally-mediated are found in bacterial strains such as Bacillus cereus and Stenotrophomonas maltophilia and in obscure non-clinical bacteria such as Aeromonas species. Metallo Beta Lactamase genes are important resistance determinants considering the fact that most of these genes are carried as mobile gene cassettes (which can easily be integrated into the chromosomes of other susceptible organisms) on class one integrons with the potential to spread to other clinically important bacteria.
And because the metallo beta lactamase genes are mainly plasmid-borne, their spread to the population of pathogenic organisms is of great concern and a menace to our ability to fight and treat a wide variety of Gram-negative infections. Opportunistic organisms from the environment are known to ubiquitously express metallo beta lactamase chromosomally, and the reason for this is still arcane. These bacteria including Bacillus species, Chryseobacterium species, Flavobacterium species, Serratia species, and Stenotrophomonas species seldom cause serious infections but yet are known to express these carbapenem-hydrolyzing enzymes in varying amounts from one bacterium to another.
Metallo beta lactamases are disseminated through genetic elements such as transposons, plasmids and integrons amongst clinically important bacteria and the extensive use of the carbapenems are known to be responsible for the worldwide spread of these enzymes. The extra-chromosomally-mediated (transposable) MBLs include: the imipenemase (IMP) MBLs, Verona-imipenemase (VIM) MBLs, Sao Paulo MBL (SPM-1), and the German-imipenemase (GIM) MBLs. Pseudomonas aeruginosa infections are treated with carbapenems such as meropenem and imipenem, and the increasing usage of these antibiotics together with other expanded spectrum drugs has resulted in the development of imipenem and meropenem-resistant P. aeruginosa and other MBLs in Enterobacteriaceae.
Metallo-beta-lactamases are Ambler Class B beta-lactamases (or carbapenemases) and they constitute mainly of the enzyme types: IMP and VIM MBLs which are found in organisms such as P. aeruginosa, A. baumannii and the Enterobacteriaceae. Other carbapenemases include: the Klebsiella pneumoniae carbapenemases (KPC) – which is an Ambler Class A enzyme, four serine carbapenemases (SME, NMC-A, IMI, and rare GES) – which are all Ambler Class A enzymes, and a last group of MBLs known as the OXA carbapenemases. The OXA-carbapenemases are weakly active against carbapenems and are inhibited by clavulanate poorly. They are largely restricted to Pseudomonas species and Acinetobacter species, and are rarely found in Enterobacteriaceae. The SME carbapenemases derive their name from Serratia marcescens and other Serratia species, and they are chromosomally encoded while the imipenem-2 (IMI) carbapenemases and Guyana extended spectrum (GES) variants together with KPC are plasmid mediated carbapenemases.
The non-metallo-carbapenemase (NMC) is also plasmid-mediated and they can be inhibited by clavulanic acid together with GES, SME, NMC and KPC carbapenemases. Of much concern is another group of novel MBLs known as the New Delhi Metallo-beta-lactamase (NDM-1) – producing bacteria which carries the NDM gene and confers resistance to carbapenems. NDM-1 is usually found in members of the Enterobacteriaceae and they differ from other beta-lactamase enzymes and are so named because they are known to originate from India and Pakistan. The NDM-1 enzyme has been identified in people who returned to the UK, USA and other parts of the world after undergoing surgery/medication in India or Pakistan, and the incidence of this type of MBL is known to have emerged in 2007.
Classification of metallo-beta-lactamases
Metallo-β-lactamases was first classified in 1980 as serine carbapenemases by the Ambler classification scheme for beta-lactamase enzymes. The classification of metallo-β-lactamases (MBLs) is based on several properties that include the substrate profile and/or affinity of the enzymes (e.g. its ability to hydrolyze any of the carbapenems such as imipenem), their sensitivity to a chelating agent (e.g. ethylene diamine tetra-acetic acid, EDTA), and their lack of inhibition by serine β-lactamases. Aside the Ambler classification of beta-lactamases, the Bush’s classification of beta-lactamase enzymes further classified MBLs into a separate group of beta-lactamases mainly based on their functional properties such as their amino acid configuration and active site of the enzymes. It is noteworthy that all metallo-beta-lactamases hydrolyze imipenem. However, the ability of the MBLs or carbapenemases to hydrolyze or breakdown imipenem varies considerably amongst the different types of MBLs available.
The classification of MBLs at a molecular level and the standardization of their molecular structure are almost impossible because the MBLs are a disparate group of proteins. Generally, MBLs can either be plasmid-mediated or chromosomally-borne. Those MBLs that are plasmid mediated harbour transferable MBL genes that can easily be transmissible to non-MBL-producing bacteria in a given environment; and such a phenomenon spur the spread of carbapenem resistant bacteria in a given habitat. The transferable MBLs (i.e. the plasmid mediated MBLs) include IMI or IMP, SME, KPC, GES, VIM (for: Verona integrons encoded metallo-β-lactamases), GIM (for: German imipenemase), SPM (for: Sao Paulo metallo-β-lactamases) and New Delhi metallo-β-lactamases (NDM). They are in the molecular Class B1 of Ambler classification of enzymes and functional group 3 of Bush’s classification of beta-lactamases.
Chromosomal metallo beta-lactamases
Chromosomally-borne MBL-producing bacteria have their resistance traits in the chromosome of the organism. The genes that mediate carbapenem resistance in these organisms are rarely transferable to susceptible bacteria in the environment. Chromosomally-borne MBLs were initially detected in environmental and opportunistic pathogenic bacteria that had no hospital origin (Walsh et al., 2005). The first metallo-β-lactamases were detected in Bacillus cereus (BCI, BCII), Chryseobacterium meningosepticum (BlaB or GOB-1), Chryseobacterium indologenes (IND-1), Aeromonas spp (CphA) and Stenotrophomonas maltophilia (L1) as chromosomally encoded MBLs.
The chromosomally mediated enzymes are also often co-regulated with serine β-lactamases. For example, two species of a bacterium expressing MBLs can be found to produce two to three different antibiotic-hydrolyzing enzymes. In particular, both Aeromonas hydrophila and Aeromonas veronii bv. sobia produce three different beta-lactamases including penicillinase (which hydrolyze the penicillins), a cephalosporinase (which hydrolyze the cephalosporins), and an MBL (which hydrolyze the carbapenems). These enzymes are usually co-expressed and over-expressed in the bacteria when high-level beta-lactam resistant mutant strains are selected in the environment. SME, IMI and NMC are some commonly encountered chromosomally encoded metallo-β-lactamases (MBLs).
SME type MBLs
SME is the acronym for “Serratia marcescens metallo-β-lactamase”; and it denotes the MBL isolated or naturally found in the organism, Serratia marcescens. These enzymes were first discovered in S. marcescens isolates from England in 1982. They are currently three (3) SME types viz: SME-1, SME-2, and SME-3. These enzymes have been sporadically observed in S. marcescens isolates throughout the United States of America and in some other parts of the world. However, no clonal spread among these isolates has been observed.
IMI type MBLs
IMI is the acronym for “imipenem hydrolyzing beta-lactamases”. IMI enzymes was first discovered in an Enterobacter cloacae isolate in the United States of America in 1984; and the enzymes has since been rarely discovered in clinical isolates of E. cloacae in the U.S.A, France and Argentina. Several types of IMI enzymes including IMI-1 and IMI-2 exist in clinical pathogens. IMI-2 was reported in China, and the enzyme was plasmid encoded.
NMC type MBLs
NMC is the acronym for “not metalloenzyme carbapenemase”. NMC-A enzyme was isolated from Enterobacter cloacae isolates in France during 1990 and the enzyme was subsequently reported from the United States of America and Argentina. It is worthy of note that NMC-A and IMI have 97 % amino acid homology and are related to SME-1 with 70 % amino acid homology. Though these enzymes can hydrolyze extended spectrum cephalosporins, their rate of hydrolysis is comparatively less. Hydrolysis of cefoxitin by these enzymes is also inefficient; and cefoxitin and imipenem can also induce the production of these enzymes in a given pathogenic bacterium.
Transferable metallo beta-lactamases
Transferable MBLs are plasmid-borne (encoded) carbapenemases that are transmissible amongst bacterial species. Pathogenic bacteria expressing these enzymes possess transferable MBL genes that can be passed on to susceptible bacteria in a given environment. Transferable MBLs can also be called mobile MBLs because of the ease with which they could be transferred from one organism to another through mobile genetic transfer elements including plasmids and transposons. Transferrable MBLs was first discovered in Japan in a Pseudomonas aeruginosa isolate which possessed an IMP gene. Transferable MBLs include KPC, GES, IMP, VIM, SPM, GIM, SIM (Seoul imipenemase) and NDM.
Transferable or mobile MBLs have also been reported in Brazil, England, Hong Kong, Australia, Portugal, Malaysia, Canada, Italy, USA, Singapore and Taiwan. Apart from P. aeruginosa isolates from which the first transferable MBLs was discovered, these multidrug antibiotic degrading or hydrolyzing enzymes can also be harboured in Acinetobacter species, Klebsiella species, Enterobacter species, Escherichia coli, Proteus species, Providencia species, Shigella flexneri, Serratia marcescens and Alcaligenes xylosoxidans. Plasmid-borne MBLs have now attained public health significance because of their global spread; and the majority of these mobile MBLs are found as gene cassettes in these organisms.
KPC type MBLs
KPC is the acronym for “Klebsiella pneumoniae carbapenemase”. KPC was first isolated in a K. pneumoniae isolate from the United States of America in 1996. The resistance gene was associated with a large plasmid. There are currently over 12 known KPC enzymes. Though predominantly located or found in K. pneumoniae isolates, KPC enzymes have also been observed in K. oxytoca, Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa and Enterobacter cloacae. KPC enzymes confer resistance to all penicillins, cephalosporins, aztreonam and imipenem. However, bacteria harbouring KPC enzymes still remain susceptible to inhibition by beta-lactamase inhibitors such as clavulanic acid.
GES type MBLs
GES is the acronym for “Guyana extended spectrum”. GES type MBLs was first observed in a K. pneumoniae isolate from French Guiana in 2000; and the enzymes of the GES family differ markedly from each other by 1-4 amino acid substitutions. The genes encoding GES family of enzymes are located in integrons on the bacterial plasmids. GES type MBLs were initially taught to be ESBLs because of their exceptional ability in hydrolyzing extended spectrum cephalosporins. Currently there are over 22 known GES type MBLs; and these enzymes have also been observed in other bacteria aside K. pneumoniae including P. aeruginosa and E. coli isolates. GES type MBLs have also been reported in Greece, Portugal, South Africa, Japan, Korea and Argentina.
IMP type MBLs
IMP is the acronym for “imipenem”. IMP type MBLs are those class of MBLs that have action on imipenem. They are transferable carbapenem resistance enzymes that were first detected in P. aeruginosa isolates from Japan in 1990. IMP type MBLs have also been reported in Acinetobacter baumannii and Serratia marcescens isolates aside P. aeruginosa from which they were first detecte. Over 37 different known IMP type MBLs occur around the world; and these enzymes are common among members of the bacterial family Enterobacteriaceae and P. aeruginosa isolates.
VIM type MBLs
VIM is the acronym for “Verona integrons encoded metallo-β-lactamases”. It is a class 1 integron associated MBLs that was first observed in a P. aeruginosa isolate from Italy in 1997. VIM type MBLs is closely related to BCII with only 39 % amino acid homology. VIM type MBLs is the second dominant group of acquired MBLs and it is known to be resistant to a range of beta-lactams including piperacillin, aztreonam, imipenem and ceftazidime. Currently there are 34 known VIM type MBLs; and VIM-2 which was reported in a clinical isolate of P. aeruginosa from France is the most dominant MBL across Europe.
SPM-1 type MBLs
SPM is the acronym for “Sao Paulo metallo-β-lactamases”. SPM-1 was first identified in a Pseudomonas aeruginosa isolate from Sao Paulo in Brazil. Genetic analysis has revealed that it is not a part of any integron but is associated with a new type of transposable structure. blaSPM-1 is a part of mobile pathogenicity island located on a plasmid. SPM-1 type MBLs also has the ability to hydrolyze several beta-lactams including piperacillin, carbenicillin, penicillin, ampicillin, and cephalothin. However, SPM-1 type MBLs is susceptible to aztreonam and clavulanic acid, a beta-lactamase inhibitor.
GIM-1 type MBLs
GIM is the acronym for “German imipenemase”. GIM-type MBLs was first isolated from Germany in 2002. GIM type MBLs was isolated from P. aeruginosa clinical isolates in Germany; and they have no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine β-lactamase inhibitors.
SIM-1 type MBLs
SIM is the acronym for “Seoul imipenemase”. SIM type MBLs was first isolated from Pseudomonas aeruginosa isolates and Acinetobacter baumannii isolates during a large scale screening of imipenem resistant isolates in Seoul.
NDM-1 type MBLs
NDM is the acronym for “New Delhi metallo-β-lactamase”. NDM-1 was first reported in 2009 from a K. pneumoniae isolate obtained from a Swedish patient of Indian origin, and who had received medical treatment in India. NDM-1 is located on a 180 kb plasmid; and it expresses high-level resistance to all penicillins, cephalosporins, aztreonam, cefoxitin, carbapenems and ciprofloxacin. NDM type metallo-β-lactamase enzyme is only susceptible to colistin. NDM-1 enzyme shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4 % homology. Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins in particular, cefuroxime, cefotaxime, and cephalothin, and also to the penicillins. However, NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2. NDM-1 not only is a new subclass of the B1 group of MBLs but also it possess novel amino acids near the active site, thus suggesting that it has a novel molecular structure from the other MBL types.
Overview of Carbapenem-Resistant Gram-negative Bacteria
Carbapenems are broad-spectrum beta-lactam drugs that include imipenem, meropenem, ertapenem and doripenem. They are the most powerful class of antibiotics used for the treatment of bacterial infections caused by multidrug resistant bacteria including those that produce extended spectrum beta-lactamases. Carbapenems have a pyrrolidine moiety as a side chain, and they possess broader spectrum of antimicrobial activity than the penicillins and cephalosporins. They enter Gram-negative bacteria through outer membrane proteins (porins) and reach the periplasmic space of the target bacteria – where they permanently acylate or cleave to the penicillin-binding proteins (PBPs) of the target organism. Carbapenems have the ability to bind specifically to multiple different PBPs, and thus inhibit the peptide cross-linking of N-acetylmuramic acid (NAM) and N-acetyl glucosamine (NAG) – which are important reactions for the development of bacterial cell wall. However, the extensive and irrational use of this potent group of drugs (i.e. the carbapenems) has resulted in the emergence of carbapenem-resistant Gram-negative bacteria – that render inefficacious the antimicrobial activity of antibiotics in the group carbapenems.
Metallo-β-lactamase (MBL) is one of such enzymes produced by Gram-negative bacteria, and which allow these organisms to be resistant to the carbapenems. The weakening of bacterial cell wall and autolysis (which are both mediated by the antimicrobial activity of carbapenems) result in the death of the target pathogenic bacteria in vivo. The carbapenems including imipenem, meropenem, ertapenem and doripenem have the broadest antimicrobial spectrum than the penicillins, cephalosporins or beta-lactamase inhibitor combinations such as amoxicillin-clavulanic acid. They are effectively used to treat bacterial diseases caused by Gram-positive and Gram-negative bacteria especially those that are multiply drug-resistant in nature. Carbapenems can also be co-administered along with other antimicrobial agents for effective treatment outcome. But the excessive use of the carbapenems alters the intestinal microflora and thus selects for carbapenem-resistant bacteria that produces carbapenemases. Carbapenemases are carbapenem hydrolyzing enzymes, and they are broadly divided into two types based on the reactive site of the enzymes. Serine carbapenemases (Class A carbapenemases) and metallo-β-lactamases (Class B carbapenemases) are the two major types of carbapenemases that hydrolyze the carbapenems.
Klebsiella pneumoniae carbapenemases (KPC) is another group of the carbapenemases which mediate antibiotic resistance in Klebsiella species particularly K. pneumoniae. The emergence of carbapenem resistance in Gram-negative bacteria including E. coli, K. pneumoniae and P. aeruginosa significantly limits possible treatment options for treating life-threatening infections caused by these pathogenic bacteria. Pathogenic bacteria that harbour genes for the production of carbapenemases portend serious public health concern globally since the carbapenems are often the last line of drugs for the management of multidrug resistant infections. The fact that no newer broad-spectrum drugs are being developed against Gram-negative bacilli infections calls for the need to preserve these antibiotics through proper use. It is of utmost importance to detect these organisms from both hospital and environmental sources so that appropriate infection control practices can be implemented to control their spread and emergence in either the hospital environment or community.
Serine carbapenemases are Ambler Class A enzymes and they are chromosomally- and plasmid-mediated enzymes i.e. they are carried by the bacterial chromosomes and can also be transmitted via plasmids found in the same organism respectively. The Class A serine carbapenemases include SME enzymes (first discovered in Serratia marcescens), IMI enzymes (for: imipenem hydrolyzing β-lactamase), KPC (for: Klebsiella pneumoniae carbapenemase), NMC (for: not metalloenzyme carbapenemase) and GES (for: Guiana extended spectrum). The serine Class A carbapenemases are commonly found in Enterobacteriaceae including Escherichia coli and Klebsiella species. They are rarely found in Pseudomonas aeruginosa isolates.
DETECTION OF METALLO – BETA LACTAMASES (MLBs)
Metallo-beta-lactamases (MBLs) should be promptly and accurately detected in clinically important Gram negative bacteria especially P. aeruginosa and the Enterobacteriaceae. This is crucial, owing to the fact that MBL- and other carbapenemases-producing bacteria are multidrug resistant (MDR), and thus have the inherent ability to breakdown (hydrolyze) and confer resistance to the carbapenems – the last line of drug for the treatment of serious Gram negative infections including those mediated by ESBLs. There are no available standardized phenotypic detection methods for the detection of MBLs amongst clinically important bacteria, and according to a recent study, MBL detection depends likely on whether the MBL gene is carried by P. aeruginosa or a member of the Enterobacteriaceae i.e. the level of the antimicrobial susceptibility patterns of the test isolates to any of the carbapenems example imipenem and meropenem.
In other words, any phenotypic screening and confirmatory tests for the accurate detection of MBLs from clinically important isolates must take into account the genus of the bacterium to be screened and confirmed for MBL production, and also the level of resistance of the test bacterium as well. The Genus Pseudomonad’s intrinsically have higher carbapenem MICs and are thought to produce MBLs more frequently than the Enterobacteriaceae. The use of PCR and sequencing techniques still remains the gold standard for the detection of MBLs, but the accessibility of these molecular techniques is still limited to only reference laboratories in the western world and this becomes a problem for hospitals that are eager towards detecting MBLs from clinically important isolates here in Nigeria. The detection of MBLs enzymes just as other beta-lactamases is usually two fold: a phenotypic (non-molecular) detection method using carbapenems (imipenem or meropenem) and a genotypic (molecular) detection method which makes use of PCR and other molecular techniques.
The phenotypic detection methods for MBLs from clinically important Gram negative bacteria make use of tests such as disk approximation, disk diffusion, micro-dilution test, E-test, and carbapenem hydrolysis test – all of which are usually performed based on routine clinical microbiology antimicrobial susceptibility test with some modifications. These tests though limiting in detecting MBLs are very easy to perform unlike the molecular detection methods using PCR. Phenotypic detection of MBLs takes advantage of the enzymes (or MBLs) dependency on zinc by using chelating agents such as EDTA or mercaptopropionic acid to inhibit or stop the activity or production of MBL in the test bacterium. Since all MBLs are affected by a loss of zinc from the active site of the enzyme (i.e. MBLs), their detection in principle, should be a straightforward procedure in which various studies have used a variety of inhibitor-beta-lactam combinations to detect bacterial strains that produce these enzymes.
Ceftazidime and imipenem are the two substrates usually used to screen for MBLs in clinically important isolates, and the ability of MBL-producing organisms to confer resistance on these two substrates (imipenem and ceftazidime) varies a great deal. Enterobacteriaceae carrying MBLs are often carbapenem susceptible or intermediate and those enzymes can be missed when using imipenem or meropenem in detecting MBLs. Thus, there is no perfect beta-lactam inhibitor combination for the accurate detection of all transferable MBLs in clinically important Gram negative bacteria. Imipenem and meropenem can also be used to screen for MBL production in bacteria since MBL positive organisms tends to show reduced susceptibility when exposed to these carbapenems – an indication that these organisms harbor MBL enzymes.
The chelating agents (EDTA, 2 – mercaptopropionic acid, sodium mercaptoacetic acid) used in the phenotypic detection of MBLs in clinical isolates may be used alone or in combination to increase the effectiveness or potentiate the activity of either the ceftazidime or carbapenem used to screen for MBL production in the test bacterium, and it is also vital to test the chelating agent alone in order to make sure that it does not cause any false-positive result by inhibiting the test organism. In addition, the modified Hodges test (MHT) has been used by a number of researchers to detect MBL production in clinically important bacteria phenotypically. The only limitation of a MHT is that it cannot distinguish between MBL and KPC, but nevertheless, the MHT is a general screening test for the detection of carbapenemases in Gram negative bacteria.
The genotypic or molecular detection methods for the presence of MBLs in clinically important Enterobacteriaceae and P. aeruginosa is very different and a little bit complex than the phenotypic detection methods described above. They are usually used to substantiate or verify the results that ensue from a phenotypic detection method. The molecular detection methods for MBL production is the “gold standard” by which these all important enzymes can be detected. These molecular detection methods include: the use of PCR, DNA probes, cloning and sequencing techniques, and they look out for genetic elements that cause resistance in the test bacterium. Molecular detection of MBL gene is a more specific test for MBL detection and it has a high level of specificity than the phenotypic tests. They are only found in reference or research laboratories and are lacking in many microbiology laboratories in hospitals across the world. But nevertheless, Gram negative bacteria (including P. aeruginosa and the Enterobacteriaceae) that have been found to produce MBL enzymes phenotypically can be forwarded to either state or national reference or research laboratories anywhere in the world where molecular techniques can be used to verify the result of any phenotypic MBL detection methods.
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