Carbapenem Resistance
Ambika Arun1, Ajay pratap singh2
1Phd Scholar, Department of Veterinary Microbiology
Indian Veterinary Research Institute (IVRI) Izzatnagar, Bareilly, Uttar Pradesh
2Assistant professor, Department of Veterinary Microbiology
College of Veterinary Science and Animal husbandry (DUVASU)Mathura, Uttar Pradesh
Antimicrobial Resistance
Various researchers have suggested that bacteria could destroy antimicrobial agents by enzymatic degradation, even before the extensive use of antimicrobial Following the introduction of sulfonamides and penicillin around 1937 and 1940, resistance to sulfonamides and penicillin were reported within a few years (around 1945). Resistance to tetracycline, streptomycin, and chloramphenicol was found in the 1950s. Methicillin was introduced in 1959 and methicillin resistant S. aureus (MRSA) was identified in 1961. Antimicrobial resistance is one of the major public health problems especially in developing countries where relatively easy availability and higher consumption of medicines have lead to disproportionately higher incidence of inappropriate use of antibiotics and greater levels of resistance compared to developed countries (WHO 1996). Among gram-positive pathogens, a global pandemic of resistant S. aureus and Enterococcus species currently poses the biggest threat. The global spread of drug resistance among common respiratory pathogens, including Streptococcus pneumoniae and Mycobacterium tuberculosis, is epidemic. Gram-negative pathogens are particularly worrisome because they are becoming resistant to nearly all the antibiotic drug options available, creating situations reminiscent of the pre-antibiotic era. The most serious gram negative infections occur in health care settings and are most commonly caused by Enterobacteriaceae (mostly Klebsiella pneumoniae), Pseudomonas aeruginosa, and Acinetobacter .
Resistance to Carbapenem antimicrobials
Carbapenems are bactericidal β-lactam antimicrobials with proven efficacy in severe infections caused by extended spectrum β-lactamase (ESBL) producing bacteria. There are a few examples, namely imipenem, meropenem, doripenem, ertapenem, panipenem and biapenem, in use worldwide as a result of the rising resistance to cephalosporin antimicrobials in the Enterobacteriaceae group. Recent emerging mechanisms of resistance accumulate through the spread of carbapenem-destroying β-lactamases leaving narrow therapeutic options.
Mode of action of carbapenems is initiated first by penetrating the bacterial cell wall and binding to enzymes known as penicillin-binding proteins (PBPs). Transpeptidase inhibition as the main enzyme target of carbapenems during bacterial cell wall synthesis. Generally, carbapenems are preferred over other types of antimicrobials in treating invasive or life-threatening infections because of their concentration-independent killing effect on the infecting bacteria. They are broad-spectrum and act against Gram-positive, Gram-negative bacteria and including anaerobes.
Mechanism of development of Resistance to Carbapenem antimicrobials
Development of resistance to carbapenems may be due to intrinsic or acquired resistance mechanisms or both. Carriage of such genes was laterally exchanged to clinical pathogens and multiple mobilisation sequences including noncoding regions were identified in short-read sequence data from many soil bacteria. Bacteria have acquired multiple mechanisms of resistance including enzymatic inactivation, target site mutation and efflux pumps. Many of the acquired carbapenemases found in Enterobacteriaceae are plasmid mediated and have several ways of spreading amongst bacterial isolates. Apart from these enzymes, there are other important mechanisms conferring carbapenem-resistance that have been observed in recent times. First, the carbapenems least susceptible to hydrolysis (especially, imipenem and meropenem) can be destroyed in the presence of plasmid AmpCs in combination with ESBL enzymes associated with porin loss making Gram-negative bacteria insusceptible to carbapenem agents. Those strains having their porins mutated or their expression modulated typically do not have potential for mobilisation but may proliferate locally within hospitals. This type of mechanism is recognisable in Klebsiella, Enterobacter species and Escherichia coli as well as other genera. The overexpression of efflux pumps and loss of OprD porin are the most common mechanism of carbapenem resistance in Pseudomonas aeruginosa, notably to imipenem. Thirdly, non-carbapenemase specific mechanisms such as mixture of porin loss in addition with specific efflux pump systems have been reported in Acinetobacter isolates.
Molecular classification of carbapenemase enzymes
A large variety of carbapenemases have been identified in Enterobacteriaceae belonging to 3 classes of β-lactamases: the Ambler classes A, B and D β-lactamases
Class A carbapenemases: A number of this class of enzymes have been identified; some are chromosomally encoded – NmcA (not metalloenzyme carbapenemase A), SME (Serratia marcescens enzyme), IMI-1 (Imipenem-hydrolysing β-lactamase), SFC-1 (Serratia fonticola carbapenemase-1), with the others being plasmid encoded – KPC (KPC-2 to KPC-13), IMI (IMI-1 to IMI-3), derivatives (GES-1 to GES-20) of GES (Guiana extended spectrum), but all actively hydrolyse carbapenems and are partially inhibited by clavulanic acid. Of these, the KPCs are the most prevalent and after a few years of its discovery, had spread worldwide and caused outbreaks in many Asian, North American and European countries as well as in Africa.
Class B carbapenemases These enzymes are mainly in the class of β-lactamases having the ability to hydrolyse carbapenems but are susceptible to inhibition by EDTA, a chelator of Zn2+ and other divalent cations. The mechanism of hydrolysis depends on interaction of the β-lactam drugs with zinc ions in the active site of the enzyme. The most common metallo-βlactamase families include the NDM-1, IMP-type carbapenemases, VIM (V erona integron-encoded metallo-β-lactamase) GIM (German imipenemase), and SIM (Seoul imipenemase). The genes encoding these enzymes are often located within a variety of integron structures and incorporated in to the gene cassettes.
Class D carbapenemases These enzymes are serine-ß-lactamases poorly inhibited by EDTA or clavulanic acid. These carbapenemases are of the oxacillinase (OXA) enzyme type, and have a weak activity against carbapenems. The enzymes are found primarily in non-fermenter organisms such, Acinetobacter baumannii, Pseudomonas aeruginosa and rarely in isolates of Enterobacteriaceae family in most countries including the United K ingdom and in the United States
Laboratory detection of carbapenem-resistant organisms
The presence of a carbapenemase can be detected by a number of methods in clinical laboratories. These include automated systems or disc diffusion, minimum inhibitory concentrations (MICs), selective agar, modified Hodge test, synergy tests (e.g., E-tests or double disc tests) and molecular methods.
Phenotype based methods
The modified Hodge test has been used extensively and is a phenotypic technique for detecting carbapenemase activity routinely used in clinical pathology laboratories. This test was recommended by the CLSI in 2009 (CLSI 2009), however, it is not specific for the detection of all carbapenemase enzymes, most significantly with isolates showing weak positive results and AmpC producers.
Double-disc synergy testing has several versions; carbapenem with -clavulanate, – cloxacillin -EDTA or 2-mercaptoproionic acid and one which utilizes a double sided Etest, imipenem versus imipenem with EDTA, and are used as a screening test for MBL producers. This method is efficient for detection of MBL carbapenemases with high resistance, but may be deficient for detecting MBL 35 producers with low resistance to imipenem .
Carba NP test, derived from the name “Carbapenemase Nordmann-Poirel”, is in use to detect carbapenemase producers in Enterobacteriaceae. The Carba NP test rapidly and reliably identifies carbapenemase producers by changes in pH values using phenol red as the indicator. The test is 100% sensitive and specific for Enterobacteriaceae and 100% specific and 94.4% sensitive for Pseudomonas species harbouring carbapenemases.
Genotype based techniques
Molecular techniques have become an efficient tool for carbapenemase detection. These are mostly focused on the detection of genes in Enterobacteriaceae including their subgroups of carbapenemases. Real-time PCR assay with specificity and sensitivity at 100% as compared with 90% phenotypic KPC activity when assessed by modified Hodge test (MHT). More recently, multiplex PCR and microarray techniques for detection of several carbapenemase genes in one test have been produced. The use of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) as a new tool detecting resistance patterns in bacteria from fresh positive blood cultures.
