The First Carbapenemase Genes Were Chromosomal Biology Essay


Carbapenemases are β-lactamase enzymes that are able to hydrolyse carbapenem (and other β-lactam-) antibiotics. Carbapenems are based on the naturally occurring product (thienamycin) of Streptomyces cattleya, a soil organism and carbapenemases, which may have originally existed to regulate cell wall production, could also have provided a selective advantage for other soil bacteria in a thienamycin containing environment. The first carbapenemase genes were chromosomal but with time, the genes for some of these enzymes have become plasmid and integron-borne and hence, more mobile. Carbapenemases are found within each of the four Ambler classes of β-lactamase, A-D. Classes A, C and D have a serine based functional site, whilst class B requires a zinc ion.

Class A carbapenemases include the NMC/IMI, SME, KPC and GES enzyme families. The NMC and IMI families which share 97% amino acid homology and have been isolated from Enterobacter cloacae, together with the SME (about 70% amino acid homology with NMC/IMI) which have been isolated from Serratia marcescens, are only seen occasionally in isolation or small clusters, probably because they are chromosomally encoded, although IMI-2 from the IMI family has been detected on plasmids in Enterobacter sp in the USA and China. Of more concern in this class are the KPC family (Klebsiella pneumonia carbapenemases). These are encoded by transferable plasmids and have been detected not only in Klebsiella sp, but also in other enterobacteriaceae including E.coli, and in Pseudomonas aeruginosa. KPC's are cause for unease because they are often found in organisms that are multidrug resistant via various mechanisms leading to limited treatment possibilities. Despite being integron-borne on plasmids, the GES family are a relative rarity, having been detected in a few P.aeruginosa and K.pneumoniae isolates at several world-wide locations.

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Class B carbapenemases - metallo-β-lactamases- contain both chromosomal and integron associated families. Chromosomal encoded carbapenemase genes have been found in Bacillus cereus (BCII), Bacteroides fragilis (CcrA) and Stenotrophomonas maltophilia (L1). With the exception of S.maltophilia which causes opportunistic infections, these carbapenemases are of little interest. The IMP, VIM, GIM and SIM genes are located within gene cassettes in integrons and move readily between gram-negative organisms, especially IMP and VIM in pseudomonads and enterobacteriaceae. The most recent family to be discovered in this class is the New Delhi metallo-beta-lactamase (NDM-1). This gene spread from India and Pakistan to the UK in 2008, becoming one of the most important mechanisms of resistance to carbapenems in 2009 in the UK. It has been found both within chromosomes and on plasmids, is seen in the UK mainly with K.pneumoniae. NDM-1 is linked in 60% of UK cases with travel to either India or Pakistan. It is associated with resistance to many antibiotic classes including aminoglycosides and fluoroquinolones, in addition to the β-lactam class. Generally these organisms are still susceptible to tigecycline and colistin.

Class C carbapenemases have only been discovered relatively recently in enterobacteriaceae and include the CMY and BER families. These are plasmid borne and therefore can transfer from organism to organism.

Class D carbapenemases contain the Oxacillin-hydrolysing (OXA) family, a large family of functionally similar enzymes, with varying amounts of amino acid similarity. These carbapenemases were first detected in strains of Acinetobacter baumanii and this organism is also where the majority of subsequent class D carbapenemases were discovered. OXA's are mostly plasmid-encoded and have been detected in K.pneumoniae and P. aeruginosa strains also. Genes encoding OXA's 54 and 55 may be chromosomal components of their host, Shewenella sp. This class, D, degrade carbapenems quite slowly and it is likely that additional resistance mechanism such as efflux pumps contribute to their success.

In vitro detection of carbapenemases is not always straight forward. Organisms showing reduced sensitivity to any carbapenem should be investigated for the presence of a carbapenemase, however phenotypic demonstration of these enzymes is not always obvious during disc diffusion or E-test MIC testing and there is the potential for them to go undetected. The Modified Hodge Test (MHT) is one common method of confirming suspected carbapenemase production. This involves laying a lawn of carbapenem-susceptible E.coli over a Muellar Hinton plate. A carbapenem disc, eg: Imipenem, is placed on the lawn and the test organism is streaked from the disc toward the edge of the plate. The plate is incubated overnight and if the test organism is producing carbapenemase, this will degrade the antibiotic along the streak and allow the sensitive E.coli to grow in these regions, producing an indented 'clover-leaf-like' zone. If carbapenemase is absent there will be no zone distortion. Variations of this method include using multiple discs (Meropenem, Imipenem and Ertapenem) and a single test organism on a single plate, or using a single disc and multiple test isolates and control organism on a single plate. This method can only detect the presence of a carbapenemase - it tells us nothing of its identity.

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Double ended E-tests are available for the detection of class B carbapenemases. Enzyme functionality of this class is dependant on the presence of the zinc ion at the active site and thus can be inhibited by the presence of EDTA which chelates zinc. These E-test strips have one end impregnated just with the carbapenemase, and the other end impregnated with both the antibiotic and EDTA. Absence of a zone (or reduced zone) around the end with only the carbapenemase, and presence of a zone around the end with the added EDTA is indicative of a metallo-β-lactamase. An expensive commercially produced KPC chromogenic screening agar is available, however both these methods are really only suitable for categorizing the carbpenemase after detection.

Automated systems such as VITEK, Microscan and BD Phoenix rely on a programmed set of rules for determination of sensitivity patterns. One study by Neil Woodford showed that OXA's were poorly detected, but detection of KPC's and metallo-β-lactamases were more reliable. Automated systems need to be set to flag up unusual isolates for supplementary testing such as the MHT , thus improving sensitivity and specificity. Ultimately PCR is the fastest and most accurate method of screening isolates for the presence of, and specific identification of a carbapenemase, but this method tends to be limited to reference laboratories, and novel enzymes may not be detected.

For many serious infections, carbapenems are the only remaining treatment option, but as carbapenemases become more mobile and more disseminated, and new ones emerge, treatment options become more limited. The mobility of some carbapenemase genes on transferable plasmids means world-wide spread has already occurred. Antibiotic development to combat these resistant bacteria is limited and as with MRSA and C.difficile, detection and prevention of transmission though good infection control practices is a better option than treating an established infection.