Antibiotic Resistance of the E. cloacae Complex
The in vitro activity of a variety of antimicrobial agents against the E. cloacae complex has been examined by numerous investigators, but the majority of studies are focused on E. cloacae, E. hormaechei and E. asburiae, which are the species most frequently isolated from clinical sources.
Table 2 shows the resistance mechanisms of species of the E. cloacae complex to each of the major classes of antibiotics in published reports.
E. nimipressuralis is a plant pathogen and has not been associated with human diseases. There are very few reports about this species; in 2010, a pseudobacteremia was reported to be caused by contaminated saline cotton at the venipuncture sites where blood was drawn. There have also been a limited number of reports regarding E. kobei infections of clinical significance; a case of nosocomial urosepsis has been described as being caused by an E. kobei strain that was Voges–Proskauer test-positive and resistant to β-lactams, ciprofloxacin, gentamicin and cotrimoxazole. The literature on E. ludwigii reports type strain EN-119T, as described above, and an imipenem-resistant E. ludwigii producing VIM-2 located on a plasmid. This microorganism was isolated from sewage water in a hospital of Mallorca (Spain), suggesting that environmental bacteria represent a reservoir for the dissemination of metallo-β-lactamase (MBL) genes.
Most isolates of the E. cloacae complex are susceptible to fluoroquinolones, trimethoprim/sulfamethoxazole, chloramphenicol, aminoglycosides, tetracyclines, piperacillin–tazobactam and carbapenems, while they are intrinsically resistant to ampicillin, amoxicillin, amoxicillin–clavulanate, first-generation cephalosporins and cefoxitin owing to the production of constitutive AmpC β-lactamase. In particular, fosfomycin seems to have a different activity against all species, because E. cloacae and E. asburiae are both naturally susceptible and resistant while E. hormaechei is only naturally sensitive.
The production of β-lactamases is the most important mechanism responsible for β-lactam resistance in most of these species. This phenotypic group of enzymes is a heterogeneous mixture of β-lactamases belonging to the four molecular Ambler classes: class A (penicillinases), class B (metalloenzymes), class C (cephalosporinases) and class D (oxacillinases).
All kinds of β-lactamases are found in E. cloacae and, to a lesser extent, in E. hormaechei and E. asburiae.
These microorganisms are capable of overproducing AmpC β-lactamases by derepression of a chromosomal gene, or by the acquisition of a transferable ampC gene on plasmids or on other mobile elements, commonly named as being AmpC plasmid-mediated. The AmpC plasmid-mediated strains are distinguished from chromosomal strains because, with only a few exceptions, they are not inducible. The AmpC plasmid-mediated strains represent a problem because the derepression of this enzyme is increasingly frequent among clinical isolates and confers resistance to third-generation cephalosporins and ureido- and carboxy-penicillins, and they are not inhibited by common inhibitors of β-lactamases, such as clavulanate, but by boronic acid and/or cloxacillin instead. Fourth-generation cephalosporins retain reasonable activity against derepressed strains, but if they are also extended-spectrum β-lactamase (ESBL) producers, they become resistant to this class of antibiotics. ESBLs and AmpC-type cephalosporinases that can hydrolyze third-generation cephalosporins such as cefotaxime or ceftazidime confer resistance more frequently to E. cloacae strains isolated in patients who previously received these antibiotics.E. hormaechei is also an AmpC producer, and it has been recently demonstrated that the plasmid–mediated AmpC blaACT-1 gene originated from the chromosome of E. hormaechei, which was previously believed to derive from E. cloacae.
ESBLs are of molecular class A, in the classification scheme of Ambler. They are β-lactamases capable of hydrolyzing penicillins, first-, second- and third-generation cephalosporins and aztreonam (but not cephamycins or carbapenems), and are inhibited by β-lactamase inhibitors such as clavulanic acid.
Moreover, recent studies suggest a high prevalence of ESBL production in these species, thus limiting the choice of antimicrobial agents capable of treating invasive infections.
From a clinical point of view, the discrimination between ESBLs and overproduced class C β-lactamases may not be critical, since the therapeutic options for infections caused by organisms that possess any of these mechanisms of resistance are similarly limited. Nevertheless, the detection of such 'hidden' ESBLs is still of epidemiological importance for the hospital environment. The detection of ESBLs by methods based on the inhibitory effects of clavulanic acid (conventional double-disk synergy test [DDST] and Vitek ESBL detection test) is expected to be difficult and is dependent on the level of chromosomal enzyme production. Studies by Tzelepi et al. suggested that the ESBL detection methods used are inadequate in cases of overproduction of Bush group 1 β-lactamases.[60,61]
In a recent report, the Vitek ESBL detection test, as well as the conventional DDST, were unable to detect the SHV-5 β-lactamase in a Klebsiella pneumoniae strain that produced a plasmid-borne AmpC-type β-lactamase; the ESBL present in that strain had been successfully detected by a DDST that combined amoxicillin/clavulanate with cefepime. In accordance with this observation, the use of cefepime increased the sensitivity of the DDST for the detection of ESBLs in Enterobacter from 16 to 61% when the disks were applied at the standard distance of 30 mm from clavulanate, and from 71 to 90% with closer application of the disks.
The findings of this study indicate that the frequency of ESBLs can easily be underestimated in Enterobacter spp. characterized by a high prevalence of derepressed variants. For such situations, the application of DDSTs that combine amoxicillin/clavulanate with cefepime may increase the possibility of ESBL detection because they are important factors to be considered in the management of antibiotic-resistant E. cloacae infections, and their spread in the community is a general concern.
E. cloacae may express ESBLs that are not closely related to the TEM or SHV type, including CTX-M- and VEB-type ESBLs.[62–64] VEB-producing Enterobacteriaceae have been predominantly found in Vietnam and Thailand; an E.cloacae outbreak was recently documented in China. Another important factor that concerns ESBLs is that they are typically plasmid-mediated, rather than chromosomal-mediated β-lactamases; this is of great concern given their easy spread.
In 2005, a study by Ho reported bloodstream infections by E. hormaechei isolates producing CTX-M-, SHV- and TEM-related enzymes. Of the E. hormaechei (n.539) isolates, 17.9% were ESBL-positive, which was frequently attributed to blaCTX-M genes. In a neonatal unit of an African hospital, CTX-M 15 E. hormaechei strains were also detected from powdered milk.
The E. cloacae complex produces not only ESBLs, but also β-lactamases with carbapenemase activity that have a broader range activity and may be grouped as either serine carbapenemases (classes A and D) or MBLs (class B) according to Ambler classification.[67,68]
For the screening of carbapenemase-producing Enterobacteriaceae, CHROMagar™ KPC (Hy-Labs, Israel) commercial medium could be used, where Enterobacter spp. appears as metallic blue colonies. The phenotypic tests for the confirmation of carbapenemases production are modified Hodge test (classes A and D), the combined disk of meropenem ± boronic acid (class A) and the combined disk of meropenem ± dipicolinic acid or meropenem/imipenem ± ethylene diamine tetra-acetic acid (class B).[70,71]
Carbapenemase-producing E. cloacae are currently relatively rare, but there are concerns about their emergence and spread. The class A carbapenemases appeared sporadically in clinical isolates since their first discovery over 20 years ago.[72,73] They have the ability to hydrolyze a broad variety of β-lactams, including carbapenems, and are usually inhibited by clavulanate and tazobactam. They can be chromosome-encoded, such as NMC-A, SME, SFC-1 and BIC-1, plasmid-encoded, such as KPC and GES, or both, such as IMI. NMC-A (not metalloenzyme carbapenemase) was detected for the first time in 1990 in Paris (France) and appeared in Argentina in 2000 and in the USA in 2003.[74–76] The chromosome-borne IMI-1 (imipenem-hydrolyzing β-lactamase) enzyme appeared for the first time in E. cloacae strains in 1984 in California and the most recently in 2011 in France, while plasmid-encoded IMI-2 was isolated in 2001 in China.[78,79] The IMI and NMC-A enzymes have been detected in rare clinical isolates of E. cloacae in the USA, France and Argentina.[76,80,81] NMC-A and IMI-1 have 97% amino acid identity. The IMI-2 enzymes were also found on plasmids in 22 imipenem-resistant E. asburiae that naturally produce a cephalosporinase, but not carbapenemase. In these strains, which were isolated from US rivers sampled from 1999 to 2001, an upstream LysR-type regulator gene was identified; this regulation can be responsible for the inducibility of IMI-2 expression. β-lactamase IMI-2 is therefore the first inducible and plasmid-encoded carbapenemase. The E. asburiae isolates were clonally related, belonging to a single pulse-field type, although they were obtained from distantly related mid-western rivers; the identification of clonally related E. asburiae isolates from distant rivers indicates an environmental and enterobacterial reservoir for carbapenemase genes.
The GES/IBC family of β-lactamases is an infrequently encountered family of enzymes that was first described in 2000 with reports of IBC-1, a novel class A ESBL that was encoded by a class 1 integron-associated gene (integron-borne cephalosporinase) from an E. cloacae isolate in Greece. This integron was located in a multidrug-resistant (MDR) transferable plasmid found in an E. cloacae clinical strain.
The most recently reported class A enzyme with carbapenemase activity is KPC, which is found mostly on plasmids in K. pneumoniae; these enzymes also hydrolyze the cephalosporins, such as cefotaxime. Although the KPC β-lactamases are predominantly found in K. pneumoniae, there are few reports of the presence of these enzymes in E. cloacae. KPC-2 and KPC-3 determinants were found in 2001–2003 in New York from a patient with renal failure and continuous bacteremia.[81,84]
Another mechanism of carbapenem resistance observed in E. cloacae is MBL production. These enzymes are capable of hydrolyzing all β-lactams, except monobactams, and are not susceptible to therapeutic β-lactamase inhibitors such as clavulanate, sulbactam and tazobactam.
MBLs can be either chromosomally encoded or plasmid-mediated, and most of them are inserted in integrons. The most commonly acquired MBL enzymes include IMP, VIM, SPM, GIM, SIM and NDM; IMP and VIM are common in Pseudomonas aeruginosa and other nonfermenting Gram-negative bacteria. The first appearance of MBLs in E. cloacae were reported in E. cloacae clinical isolates from various geographical areas, with IMP-1 from Turkey, IMP-8 from Taiwan, VIM-2 from Korea, VIM 1–4 from Italy[89–91] and VIM-12 from Greece.
Even if these papers describe the presence of the enzymes, alone or associated with other determinants, they are probably under-reported due to the difficulties related to their recognition by standard susceptibility tests; in some cases, only ertapenem showed a reduced susceptibility phenotype. These findings are concordant with some reports that analyzed the microbiological features of VIM-1-producing Enterobacteriaceae, in which ertapenem was shown to be a better indicator for phenotypic carbapenemase detection.[93,94] In an Italian study, most of the VIM-1-producing E. cloacae had imipenem and meropenem MICs within the range of susceptibility, while ertapenem MICs were between 0.25 and 8 mg/l. The patients included in this study, who were treated with carbapenem, had therapy failure, which led to the investigation of their mechanisms of resistance. All strains were VIM-1 producers, and the discrepancy with phenotypic tests was explained by low expression levels of the blaVIM-1 gene that is related to an inactivated P2 promoter and to a weak activity of another promoter, P1, which drives the expression of the gene cassette inserted into the class 1 integron, which was detected in all E. cloacae isolates carrying VIM-1.
The latest MBL detected is New Delhi MBL found in K. pneumoniae and Escherichia coli isolates from a patient coming from India and hospitalized in Sweden in 2008. This enzyme hydrolyzes all β-lactam antibiotics except for aztreonam, which is usually inactivated by coproduced ESBLs or AmpC β-lactamases. The rapid spread of this gene, which has already been described in several species of Enterobacteriaceae, has also been detected in E. cloacae worldwide.[96,97]
Acquired class D β-lactamases possessing carbapenemase properties were previously identified mainly in Acinetobacter spp. and occasionally in Enterobacteriaceae. The chromosome-encoded oxacillinase OXA-23 was previously described for Proteus mirabilis, and the oxacillinase OXA-48 was first identified in a K. pneumoniae isolate from Turkey with a worldwide spread. In addition to K. pneumoniae, E. coli, Citrobacter freundii and Providencia rettgeri, OXA-48 has been identified in E. cloacae in different countries.[98–101]
Finally, the resistance to carbapenems may be due to the hyperproduction of an AmpC-type cephalosporinase or ESBL combined with decreased drug permeability through the outer membrane, and carbapenem-hydrolyzing β-lactamases. The hyperproduction of chromosomal cephalosporinase combined with porin alterations confers imipenem resistance to clinical isolates of E. cloacae.[103,104]
The E. cloacae complex has also acquired resistance to another widely used class of antibiotics – fluoroquinolones – which are extensively used in clinical therapy of sustained Enterobacteriaceae infections. These antibiotics act by interacting with type II topoisomerases (DNA gyrase and topoisomerase IV). Related to this mechanism of action, bacteria have developed resistance mechanisms consisting of target mutations GyrA/GyrB for DNA gyrase and ParC/ParE for topoisomerase IV, or in reduced access to the target itself, by either decreased permeability or augmenting expression of efflux pumps, such as AcrAB and MexAB.[105,106] The genes encoding these mechanisms are of chromosomal origin, but since 1998, plasmid-mediated quinolone-resistance genes have been reported in clinical Enterobacteriaceae isolates. These genes are qnr (qnrA, qnrB, qnrS, qnrC and qnrD), aac(6')-Ib-cr and a plasmid-mediated efflux pump qepA, which confer low-level resistance to quinolones[108–110] and have an additive effect on the level of resistance caused by other mechanisms. The literature reports quinolone resistance in E. cloacae and E. hormaechei (Table 2).
From the epidemiological point of view, in the last few years, quinolone resistance has increased in E. cloacae isolates worldwide.[108,109,111] The qnr genes were found to be cocarried with various ESBLs or AmpC-type β-lactamases on the same plasmid,[112–114] as well as with MBLs.
Unfortunately, increasing in vitro resistance of ESBL producers to fluoroquinolones is limiting the role of these important classes of antibiotics for the future. In a recent report, the association of qnrB2 with blaKPC-2 carbapenemase was described on a single plasmid in two genetically unrelated clones of E. cloacae and this report highlights a very worrying problem for future treatment of these infections.
A nationwide outbreak in The Netherlands was caused by E. hormaechei strains carrying a conjugative plasmid pQC, with several complex integrons containing qnrA1, aadB, blaCTX-M-9 and tetA genes.
With regard to another class of antibiotics – the aminoglycosides – the major mechanism of resistance of the Enterobacteriaceae is usually attributable to aminoglycoside-modifying enzymes that are often plasmid-encoded, but may also be associated with transposable elements. Plasmid exchange and dissemination of transposons facilitate the rapid acquisition of resistance phenotypes. These enzymes are assigned to three groups: acetyltransferases (acetylation of an amino group/AAC), phosphotransferases (phosphorylation of a hydroxyl group/APH) and adenylyltransferases (adenylylation of a hydroxyl group/AAD or ANT).
Among these, the aminoglycoside AAC(6')-Ib is the most common cause of amikacin resistance among members of the Enterobacteriaceae family. In a previous study, it was observed that over 40% of the E. cloacae isolates had the aac(6')-Ib gene, although many of the isolates with this gene were susceptible to amikacin and gentamicin, which were the most active of all drugs tested. The association between aac(6')-IIc and blaVIM-1 has been reported in the same integron previously described in P. aeruginosa and E. coli, suggesting extensive spread of resistance genes between various Gram-negative species.
A valid alternative to carbapenems and fluoroquinolones is tigecycline, especially against clinical isolates exhibiting MDR phenotypes. Tigecycline, a novel broad-spectrum glycylcycline antibiotic derivative of minocycline, has the ability to evade common mechanisms of resistance to tetracyclines expressed in Gram-negative and Gram-positive bacteria. The vast majority of studies evaluating the in vitro susceptibility of tigecycline against species belonging to the E. cloacae complex are focused on E. cloacae, while there are no data on other species of the E. cloacae complex. A recent paper reporting results from studies on 23,918 Gram-negative isolates from intensive care units worldwide between 2004 and 2009 demonstrated excellent activity of tigecycline against 3789 E. cloacae isolates collected, with a susceptibility >90% for all regions, while the largest variation in susceptibility was seen for minocycline (from 79.2% in North America to 59.0% in the Middle East). Resistance to minocycline increased from 2004 to 2009 between 24 and 30.8% among isolates of E. cloacae collected from North America, Europe, the Asia–Pacific rim and Latin America. Middle East and Africa were excluded because of the low numbers of isolates.
Although tigecycline has good in vitro activity against E. cloacae, a few clinical strains of E. cloacae with decreased susceptibility to tigecycline have been recently described, resulting from their RamA-mediated overexpression of the AcrAB efflux pump.[122,123]
A recent case report describes a tigecycline-resistance induction under tigecycline therapy in an E. hormaechei strain isolated from a patient after liver transplantation.
Over the last few years, an old antibiotic, colistin, has been reintroduced in clinical therapy because it is active against several MDR aerobic Gram-negative bacilli, including Enterobacter spp.. The limited usage of colistin in the 1980s was due to reports of a high incidence of toxicity,[126,127] which may have been due to inappropriate patient selection and monitoring. Early clinical papers report the results of pharmokinetic studies to reduce and optimize the doses of colistin, which remains a valid alternative for the treatment of infections caused by MDR enterobacteria.
The data on the mechanisms of resistance to polimixins in Enterobacteriaceae suggest that they are due to the changes in negatively charged surface lipopolysaccharides induced by the regulatory loci pmrA and phoP, generating resistance to these antibiotics. Previously published data report a case of heteroresistance to colistin in E. cloacae isolates and two colistin-resistant, VIM-1-producing E. cloacae strains isolated from a patient with a complicated urinary tract infection.[91,131]
Future Microbiol. 2012;7(7):887-902. © 2012 Future Medicine Ltd.