Carbapenemases in Enterobacteriaceae: Detection and Antimicrobial Therapy

1Carbapenem antibiotics are generally considered the most effective antibacterial agents for the treatment of multidrug-resistant bacterial infections. However, with the widespread use of carbapenem antibiotics, the prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has increased rapidly, and has become a serious threat to public health. The production of carbapenemases is the major mechanism underlying carbapenem resistance in CRE throughout the world. Carbapenemases are a kind of β-lactamase that can hydrolyze carbapenem antibiotics. According to the Ambler classification method, carbapenemases can be divided into classes A, B, and D. Class A and class D carbapenemases are serine β-lactamases, and class B carbapenemases are metallo-β-lactamases (MBLs). There is a large overlap between CRE and carbapenemase-producing Enterobacteriaceae (CPE), but the difference is that they were named according to the carbapenem-resistant phenotype and the resistance mechanism (carbapenemase production), respectively. The correct distinction of CRE and CPE and the rapid detection of CPE are important in the treatment and management of clinical infections. This article summarizes the epidemiology of CRE, the detection of CPE, and the status of clinical treatments.

2The widespread distribution of CRE is mainly attributable to their production of carbapenemases and the plasmid-mediated horizontal transmission of the encoding genes. The prevalence of CRE and the carbapenemase species involved are highly dependent upon the geographic region.

3In 2001, the United States first reported a Klebsiella pneumoniae (KPN) strain carrying a plasmid-mediated carbapenemase gene encoding a protein later designated K. pneumoniae carbapenemase (KPC). From then on, blaKPC have spread widely in the United States and South America. And the outbreaks of KPC-producing Enterobacteriaceae are reported in majority of European regions successively. In China, the first KPC-producing CRE strain was identified in 2007, and since then, blaKPC–2 has become the most widely spread carbapenemase gene. KPN was the main clinically isolated CRE producing KPC. Among the KPC-producing KPN, multilocus sequence typing (MLST) of most strains is clonal complex 258 (CC258), which indicated that CC258 obtained a KPC-encoding gene in the early epidemic of CRE and spread rapidly. The predominant sequence type (ST) in China is ST11, and ST258 is predominant in the United States while ST340, ST437, and ST512 predominate in other countries. Therefore, clonal transmission is considered the main mechanism by which KPC-producing KPN is disseminated.

4In 2009, blaNDM-associated carbapenem-resistant KPN was first reported in India. Since then, blaNDM has been detected in most species of Enterobacteriaceae. NDM-type β-lactamase mainly spread in Asia like India, Pakistan, Bangladesh, especially in China. In recent years, NDM has become the second commonest carbapenemase found among CRE in China, and blaNDM is more prevalent in Escherichia coli. Due to the horizontal transfer of epidemic broad-host-range plasmids, a high diversity of blaNDM-associated E. coli has been detected, among which ST131, ST167, and ST410 are the dominant types. Besides, blaIMP have spread throughout Japan since the IMP-1 was first discovered in Okazaki. At present, IMP-producing Enterobacteriaceae were found in Japan and Taiwan, China with the highest frequency. In other countries, the outbreaks or reports of blaIMP are sporadic. As for VIM, Greece is the epicenter of VIM-producing Enterobacteriaceae. Certainly, there are significant outbreaks in other parts of Europe such as the United Kingdom, Belgium, Spain, Italy, Hungary, and some Asian regions such as Taiwan, China, and South Korea. Moreover, the sporadic outbreaks of VIM-producing Enterobacteriaceae are globally reported.

5The class D β-lactamases, which function by splitting oxacillin, are designated oxacillinases (OXA). In 1985, the first OXA-encoding gene was found in an Acinetobacter baumannii isolate from the United Kingdom and designated blaOXA–23. Since then, a number of OXA family members have gradually been detected in the Enterobacteriaceae, including OXA-23-like, OXA-48-like, OXA-40-like, OXA-51-like, and OXA-58-like. The commonest class D β-lactamases is OXA-48, which was first identified in a KPN isolate from Turkey in 2001. OXA-48 includes classical OXA-48 and its variants, OXA-181 and OXA-23. CRE producing OXA-48 are mainly concentrated in European countries (France, Germany, Netherlands, Italy, the United Kingdom, and so on), Middle East (Turkey), and Mediterranean countries, including North Africa (mainly Morocco, Tunisia, Egypt, and Libya). Figure 1 has shown the global distribution of CRE that produce various carbapenemases.

6In the past few years, cases of multiple carbapenemases in the same Enterobacteriaceae isolate have been reported. For example, blaNDM–1 and blaIMP–4 coexisted in KPN, Enterobacter cloacae or Citrobacter freundii carried both blaKPC and blaNDM. Besides, there was a Klebsiella oxytoca isolate coexpressing three carbapenemases, KPC-2, NDM-1, and IMP-4, which was identified in 2017, and the plasmids containing these three resistance genes have emerged in most other members of the family Enterobacteriaceae, including E. coli, E. cloacae, and Klebsiella species.

7Initial susceptibility testing like broth microdilution techniques, the Kirby–Bauer disk diffusion method and automatic analysis systems were standardized and simple. But using the screening breakpoints recommended by the CLSI or EUCAST guidelines will miss the inefficient carbapenemases like KPC variants and OXA-48. Automated systems may cause discrepancies in the detection of all types of carbapenemase producers. Therefore, phenotypic assays and molecular-based techniques are the two main methods currently used to detect carbapenemases.

8The modified Hodge test (MHT) is a common phenotypic method for the detection of CPE. It is based on whether the growth of the indicator strain is enhanced at the junction of the inhibition zone and the growth line produced by the indicator strain and the test strain, respectively, and estimates whether the test strain has an inactivation effect on antibacterial drugs. The method has high sensitivity and specificity in detecting KPC-producing CRE but poor sensitivity in detecting class B β-lactamases (<50%). However, this limitation can be overcome by the addition of Triton X-100, which was proposed and called the Triton Hodge test. This method increased the sensitivity of the detection of NDM-producing clinical isolates to >90% and improved its performance in detecting other carbapenemases at the same time. But the false-positive and false-negative results will affect clinical judgment.

9subsequently developed a colorimetric assay, the Carba NP test, which is faster and has lower false-positive rate than MHT. In this test, the change in the pH of the reaction system caused by the carbapenemase hydrolysis of imipenem is monitored as the concomitant change in the color of phenol red, which is judged subjectively by the operator in the laboratory. Moreover, this method could preliminarily identify carbapenemases types based on tazobactam and EDTA. And then replaced phenol red with bromothymol blue as the pH indicator when they developed the Blue-Carba test, which improved the assay sensitivity from 93.3 to 100%. proposed an electrochemical method derived from the traditional assay, and designated it the Bogaerts–Yunus–Glupczynski (BYG) Carba test. This test reduces the time required from 2.5 h to about 30 min, and resulting from the real-time curve results, this test offers a real-time objective measurement of carbapenemase-producing isolates. Various commercialized products are also available, such as Rapidec Carba NP (bioMérieux), Rosco Rapid Carb Screen, and the Rapid Carb Blue Kit. A study suggested that most manual and commercial rapid colorimetric assays are insufficiently sensitive for the detection of OXA-48-type producers. In 2018, another study demonstrated that the MBT STAR-Carba kit (Bruker Daltonics), which is based on bicarbonate, displays higher sensitivity in the detection of OXA, but still cannot avoid undetected errors.

10The carbapenem inactivation method (CIM) is another effective phenotypic test. This method determines the carbapenemase activity of the tested bacteria by measuring the diameter of the inhibition zone of E. coli ATCC 25922 after the carbapenem disk is inactivated by the test bacterium. The results are highly consistent with the presence of carbapenemase genes, including those encoding KPC, NDM, VIM, IMP, OXA-48, and OXA-23, detected with polymerase chain reaction (PCR) (100% agreement for Enterobacteriaceae). The modified CIM (mCIM) became the CLSI-recommended method in 2017. A study indicated that both the sensitivity and specificity of mCIM were 100%. Because of its simplicity, clear criteria, cost-effectiveness, and availability in any laboratory, the mCIM has become a useful tool in microbiology laboratories.

11Many tests that rely on directly monitoring the hydrolysis of β-lactamases to detect CPE have been reported, including a spectrophotometric method, which is regarded as a reliable detection assay. But extracting the carbapenemases from the bacterial cells is time-consuming, and there were various factors reducing the veracity of the results. To overcome these limitations, developed a dual-wavelength measurement which could measure the hydrolytic activity of carbapenemases using bacterial cells directly. On the one hand, this method is time saving (about 40 min for preparation and incubation, but the time of detecting OXA should be prolonged appropriately). On the other hand, this method showed higher sensitivity and specificity than carbaNP at the same incubation time, and obtained consistent results upon mCIM. However, the requirement for a specific instrument (spectrophotometer) and the small sample size limit its clinical application.

12In 2011, proposed that matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) could be used to screen CPE by detecting the by-products of the hydrolyzed carbapenem. Since then, other groups have developed various MALDI–TOF-based methods to improve the sensitivity of the procedure, reduce the detection time, and facilitate the interpretation of the results. For example, aiming at the low sensitivity mainly resulting from the false-negative results obtained with OXA-48-type producers, added NH4HCO3 to the reaction buffer, which improved its sensitivity from 76 to 98%. To save time, developed a MALDI–TOF-based method that directly detects resistant Enterobacteriaceae from primary culture plates in <30 min and ensures high sensitivity and specificity. In 2018, a survey demonstrated that a MALDI–TOF-MS-based ertapenem hydrolysis assay rapidly and accurately detected the carbapenemase activity of Enterobacteriaceae strains in positive blood cultures. Although the costs of measurement using MALDI–TOF MS are low, the equipment remains expensive, which limits the wide application of this method in clinic.

13As well as all these methods, carbapenemase-inhibitor-based disc tests have been shown to detect carbapenemases. For example, combining boronic acid with an ertapenem or meropenem disk has been applied in detecting production of KPC. Adding ethylenediaminetetraacetic acid to a carbapenem disk makes it a useful compound in detecting MBLs. Ote et al. developed an immunochromatographic assay to directly detect OXA-48-like carbapenemase using a monoclonal antibody, and the results were obtained in a very short time. A bioluminescence-based carbapenem susceptibility detection assay was reported in 2018 that allows carbapenemase-producing CRE and non-carbapenemase-producing CRE to be distinguished with a sensitivity of 99% and a specificity of 98%.

14Tests based on molecular techniques are considered the gold standards for the identification of carbapenemase genes, the advantages and limitations have been summarized in Table 1. PCR is the most commonly used traditional molecular genotyping method. However, the traditional PCR method for identifying a single gene is time-consuming. Therefore, multiple PCR that was time-saving with high levels of sensitivity and specificity was proposed and developed. From 2006 to 2012, the multiplex real-time PCR systems have been initially established for the rapid detection of most carbapenemases like KPC, OXA-48, VIM, IMP, and NDM. Furthermore, various modified methods were proposed to overcome the inaccuracy caused by the diversity of OXA-48-like carbapenemases, such as a real-time PCR assay based on a high-resolution melt analysis, and a multiplex PCR assay using peptide–nucleic acid probes, which could identify resistance genes in a mixture of Enterobacteriaceae isolates with highly efficient.

15As well as the methods described above, several other molecular methods are used to detect CPE. For example, combined nested PCR, real-time PCR, and microfluidics to identify the common carbapenemases genes. A PCR-based method in a cartridge format developed to detect CPE in rectal swabs, which is run on the GeneXpert platform, displayed high sensitivity (96.6%) and specificity (98.6%) within a short time (32–48 min). developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB), which was highly efficient (100% sensitivity and specificity). In 2018, the microfluidic chip technology which allows the rapid detection of pathogens and their resistance genes was used to detect carbapenem-resistance genes, with high sensitivity and specificity (both >90.0%), and fully met the requirements for clinical diagnoses. Verigene Gram-negative blood culture assay, the microarray-based commercialized products, was available to identify the carbapenemases. But the materials cost is a little bit expensive approximately $60–80 per test. In addition, whole genome sequencing is the most reliable method for the detection of carbapenemase genes, but the high cost, long turnaround time, and difficult data management limit the routine clinical application of this method. also developed a novel multiplex PCR amplification reaction to directly and rapidly identify the epidemic CRKP ST258/ST11 strain. The advantages and limitations of common detection methods have been shown in Table 1.

16To the bests of our knowledge, almost all β-lactam antibiotics have limited effects on the treatment of CRE infections, and carbapenemases cannot be inhibited by traditional β-lactamase inhibitors. Some restricted drugs, such as polymyxins, tigecycline, and fosfomycin, may be active. A proportion of CRE strains producing KPC and OXA-48 are also sensitive to aminoglycosides (gentamicin and amikacin). However, there are significant deficiencies in the use of monotherapy to treat CRE infections with these antibiotics. Polymyxin has significant nephrotoxicity and neurotoxicity, and the optimal dose for treatment is unknown. This antibiotic has also been challenged by the emergence and global spread of mobilized colistin resistance (mcr) determinants. The presence of both mcr-1 and various blaNDM has been reported in Enterobacteriaceae isolates. The increased mortality risk conferred by tigecycline has led to warnings by the Food and Drug Administration. Furthermore, reports of clinical tigecycline resistance were published soon after its first use in medical practice. The resistance mechanisms that have been reported including mutations in tet and the increased expression of RND efflux pumps. Besides, tigecycline tends to inducing resistance during therapy. The therapeutic effects of aminoglycosides in CRE infections can be affected by rmtB which confers high-level and widespread resistance. The efficacy of fosfomycin is limited and resistance to this drug develops rapidly during treatment. Moreover, fosfomycin-modified genes play the key role in fosfomycin resistance. It is noteworthy that a carbapenem-, colistin-, and tigecycline-resistant E. coli strain carrying the fosA3 was reported in China in 2018, which poses a great threat to public health.

17For the reasons described above, several methods have been proposed to enhance the efficacies of these antibiotics, including aerosolized antibiotics for treatment with colistin and higher maintenance doses of colistin and tigecycline. These regimens did improve the therapeutic effects, but convincing evidence is sparse. In this context, combination therapies have been recommended to treat multidrug-resistant CRE infections. Not only the retrospective studies but also the in vitro tests and clinical applications have proved that the combination therapies were effective for the treatment of CRE. And the mortality rates associated with combination therapies especially the carbapenem-containing combinations were lower than those associated with monotherapy. By combining previous researches on combination therapies, several regimens were proposed in Table 2. However, the mechanistic basis of the synergy has not yet been established for most commonly used combination therapies.

18As well as the antibiotic combination treatments, novel β-lactamase inhibitors and antimicrobial therapeutics were developed to treat CRE infections and eliminate colonization. Avibactam (AVI) is a novel β-lactamase inhibitor that inhibits KPC, ESBL, AmpC, and OXA-48. Ceftazidime–AVI (CAZ–AVI) has been used in clinical treatments in the United States since 2015 and was recommended by CLSI in 2018. These combination is effective not only for strains producing KPC and OXA-48, but also for hypervirulent KPN carrying blaKPC–2. CAZ-AVI combined with ertapenem also successfully treated a patient infected with NDM-producing KPN. And clinical reports indicated that CAZ-AVI showed commendable therapeutic effect in treating complicated urinary tract or intra-abdominal infections. Comparing with colistin, CAZ-AVI showed better efficacy, lower mortality, and fewer side effects in treating KPC-producing CRE. However, CAZ–AVI-resistant isolates have been reported since 2015. To broaden the antibacterial spectrum, aztreonam–AVI was proposed, and effectively inhibited a variety of class A, B, and D carbapenemases. Another two novel carbapenem-β-lactamase inhibitor combinations, imipenem–relebactam and meropenem–vaborbactam, were developed to treat CPE infections. And the latter has been recommend by FDA1. In vitro data have indicated that the two combinations are highly active against KPC-producing Enterobacteriaceae but poorly susceptive against MBLs and OXA-type carbapenemases. And exact efficacy and safety must be defined with further clinical data. Besides, meropenem–nacubactam during clinical development have shown promising in vitro activity against KPC and MBL-producing CRE. Moreover, cefepime–zidebactam could inhibit CRE producing carbapenemases of classes A, B, and D, other cefepime-β-lactam enhancer such as cefepime–enmetazobactam (AAI101)/WCK-5153, etc. which were in earlier stages of development may represent a novel carbapenem-sparing option. Several other new drugs such as plazomicin, eravacycline, and cefiderocol developed to treat CRE infections are in various stages of development, among which plazomicin performed more potent effect and lower side effects than other aminoglycosides and eravacycline showed favorable clinical response and had well pharmacokinetics, tolerability, and in vitro activity. The application of cefiderocol needs further clinical data. In 2018, the injection products of plazomicin and eravacycline have been recommend by FDA2. However, due to the emergence of resistant isolates, enough attention should be paid to the development of drug resistance. The advantages, limitations, and mechanisms of resistance of novel antimicrobial therapeutics have been shown in Table 3.

19As well as novel drugs, various strategies for the management of carbapenem resistance have recently emerged. For example, based on studies of fecal microbiota transplantation (FMT) and enteric pathogens, FMT was hypothetically suggested to be used as a clearance method for CRE colonized patients, but the feasibility requires further study. Based on research into the mechanisms of antibiotic cytotoxicity, novel synthetic tools developed for the precise removal of genomic islands have been proposed to replace antibiotic treatments. Immunological-based therapies, such as monoclonal antibodies targeting poly-(-1,6)-N-acetyl glucosamine and cationic antimicrobial peptides, are also under investigation as substitutes for traditional antibiotics. The ability of predatory bacteria to reduce the primary pathogen in mammalian system has been demonstrated, which suggested the application prospect in clinic. The advantages and limitations of these main novel strategies have been summarized in Table 4.

20In recent decades, CRE have spread widely in various medical institutions around the world, and due to the time-consuming detection methods and limited treatment regimens, the mortality rates among patients are high. Therefore, the timely and accurate detection of CRE, especially CPE, is essential for the clinical treatment and prevention of infections. A variety of phenotypic methods and gene-based methods are available for the rapid detection of carbapenemases, and these are expected to be used routinely in clinical microbiology laboratories. At present, novel antibacterial drugs and emerging strategies which have been recommend or during development, with good activity and safety profiles, are expected to be applied to the clinical treatment of these infections in the near future.

21HD designed the study. XC and HZ performed data analysis and prepared the manuscript. All authors approved the final manuscript.

22The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.