The emergence and spread of Carbapenemase producing Enterobacteriaceae (CPE) is undoubtedly a huge public health problem, because CPE is often resistant to several antibiotics and has few antibiotic choices[1, 3, 17, 18].The production of carbapenemase is the most important mechanism of carbapenem resistance. Other mechanisms, such as overproduction or broad-spectrum of AmpC β- The production of lactamase can play a role together with the lack of outer membrane protein and the overproduction of some efflux pumps, thus conferring carbapenem resistance[19]. Previous study indicated that CPE was the primary type of CRE and the ratios varied from 77.3–91.3%[20–23]. Therefore, the rapid detection and identification of CPE is essential to help physicians quickly implement appropriate infection control measures, to adapt antibiotic treatment rapidly and to optimize care strategies and outcomes.
Generally, carbapenemases include Klebsiella pneumoniae carbapenemase (KPC), imipenemase (IMP), New delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-β-lactamase (VIM), and oxacillinase (OXA)-48-like enzymes, et al[17, 24]. In terms of treatment, the choice of drugs to treat CRE infection depends on specific carbapenemases[25]. For KPC and OXA-48-like enzyme, ceftazidime-avibactam can be the preferred agents[23, 26, 27]. If Enterobacterales isolates produce NDMs (or any other metallo-β-lactamase), the preferred antibiotic is ceftazidime avibactam combined with aztreonam[28, 29]. Therefore, given the distinctive features of different carbapenemases, rapid and reliable discrimination between these carbapenemases will provide valuable information for appropriate treatment.
In this study, 96.7% (237/245) of clinical CRE were carbapenemase-producing Enterobacterales strains, indicating the carbapenemase production is the prominent mechanism of CRE in Guangdong, China. 58.2% of Klebsiella pneumoniae producing KPC carbapenemase was the most common CRE. NDM-producing Klebsiella pneumoniae accounted for 30.4%. Significantly, NDM-type are the primary carbapenemase among Escherichia coli and Enterobacter cloacae strains, accounting for 46 (93.9%) and 20 (83.3%) respectively. In Chinese adults, KPC is reported as the most predominant carbapenemase (81%) of CP-kpn, while NDM-kpn is more prevalent in infants and neonates (61% ~87.2%)[30–32]. A study found that NDM accounted for 73.8% of Enterobacter cloacae carbapenemase, far lower than our research results, indicating that CPE has different molecular and epidemiological characteristics in different geographical regions[33]. Therefore, it is very important to monitor and master the characteristics of enzyme production of strains in each region. Moreover, carbapenemase co-producing strains poses an important diagnostic challenge. It is worth noting that in our study, seven isolates co-produced two types of carbapenemases, including NDM + IMP (n = 5), KPC + NDM (n = 1) and OXA-48 like + IMP, n = 1), which were also accurately identified by the two methods. Considering that the strains producing multiple carbapenemases at the same time are more and more common, for example, in Guangdong, we need to pay special attention to the simultaneous carrying of NDM + IMP.
Carba 5 and K-Set are two commercial immunological enzyme detection reagents for the detection of five major carbapenemases (KPC, NDM, VIM, IMP, and OXA-48). The Carba 5 is a multiplex immunochromatographic test that can detect the five major carbapenemases simultaneously in a single test cassette. K-Set is a monoplex immunochromatographic test that can detect the five major carbapenemases separately or simultaneously. In addition to being easy to use, they also showed results in 15 minutes. Our research shows that, compared with PCR method, K-Set is very suitable for identifying five major carbapenemases, with sensitivity and specificity of 100%. Another study has also shown that this kit could classify almost all carbapenemase-producing strains within its detection range for its high sensitivity (99.28%) and specifificity (100%)[18]. In the study results of Mustafa Sadek, the sensitivity and specificity of K-Set for detecting the 5 major carbapenemases were 100% and 98.8%, which are almost consistent with the results in the present study, respectively[34]. The sensitivity of 99.6% and specificity of 100% obtained by Carba 5 in this study also confirmed that it is a relatively accurate method for detecting carbapenemase, which is consistent with other studies [15]. A multicenter study in the United States shows that Carba 5 has 100% sensitivity and specificity in detecting five major carbapenemase families[35].
Recently, various KPC variants resistant to ceftazidime-avibactam have emerged in clinical settings. The most critical phenotypic features of KPC variants are their resistance to ceftazidime-avibactam and their restoration of susceptibility to meropenem or imipenem, which is mainly due to amino acid substitutions and conformational changes in the carbapenemase active site[36–39]. KPC-33, one of the new subtype of KPC have been detected in several regions and countries, is easy to be overlooked due to the inconspicuous characteristic of carbapenem resistance[36, 39]. In our study, we also identified a KPC variant, called KPC-145, whole genome sequencing confirmed this Klebsiella pneumoniae isolate (CZHKP-07) harbored a novel blaKPC gene with a point mutation (A to G) at nucleotide position 787 compared with the blaKPC−33 gene, this mutation resulted in the amino acid substitution of threonine to alanine (T263A), assigned by GenBank as blaKPC−145 (accession number: OP626310). However, this KPC variant was accurately detected by the K-Set, but failed detected by Carba 5. Currently, it has been documented that Carba 5 has poor diagnostic ability for KPC-33 variant[40]. It is believed that K-Set has certain ability to detect KPC variants, but we'll need more data to confirm that in the future. Thus, clinical laboratories must find appropriate detection methods to identify these strains, and should constantly improve existing detection methods to better detect new KPC mutations.
There are some limitations in this study. First, the richness of Enterobacter species in this study is limited. The present study focus only on K. pneumoniae, E. coli, and E. cloacae, which are the most common species in clinic. Second, the quantity and variety of carbapenemases is limited in the present study. No VIM-carbapenemase is determined in our experience while only 2 IMP-producing strains and 1 OXA-producing strains are included. Of particular concern, more IMP, VIM and OXA-producing isolates should be tested in further study.