Comparison between detection power of MBT STAR-Carba test and KBM CIM Tris II for carbapenemase-producing bacteria

Introduction

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a valuable analytical technique for microbial identification owing to its speed and simplicity [1]. MALDI-TOF MS has recently been utilized for detecting carbapenemase activity [2]. The commercially available MBT STAR-Carba test can identify carbapenemase activity by detecting the loss of native carbapenems and/or production of carbapenem hydrolysis products after incubation with carbapenem molecules. Carbapenemases are classified into three groups according to the Ambler classification system: (i) Ambler class A includes KPC and some GES variants, (ii) Ambler class B consists of metallo-β-lactamases such as VIM, IMP, and NDM, and (iii) Ambler class D includes OXA-48 [3]. While studies have confirmed the effectiveness of the MBT STAR-Carba test, the predominant types of carbapenemases differ between countries and regions [4], [5], [6]. This study evaluated the MBT STAR-Carba test on carbapenemase-producing bacteria isolated from Kobe City, Japan, and compared its performance with the KBM CIM Tris II kit (Kohjin Bio Co., Ltd., Saitama, Japan), which has a shorter reaction time than the modified carbapenem inactivation method (mCIM), and can be used for Acinetobacter and Pseudomonas species as well as Enterobacterales, and assessed its detection capabilities using modified procedure parameters [7].

Materials and methods

Results and discussion

All strains, except those harboring bla_GES-5, bla_GES-24, or bla_OXA-66 tested positive for carbapenemase using the KBM CIM Tris II kit (Table 1). For strains that were not active for 30 min, the incubation time was extended, and the respective positive (hydrolyzing) and negative (non-hydrolyzing) control strains at 30 min were used for the calculation. IMP-1, IMP-6, NDM-1, NDM-5, KPC-2, and OXA-181 were detected after an incubation time of 30 min in the MBT STAR-Carba test (Figure 1). IMP-1 shows high degradation activity for imipenem and meropenem, while IMP-6 shows higher degradation activity for meropenem, but not for imipenem, owing to the different susceptibilities of IMP-1 producers to carbapenem antibiotics [14]. Nonetheless, the average logRQ values on IMP-1 and IMP-6 were 0.99 and 0.94, respectively, suggesting no difference in hydrolyzing activity in the MBT STAR-Carba test. Among the GES-type β-lactamases, those with a replacement of glycine at position 170 with an asparagine or serine (such as GES-5 and GES-24) exhibit carbapenem-hydrolyzing activity [15]. Three GES-5 producing Serratia marcescens strains caused an intensive care unit outbreak that was negative for KBM CIM Tris II and harbored bla_GES according to PCR screening [13]. MBT STAR-Carba activity showed differences among the three strains after 30 min of incubation (Table 1). Two strains (KA0048 and KA0054) tested positive at 30 min. Meanwhile, MBT STAR-Carba activity of the KA0044 strain was negative at 30 min but turned positive after 2 h of incubation (Figure 1). GES-24 producing R. ornithinolytica and K. pneumoniae strains were negative for KBM CIM Tris II. However, MBT STAR-Carba activity of these strains increased after 2 h. There was no change in the activities of the strains used as negative or positive controls, and ESBL strains when the incubation time was extended (Figure 1). Therefore, some GES-type carbapenemases are difficult to detect by reaction with inhibitors or KBM CIM Tris II, while the MBT STAR-Carba test enabled their detection after incubation for over 30 min.

Figure 1:

Graph showing the dependence of the determined carbapenemase activity level (normalized logRQ) on type of β-lactamase as well as the incubation time.

Two Acinetobacter baumannii strains (MDRA1 and MDRA2) isolated from two patients in the same hospital were resistant to ciprofloxacin (>32 μg/mL) and amikacin (>256 μg/mL) and showed low-susceptibility to carbapenems. These strains confirmed the presence of the bla_OXA-51-like variant, bla_OXA-66. ISAba1 was present upstream of bla_OXA-66 in two isolates, leading to bla_OXA-66 expression. We detected the induction activity of bla_OXA-66 against carbapenems by incubation for 2 h in the MBT STAR-Carba test.

Thus, there were differences in the detection of carbapenemase between the KBM CIM Tris II and MBT STAR-Carba tests. The KBM CIM Tris II and MBT STAR-Carba tests test for carbapenemase activity with meropenem and imipenem, respectively; differences in responsiveness between these tests may be due not only to differences in antimicrobial reactivity, but also to a combination of factors, including bacterial species and expression levels.

In summary, we revealed that the MBT STAR-Carba test can rapidly detect not only major carbapenemases, prevalent IMP-1 and IMP-6 in Japan, but also GES-type and OXA-51-like carbapenemases that are difficult to detect by reaction with inhibitors or KBM CIM Tris II. The MBT STAR-Carba test will help to rapidly investigate the presence of carbapenemases in clinical settings and environmental investigations.