The worldwide dissemination of KPC-type carbapenemase-producing Enterobacterales requires rapid identification methods for timely decision-making in critically ill patients. the indirect MALDI-TOF detection method through the detection of the 11,109 Da peak provides a rapid option for identifying the presence of these enzymes.

This study confirmed that MALDI TOF VITEK MS provides quick results due to the joint species identification of the microorganism and the 11,109 Da peak in the generated spectrum, indicating the indirect presence of KPC-type carbapenemase-producing microorganisms associated with Tn4401a. Thus, the applicability of this methodology may be dependent of the epidemiology and the genetic background of the KPC producing isolates in a specific setting. This rapid result should directly impact therapeutic decision-making for the initial management of patients, decreasing the time of hospitalization with significant savings in time and costs of diagnosis [3, 10].

The MALDI TOF VITEK MS diagnostic performance for identifying KPC found in the present study showed a sensitivity for the indirect MALDI-TOF detection method similar to values reported in other studies describing sensitivities ranging from 85.1 to 98.7% and specificities ranging from 99 to 100% [17, 19, 20, 26, 27]. The advantage of the present study, compared to others that have evaluated the indirect MALDI-TOF VITEK MS detection method, is that the isolates used were from different institutions, decreasing the possibility of a bias effect due to the potential clonality of nosocomial isolates from a single institution [19, 20].

In this study, seven isolates that were negative for KPC were identified as positive by the indirect MALDI TOF method. False positives may be explained by the presence of a protein of similar molecular weight as P019 with an analogous peak in the spectrum. In addition, this finding could also be explained by the independence in mobilization demonstrated between the tn4401a transposon, the ISKpn31 sequence and its possible presence in plasmids not related to the presence of KPC-type enzymes (an aspect that should be verified by whole genome sequencing analysis) [28, 29]. Several studies in Colombia have described the co-circulation of blaKPC genes associated with Tn4401 transposon isoforms a and b and with elements other than Tn4401 [8, 30]. This scenario suggests that identifying the presence or absence of the 11,109 Da peak in the MALDI-TOF MS spectrum is helpful according to the epidemiological setting of the blaKPC gene associated with the Tn4401a transposon. In the case of settings where other mechanisms mediate resistance to carbapenems or there is a circulation of different transposons carrying blaKPC, the presence of the 11,109 Da peak by MALDI TOF Vitek MS can confirm the presence of pKpQIL KPC-type carbapenemases, with a high positive predictive value, however, a negative result does not rule out resistance to carbapenems, and additional methods for detecting carbapenemases are needed.

The evaluation of the diagnostic performance for the mCIM/eCIM carbapenem inactivation test showed a sensitivity of 89.58% and a specificity of 97.14%, like those reported for other studies [31]. Although this technique has the benefit of differentiating metallo-carbapenemases and is less expensive [23], it is laborious and slow, involving a 4-hour incubation time during the inactivation step and a subsequent overnight incubation.

Rapidec CARBA NP also showed high sensitivity and specificity values (100% and 95.7%, respectively), like those described in other studies [31]. These performance values, together with the rapid identification of carbapenemase-producing bacteria, make this test a good option in the clinical setting for an initial approach to carbapenemase identification; however, since this technique does not differentiate the type of enzyme, it may require the use of additional discriminatory tests, especially in settings like Colombia where both metallo-carbapenemase and serine carbapenemases are endemic. In addition, when using this test, it is necessary to consider that low sensitivity for OXA48-type carbapenemase identification has been reported, and its visual interpretation, based on color variation, may have a degree of subjectivity [31, 32]. OXA-48-producing Enterobacteriaceae have a very low prevalence in Colombia but are endemic in other setting. In our study, in 10 out of 168 isolates, there was no agreement between the three observers when using RAPIDEC CARBA NP, and the kappa index was 0.91.

The comparison of the three methods used for the detection of the production of KPC-type carbapenemase showed good agreement and a similar AUC of the ROC curves. For rational use and considering the characteristics of the evaluated tests, a diagnostic algorithm may be implemented to establish the presence of carbapenemase-producing isolates. This algorithm should consider an early screening test to be the indirect MALDI TOF detection method, which allows the detection of the 11,109 Da peak together with the identification of the bacteria at no additional cost in an average time to result of 30 min, when a positive result is obtained, the presence of a KPC-type carbapenemase can be confirmed and reported with a high positive predictive value. Due to the low negative predictive value, a negative result by MALDI-TOF should be followed by performing the RAPIDEC CARBA NP test, which allows confirmation of carbapenemase presence or absence in a maximum time of 2 h. A negative result rules out the presence of any enzyme, but a positive result should be followed by eCIM/mCIM tests to differentiate metallo-type carbapenemases from other carbapenemases or by a rapid carbapenemase detection methods such as those based on immunochromatography or molecular methods [33].

According to the susceptibility profile, all carbapenemase-producing bacteria and, in particular, the KPC-type bacteria carrying the Tn4401a transposon were resistant to all carbapenems: meropenem, ertapenem, and imipenem [23]. As expected, most of the KPC and OXA carbapenemase-producing isolates evaluated were susceptible to ceftazidime/avibactam and aztreonam [34]. Nevertheless, 9% of the isolates presented resistance to these drugs, suggesting the presence in our setting of additional resistance mechanisms, such as mutations in ampC, blaKPC−3 carriers with omega-loop mutations [35, 36], changes in PBPs, or increased efflux pumps [37]. Among the metallo carbapenemases-producing isolates evaluated, 13% presented an unusual profile of susceptibility to ceftazidime/avibactam, considering that the most common mechanism of acquired resistance to this antibiotic is the production of avibactam-refractory beta-lactamases such as class B enzymes [38]. On the other hand, a resistance profile was observed for aztreonam in most of the isolates carrying the blaKPC gene, as expected. In contrast, the isolates producing metallo-carbapenemases showed a more heterogeneous profile, suggesting that metallo-carbapenemases do not hydrolyze aztreonam, and their action may be affected by the existence of additional serine-type beta-lactamases [39, 40].

The present study described and validated MALDI-TOF MS technology as a rapid and efficient method for the identification of KPC-producing Enterobacterales carrying the Tn4401a transposon; furthermore, its inclusion as part of a diagnostic algorithm would allow savings in time and costs by rapidly detecting KPC-type carbapenemases simultaneously with microbial identification. However, an important limitation of the method evaluated is its inability to detect carbapenemases other than those carrying by the Tn4401a transposon, a situation that is conditioned by the prevalence of the KPC gene associated with the a isoform of the Tn4401 transposon. Therefore, prior to the routine use of the methodology, it is important to know the epidemiology and genetic background of clinical isolates. Additionally, according to the timing of collection of the study strains (2012–2015) it is possible that they do not represent the current epidemiological situation in our setting with respect to the circulation of microorganisms and their resistance mechanisms.

In conclusion a positive result should be used as a first step for the indirect detection of KPC-producing associated to Tn4401a in Enterobacterales, while an additional confirmatory test must confirm negative results.