1. Introduction
Carbapenem-resistant Enterobacterales (CRE) have been listed as an urgent threat [
1]. CREs are resistant to at least one of the carbapenem antibiotics (ertapenem, meropenem, imipenem, or doripenem) or produce a carbapenemase, which is an enzyme that can hydrolyse β-lactam and allows bacteria to be resistant to carbapenem antibiotics [
2]. Carbapenem-resistant
Klebsiella pneumoniae (CRKP) is the predominant pathogen among epidemic and endemic CRE infections that carries a high mortality rate [
3,
4]. CRKP includes both carbapenemase-producing (C-CRKP) and non-carbapenemase-producing (NC-CRKP) strains. C-CRKP confers carbapenem resistance through carbapenemase production [
5]. NC-CRKP confers carbapenem resistance through a combination of chromosomal mutations (e.g., porin gene mutation, overproduction of efflux pump, and/or alteration in penicillin-binding protein) and acquired non-carbapenemase resistance mechanisms (acquisition or upregulation of a β-lactamase such as extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase) [
2,
6].
Based on our previous study and unpublished data from the Infection Control Department (ICD) of the University of Malaya Medical Centre (UMMC), most of the CRKP isolated in our hospital setting were C-CRKP [
7]. Changing trends of genotypic characteristics among C-CRKP leading to different phenotypic traits have been observed. In 2013, the predominant carbapenemase gene of C-CRKP was
blaOXA-48 (70.6%), followed by
blaKPC-2 (29.4%),
blaNMC-A (11.8%)
, blaIMP-8 (5.9%), and
blaNDM-1 (5.9%) genes [
8]. In 2014, only two carbapenemase genes were detected. The
blaNDM (56.0%) was the predominant gene, followed by
blaOXA-48 (36.0%) [
9]. In 2015, the predominant carbapenemase gene of C-CRKP had reverted to
blaOXA-48 (79.1%), followed by
blaNDM (40.9%) and
blaOXA-232 (0.9%) [
9]. In 2016, the predominant carbapenemase gene was still
blaOXA-48 (75.6%), followed by
blaNDM (22.2%) [
10]. Both
blaOXA-48 (43.8%) and
blaNDM (43.8%) were the predominant carbapenemase genes of C-CRKP detected in 2017 [
10]. However, there is a lack of epidemiology data on NC-CRKP or their mechanisms of resistance in our hospital.
A retrospective study conducted in South Texas between 2011 and 2019 reported that the majority of CRE (59.0%, 58/99) isolates were non-carbapenemase-producing strains [
11]. A cohort study conducted in Maryland from 2016 to 2021 also revealed that 54.0% (327/603) of CRE isolates were non-carbapenemase-producing strains, with
Klebsiella pneumoniae as the predominant pathogen [
12]. A study conducted in Taiwan from January 2013 to December 2018 revealed that 86.9% (86/99 isolates) of the CRE bacteremia specimens were non-carbapenemase-producing strains [
13]. Additionally, a study conducted by six Singapore public sector hospitals between December 2013 and April 2015 reported that 35.3% (88/249 subjects) of their recruited subjects had non-carbapenemase-producing CRE [
14]. Nonetheless, little is known about the pathogenicity, persistence, and clinical outcomes of the NC-CRKP in Malaysia.
A review study conducted in the United States reported that NC-CRKP were less transmissible than C-CRKP and can be easily eliminated in vivo by the immune system [
15]. A cohort study of 83 patients with monomicrobial CRE bacteremia conducted in the United States in 2013-2016 reported that carbapenemase-producing CRE was more virulent with a higher mortality rate (32.4%) than non-carbapenemase-producing CRE (13.0%) [
16]. Nonetheless, the burden of NC-CRKP should not be underestimated as a study carried out between 2008-2011 in Italy reported that the mortality rate of patients with NC-CRKP (37.9%) was similar to that of KPC-producing (38.9%) CRKP [
17]. A multicentre study in Taiwan also reported a high 14-day mortality rate (27.3%) among 99 NC-CRKP patients [
18]. In this study, we sought to determine the prevalence, genotypic characteristics, and phenotypic profile of NC-CRKP as well as the risk factors of NC-CRKP associated with all-cause in-hospital mortality in a tertiary teaching hospital in Malaysia.
2. Results
2.1. Bacterial Strains Collection
From 2013 to 2019, a total of 381 CRKP were collected from patients’ clinical and screening samples. An increase in NC-CRKP strains has been observed over the seven years, as shown in
Figure 1. In this study, 54 NC-CRKP were isolated from UMMC patients from January 2013 to October 2019. In 2013 and 2014, 5.9% and 4.0% of the CRKP isolated from UMMC patients were NC-CRKP. An increase in the rate of NC-CRKP strains was observed from 5.2% in 2015 to 6.7% in 2016, even though the reported CRKP cases had decreased from 115 cases in 2015 to 45 cases in 2016. The rate of NC-CRKP strains had increased to 21.9% in 2017 and 26.2% in 2018, but a slight decline to 24.0% in 2019.
The rate of NC-CRKP isolation was calculated as a percentage (%) of the total number of NC-CRKP strains isolated per year over the total number of CRKP strains.
2.2. Determination of MIC Profiles
Based on the broth microdilution method (
Table 1), all strains were resistant to ertapenem and ciprofloxacin. Broth microdilution also revealed that 61.1% and 33.3% of the strains were susceptible to imipenem and meropenem, respectively. The highest MIC value of ertapenem, imipenem, meropenem, and ciprofloxacin was >256 µg/mL, 128 µg/mL, 256 µg/mL, and >512 µg/mL, respectively. Only two strains were colistin-resistant with a MIC of 32 µg/mL, while the remaining were intermediate to colistin (The CLSI guidelines do not give any interpretive MIC breakpoint for susceptible). For carbapenem resistance among all 54 NC-CRKP, 16 strains (29.6%) were mono-resistant to ertapenem (
Table 2).
2.3. Antimicrobial Susceptibility Data
All 54 strains were resistant to amoxicillin/clavulanate, ampicillin, and cefuroxime (
Table 3). All strains were resistant to at least three antimicrobial classes and were therefore classified as multidrug-resistant (MDR) strains [
19].
2.4. Determination of Resistance Genes and Porin-Associated Genes
None of the 54 NC-CRKP harboured the five major carbapenemase genes targeted for detection in this study, which were
blaNDM,
blaOXA-48,
blaIMP,
blaVIM, and
blaKPC (
Table 4). Each strain harboured at least one non-carbapenemase β-lactamase gene targeted for detection in this study. Resistance genes found among the 54 NC-CRKP isolates were
blaDHA,
blaTEM,
blaSHV,
blaCTX-M, and
blaOXA genes (
Table 4). In total, deletion of porin-associated genes was detected in 46.3% (25/54) of the NC-CRKP (
Table 2 and
Table 4). None of the 54 strains had ompK37 porin loss.
2.5. MIC Reduction Assay with Efflux Pump Inhibitor Tests
For the MIC of imipenem, meropenem, and ertapenem after the addition of PAβN, no significant reduction was observed (
Table 5). Hence, no efflux contribution of PAβN-inhibited efflux pumps to carbapenem resistance was detected among the 54 strains. However, the PAβN-inhibited efflux pump contributed to the resistance to ciprofloxacin, as a four-fold decrease in the MIC of ciprofloxacin was observed in five strains, after the addition of PAβN (
Table 5). In addition, 46.3% (25/54 strains) of the NC-CRKP showed a significant increase (≥ 4-fold) in the MIC of at least one carbapenem in the presence of PAβN (
Table 5 and
Table A1 in
Appendix A). After the addition of PAβN, 44.4% (24/54 strains) of the NC-CRKP showed a two-fold increase in the MIC of at least one carbapenem, while the remaining strains (9.3%, 5/54 strains) showed no change in the MIC of carbapenem.
2.6. Pulsed-Field Gel Electrophoresis
For PFGE dendrogram analysis, one untypeable strain (Strain no.: 285) was excluded. Among 53 NC-CRKP, 46 pulsotypes were detected and assigned as KP 1 to KP 46. These 46 pulsotypes were grouped into six clusters (Clusters A, B, C, D, E, and F) based on an 80.0% similarity cut-off (
Figure 2). All six clusters harboured the
blaSHV gene.
Cluster D was the dominant strain cluster (with 24 strains) detected from 2013 to 2019. Cluster D was resistant to ETP, CIP, AMC, AMP, CXM, CTX, CAZ, and CRO. Loss of ompK36 porin was detected among 11 strains in cluster D. Cluster D was comprised of pulsotypes KP 18 to KP 34. KP 24 was the predominant pulsotype found in six patients admitted to UMMC from August to December 2017. Pulsotype KP 24 harboured blaSHV and blaCTX-M genes. Pulsotype KP 24 was resistant to ETP and CIP. A loss of ompK36 porin was detected among three strains in pulsotype KP 24.
Cluster A composed of pulsotypes KP 1 to KP 3 isolated from 2017 to 2018. Cluster A was resistant to ETP, CIP, AMC, AMP, CXM, CTX, and GM. A loss of ompK36 porin was detected in two strains from cluster A.
Cluster B was characterised by pulsotypes KP 5 to KP 8 isolated from 2017 to 2018. In addition to the blaSHV gene, cluster B harboured blaTEM and blaCTX-M genes. Cluster B was resistant to ETP, CIP, AMC, AMP, CXM, CTX, CAZ, CRO, and MEM, while susceptible to GM. A loss of ompK36 porin was detected in two strains from cluster B.
Cluster C was composed of pulsotypes KP 14 to KP 16 isolated from 2018 to 2019. Cluster C was resistant to ETP, CIP, AMC, AMP, CXM, and CAZ, while susceptible to GM. Only one strain in cluster C had ompK36 porin loss.
Cluster E was characterised by pulsotypes KP 42 isolated in 2018 and KP 43 isolated in 2019. Cluster E was resistant to ETP, CIP, AMC, AMP, CXM, CTX, CAZ, and CRO, while susceptible to GM and IPM. Cluster E encountered ompK36 porin loss.
Cluster F was composed of pulsotypes KP 44 isolated in 2015 and KP 45 isolated in 2019. In addition to the blaSHV gene, cluster F harboured blaTEM and blaCTX-M genes. Cluster F was resistant to ETP, CIP, AMC, AMP, CXM, CTX, CAZ, CRO, IPM, and MEM, while susceptible to GM. Only one strain (pulsotype KP 45) in cluster F had ompK35 porin loss.
Additionally, 15 strains that were isolated in 2014 (pulsotype KP 39), 2015 (pulsotype KP 17 and KP 46), 2016 (pulsotypes KP 4), 2018 (pulsotypes KP 9, KP 10, KP 11, KP 13, KP 35 and KP 36) and 2019 (pulsotypes KP 12, KP 37, KP 38, KP 40 and KP 41) showed distinct pulsotypes. These strains could not be grouped into clusters A to F. All 15 strains were resistant to ETP, CIP, AMC, AMP, CXM, CTX, and CRO. A loss of both ompK35 and ompK36 porins was detected in pulsotype KP 17. Pulsotype KP 41 had ompK35 porin loss. Pulsotypes KP 4, KP 12, KP 13, and KP 46 had ompK36 porin loss.
2.7. Clinical Data and Statistical Analysis
Of the 54 NC-CRKP patients, two patients were excluded from the statistical analysis due to the restricted access to highly confidential patients’ demographic and clinical data. All 52 infected/colonised patients had invasive devices in situ before NC-CRKP was isolated. The all-cause in-hospital mortality rate was 46.2% (24/52). 23 (44.2%) patients were infected with NC-CRKP, while others were colonised. Since antimicrobial treatment was only prescribed for infected patients, the independent variables like empiric treatment and definitive therapy were included only in the infection model to analyse the all-cause in-hospital mortality risk among 23 NC-CRKP infected patients.
When considered separately in determining the risk factors of NC-CRKP association with all-cause in-hospital mortality, a total of two parameters, including previous cephems/cephalosporins exposure and previous carbapenem exposure, were found to be significant with
p < 0.050 (
Table 6).
Independent variables with a
p-value of 0.150 or less in the univariate analysis were selected for the multivariate binary logistic regression model to evaluate the risk for all-cause in-hospital mortality among NC-CRKP-infected/colonised patients (
Table 7). The binary logistic regression model achieved an overall correct classification of 82.7% with a χ
2 value of 32.804 and a
p-value of less than 0.010 (
p = 0.003). In the multivariate binary logistic regression analysis, no statistically significant risk factor was found.
3. Discussion
This study showed an increasing trend of NC-CRKP prevalence from 1 strain in 2013 to 12 strains in 2019 and attained the highest (17 strains) isolation in 2018. There was a notable rise in NC-CRKP strains over these seven years, especially from 2016 (3 cases) to 2017 (14 cases). Among 52 NC-CRKP-infected/colonised patients, the all-cause in-hospital mortality rate was 46.2%. The mortality rate of NC-CRKP-infected/colonised patients in our study was higher as compared to the United States (13.0%) [
16], Italy (37.9%) [
17], and Taiwan (27.3%) [
18]. For NC-CRKP-infected patients (44.2%, 23/52) and NC-CRKP-colonised patients (55.8%, 29/52), the all-cause in-hospital mortality rate was 52.2% and 41.4% respectively. Based on the CCI score, 92.3% (48/52) of the infected/colonised patients had comorbidity with a score of ≥1. For NC-CRKP-infected/colonised patients with CCI scores of ≥5 (severe; 61.5%, 32/52), the all-cause in-hospital mortality rate was 53.1% (17/32). Compared with severe and moderate CCI patients, patients with lower CCI scores (mild/no comorbidity) had a higher survival rate. Previous evidence suggested that NC-CRKP were confined to individuals and environments with very high levels of antimicrobial selection pressure [
20], especially those with heavy use of carbapenem [
17]. In the present study, NC-CRKP-infected/colonised patients with previous carbapenem exposure had a higher all-cause in-hospital mortality rate (63.0%, 17/27) than patients with previous cephems/cephalosporins exposure (33.3%, 12/36). Of 27 (51.9%) NC-CRKP-infected/colonised patients with previous carbapenem exposure, 75.0% (6/8) of the infected patients died, while 57.9% (11/19) of the colonised patients died. Among the 36 infected/colonised patients with previous cephems/cephalosporins exposure, 47.1% (8/17) of the infected patients died, whereas 21.1% (4/19) of the colonised patients died. NC-CRKP may be more frequently acquired endogenously through antimicrobial selective pressure as they were resistant to carbapenem via the non-enzymatic carbapenem-resistant mechanisms, while C-CRKP was more frequently acquired exogenously through horizontal gene transfer [
15]. The horizontal gene transfer by mobile genetic elements contributes to the persistence of carbapenemase genes among C-CRKP in hospitals despite aggressive infection control [
21]. However, antimicrobial selective pressure also predisposes the microorganism to be more susceptible to horizontal gene transfer events, which promotes penetration of the mobilome into new bacterial hosts and leads to the dissemination of antimicrobial resistance [
22,
23].
Broth microdilution revealed that all 54 strains were resistant to ertapenem, while 61.1% and 33.3% of the strains were susceptible to imipenem and meropenem, respectively. This was similar to another NC-CRKP study in which the loss of susceptibility was more remarkable for ertapenem, followed by meropenem and imipenem [
24]. All 54 strains were resistant to ETP, CIP, AMC, AMP, and CXM, and hence fulfiled the criteria as MDR strains. Nineteen strains were resistant to all three carbapenems. Seventeen strains were resistant to meropenem and ertapenem, while two strains were resistant to imipenem and ertapenem. Additionally, 29.6% (16/54) of the strains were mono-resistant to ertapenem. A cohort study in the United States reported that ertapenem-mono-resistant CRE rarely has carbapenemase genes [
25]. In addition to being resistant to ETP, CIP, AMC, AMP, and CXM, these 16 ertapenem-mono-resistant strains were also resistant to CTX and harboured the
blaSHV gene. Only 6 strains out of these 16 strains had ompK36 porin loss, while ompK35 and ompK37 porin loss were not detected in these 16 strains. This suggested that ertapenem and cefotaxime resistance were not due to the loss of ompK35 porin. A study reported that the loss of ompK35 porin alone may not confer high-level resistance to ertapenem and may not affect susceptibility to imipenem and meropenem [
24]. Additionally, a previous study also reported that a decrease in expression of ompK36 porin, but not ompK35 porin, was statistically associated with individual carbapenem resistance as the former facilitates the penetration of cefotaxime, cefoxitin, and carbapenem [
26]. It is noteworthy that ertapenem resistance normally arises from a combination of non-carbapenemase β-lactamase with altered porins and can be controlled by non-ertapenem carbapenem [
27,
28]. The majority (68.8%, 11/16) of these ertapenem-mono-resistant strains belonged to the dominant cluster D in PFGE.
Among the 54 NC-CRKP in this study, both
blaSHV and
blaCTX-M were the predominant genes between 2013–2016, but
blaSHV was the predominant gene between 2017–2019. Overall, the most prevalent non-carbapenemase β-lactamase gene among the 54 NC-CRKP in this study was
blaSHV (53/54, 98.1%), followed by
blaCTX-M (46/54, 85.2%)
, blaTEM (24/54, 44.4%),
blaDHA (8/54, 14.8%),
blaOXA-1 (3/54, 5.6%), and
blaOXA-9 (3/54, 5.6%) genes. Although
blaSHV is ubiquitous in
K. pneumoniae, previous studies reported the absence of this enzyme [
29,
30]. While
blaCTX-M is an ESBL gene, not all
blaTEM and
blaSHV genes are necessarily ESBL genes [
31]. Hence, further sequencing is needed to confirm whether they are narrow- or extended-spectrum β-lactamase genes. On the other hand, the
blaOXA-1 and
blaOXA-9 genes are narrow-spectrum class D β-lactamase genes [
32]. Among all 54 MDR strains, 85.2% (46/54) of the strains carried the
blaCTX-M ESBL gene (5 strains co-harboured
AmpC β-lactamase gene), while 5.6% (3/54) of the strains carried
AmpC β-lactamase gene. The remaining five strains (9.3%) carried other non-carbapenemase β-lactamase genes. However, the spectrum could not be confirmed as the sequencing of
blaTEM and
blaSHV genes were not performed. The
blaDHA was the only
AmpC β-lactamase gene detected in 14.8% (8/54) of the NC-CRKP. The
blaDHA is generally regarded as plasmid-encoded due to the absence of the chromosomal
blaAmpC gene in the genome of
K. pneumoniae [
33]. Plasmid-mediated
AmpC β-lactamase genes such as
blaDHA-1 are inducible by β-lactam [
34] and can be expressed in high levels constitutively while downregulating inflammation to depress the immune response [
35]. Additionally, previous studies reported that the majority of NC-CRKP had porin deficiency [
18,
36] which may cause nutrient uptake impairment and lower metabolic fitness [
37]. Nevertheless, only 46.3% (25/54) of the NC-CRKP in this study had porin loss. The loss of ompK35 or ompK36 porins was detected in 5.6% and 42.6% of the NC-CRKP respectively. None of the 54 strains had ompK37 porin loss. Among the 25 strains that exhibited porin loss, 84.0% (21/25) of the strains carried the
blaCTX-M ESBL gene (2 strains co-harboured
AmpC β-lactamase gene), while 4.0% (1/25) of the strains carried
AmpC β-lactamase gene. The remaining three strains (12.0%) carried other non-carbapenemase β-lactamase genes targeted for detection in this study. They were either narrow- or extended-spectrum β-lactamase genes as the sequencing of
blaTEM and
blaSHV genes were not conducted in this study to confirm their spectrum.
In addition to ETP, CIP, AMC, AMP, and CXM, all
blaTEM-producing strains were resistant to CTX, while all
blaCTX-M-producing strains were resistant to CAZ and CTX. All strains that harboured the
blaDHA gene co-carried the
blaSHV gene and were resistant to ETP, CIP, AMC, AMP, CXM, CAZ, CTX, and CRO. All strains that harboured the
blaOXA-9 gene also carried the
blaSHV and
blaCTX-M genes. Hence, all
blaOXA-9-producing strains were resistant to ETP, CIP, AMC, AMP, CXM, CAZ, CTX, CRO, and MEM. None of the
blaOXA-9-harbouring strains had porin loss. All strains that harboured the
blaOXA-1 gene co-carried
blaSHV gene and were resistant to ETP, CIP, AMC, AMP, CXM, and CTX. Strains exhibiting ompK35 porin loss (5.6%, 3/54 strains) also harboured
blaTEM,
blaSHV, and
blaCTX-M genes. They were resistant to all three carbapenems (ETP, IPM, MEM), CIP, AMC, AMP, CXM, CAZ, CTX, and CRO, but susceptible to GM. A loss of ompK35 porin usually contributes to the emergence of antimicrobial resistance in ESBL-producing strains and may favour the selection of additional mechanisms of resistance, including the loss of ompK36 and/or active efflux [
38]. In addition to ETP, CIP, AMC, AMP, and CXM, all imipenem-resistant strains were resistant to CAZ, while all meropenem-resistant strains were resistant to CAZ and CTX. All gentamicin-resistant strains were resistant to ETP, CIP, AMC, AMP, CXM, and CTX. All imipenem-resistant strains and gentamicin-resistant strains harboured the
blaSHV gene.
In this study, the PAβN-inhibited efflux pump contributed to the resistance to ciprofloxacin, as a four-fold decrease in the MIC of ciprofloxacin was observed in five strains, after the addition of PAβN. PAβN is one of the most extensively studied compounds which acts as a peptidomimetic efflux pump inhibitor that combines with fluoroquinolone antibiotics to overcome efflux-mediated multidrug resistance [
39]. However, PAβN did not reduce carbapenem resistance among all these 54 NC-CRKP. Therefore, the efflux system was unlikely to be involved in carbapenem resistance, which was in accordance with previous studies [
40,
41,
42]. In addition, 46.3% (25/54 strains) of the NC-CRKP showed a significant increase (≥4-fold) in the MIC of at least one carbapenem in the presence of PAβN. According to Saw et al. (2016), the effect of PAβN on the MIC of carbapenem was caused by the altered expression of outer membrane porins, as their clinical isolates (15.1%, 13/86 strains) with increased ertapenem resistance in the presence of PAβN had altered porin expression, whereas those (3.5%, 3/86 strains) with no differences in ertapenem MIC value after the addition of PAβN did not. Careful evaluation of new efflux inhibitors is necessary to ensure that antimicrobial-resistant bacteria do not develop increased resistance to clinically important antimicrobials [
42].
Since all the strains isolated had at least one non-carbapenemase β-lactamase gene targeted for detection in this study and no active efflux contribution of PAβN-inhibited efflux pumps to carbapenem resistance was detected, other potential chromosomal mutations such as reduced expression or alteration in outer membrane porin or penicillin-binding protein may have occurred in NC-CRKP to develop carbapenem resistance, as only 46.3% (25/54) of them had porin loss. The majority (53.7%) of the isolated strains in this study did not have porin loss, unlike a multicenter study in Taiwan where only 5.1% (5/99 isolates) of their NC-CRKP isolates preserved expression of both ompK35 and ompK36 porins [
18]. The majority of their NC-CRKP conferred carbapenem resistance through a combination of porin loss and acquired non-carbapenemase resistance mechanisms [
18].
For PFGE dendrogram analysis, 46 pulsotypes were detected among 53 NC-CRKP. They were grouped into six clusters based on an 80.0% similarity. Cluster D was the dominant strain cluster found in 24 strains (pulsotypes KP 18 to KP 34) isolated from 2013 to 2019. No clonal transmission was identified as all the profiles were unique. This was consistent with the recommendations for the control of carbapenemase-producing
Enterobacteriaceae published by the Australian Commission on Safety and Quality in Health Care, in which NC-CRKP-infected/colonised patients represented a lower infection control risk and did not warrant attention unless cross-transmission is demonstrated [
20]. There was only sporadic isolation of pulsotypes KP24 (six strains), KP26 (two strains), and KP32 (two strains). No circulation of a specific pulsotype occurred in our study setting. Nonetheless, the high diversity observed may be indicative of the presence of the genetic events that contributed to the clone emergence and adaptation to the hospital’s environment.
The possible risk factors previously reported for in-hospital mortality among CRKP-infected/colonised patients were the usage of mechanical ventilation [
8] and delayed definitive treatment [
9]. Additionally, patients with NDM-producing CRKP had previously been found to have a lower hazard ratio for in-hospital mortality as compared to the OXA-48 variant [
10]. In this study, no statistically significant risk factor was found in the multivariate binary logistic regression analysis due to the limited sample size. Therefore, all possibilities remained. A larger sample size should be included in future studies to increase the robustness of the statistical analyses. Additionally, the role of structural mutation such as reduced expression or alteration of porin or penicillin-binding protein should be investigated further to assess the mechanism of carbapenem resistance of the remaining strains (53.7%). Since only the five clinically important major carbapenemase genes (
blaNDM,
blaOXA-48,
blaIMP,
blaVIM, and
blaKPC) were targeted for detection in this study, future studies may also target for detection of rare carbapenemase genes (such as
blaIMI,
blaNMC,
blaFRI,
blaGES,
blaBKC,
blaSFC,
blaSME,
blaGIM,
blaTMB,
blaLMB,
blaKHM,
blaSFH,
blaAIM,
blaOXA-372,
blaCMY,
blaACT, or
blaBIC) [
43] among the isolates in this study.