Co-occurrence of genes encoding carbapenem resistance and aminoglycoside resistance in clinical isolates of Enterobacterales

Abstract

Introduction: This study aimed to detect the co-occurrence of carbapenem resistance genes along with aminoglycoside-modifying enzyme (AME) genes in clinical Enterobacterales isolates to understand the distribution of multiple resistance genes among clinical isolates.
Methods: This prospective study was conducted for six months (November 2024 to April 2025) in the department
of microbiology of a tertiary care hospital. A total of 30 blood culture isolates were identified as resistant
to both carbapenem and aminoglycoside antibiotics using the automated VITEK 2 compact system. The genes
responsible for carbapenem resistance (bla_NDM, bla_OXA-48, bla_KPC, bla_IMP, and bla_VIM) were detected by multiplex realtime PCR, and the aminoglycoside-modifying enzyme genes [APH(3’)-Ia, APH(2”)-Ib, AAC(3)-IIc, AAC(6’)-Ib, and ANT(3”)-I] were detected by the conventional polymerase chain reaction method. All the clinical data, patient demographics, and molecular findings were entered in an MS Excel spreadsheet version 14.0.4734.1000 and analyzed using GraphPad/PRISM software version 10.5.0.
Results: Of the 30 Enterobacterales isolates, Klebsiella pneumoniae was the most common isolate (66.7%).
Molecular detection revealed bla_NDM in 40% isolates and bla_OXA48 in 10% isolates. The majority of the AME genes
were in combination. The most common combination of the AME gene was AAC(6’)-Ib+ AAC(3)-IIc+ ANT(3”)-I +
APH(3’)-I detected in 4 (13.3%) isolates. The most common combination of carbapenem and aminoglycoside
resistant genes was bla_NDM + bla_OXA48 + AAC(6’)-Ib+ AAC(3)-IIc+ ANT(3”)-I+ APH(3’)-I (13.3%). The bla_OXA-48 gene had a statistically significant association with AAC(6’)-Ib, ANT(3”)-I, and APH(3’)-I (p <0.05).
Conclusion: The Co-occurence of carbapenem resistance and aminoglycoside-modifying enzyme genes in clinical
Enterobacterales isolates limits the therapeutic option.

Introduction

The increasing spread of multidrug-resistant Enterobacterales is a major global public health concern (1,2). Carbapenem resistance is caused either by the production of carbapenemase enzymes or by mutation in porin channels, which has led to limited treatment options (3-5). The Centers for Disease Control and Prevention (CDC) defines carbapenem resistance as non-susceptibility to at least one or more carbapenem or production of the carbapenemase enzyme (6). The Clinical and Laboratory Standards Institute (CLSI) recommends the modified carbapenem inactivation method (mCIM), EDTA-modified version (eCIM), and Carba NP for the detection of carbapenemase enzyme production (7). Carbapenemase enzymes are produced by different carbapenemase encoding genes like: Ambler class B metallo-β-lactamases (MBLs)- bla_NDM, bla_VIM, and bla_IMP; Class A serine β-lactamases- bla_KPC, and class D oxacillinase- bla_OXA-48 (8). Aminoglycoside resistance occurs primarily due to the production of aminoglycoside-modifying enzyme genes such as aminoglycoside acetyltransferase (AAC), aminoglycoside nucleotidyltransferase (ANT), and aminoglycoside phosphotransferase (APH), which is encoded by AAC, ANT, and APH genes, respectively (9-11). These resistance genes are frequently present on mobile genetic elements (MGEs) (for example, transposons, plasmids, and integrons), which promote horizontal transfer in bacterial populations (12). Few studies have indicated that clinical isolates with carbapenem resistance genes (i.e., bla_NDM and bla_OXA) have plasmids that also harbor multiple resistance genes, leading to the limitation of effective treatment options (5,13). This study aimed to evaluate the coexistence of genes conferring resistance to carbapenems and aminoglycosides in clinical isolates of Enterobacterales.

Materials and Methods

This prospective study was conducted for a period of six months from November 2024 to April 2025 in the department of microbiology of a tertiary care hospital.

Inclusion Criteria: All clinical isolates of Enterobacterales that were isolated from positive blood culture samples of patients admitted to the main intensive care units (ICUs).

Exclusion Criteria: All non-Enterobacterales isolated from positive blood culture samples, Enterobacterales obtained from samples other than blood, or obtained from patients admitted to ICUs other than the main ICU and wards.

Those Enterobacterales that were screened to be resistant to both carbapenem and aminoglycoside antibiotics phenotypically, as per CLSI 2024 guidelines, were included in the study (7). Based on the screening, 30 isolates were included, and DNA extraction of the isolates followed by molecular detection of carbapenem-resistant genes and aminoglycoside-modifying enzyme genes was done. Demographic data, clinical parameters, and outcomes of the patients were recorded and analyzed.

Sample collection and processing

Blood samples from patients diagnosed with septicemia were collected in aerobic blood culture bottles (BACT/ALERT FA and FN Plus BC bottles) under aseptic conditions. The culture bottles were incubated inside the BACT/ALERT 3D system (BioMérieux, France) until they flagged positive or upto a maximum of 5 days (14). The blood culture bottle that flagged positive was subcultured on MacConkey and blood agar plates. After incubating the plates overnight at 37°C, the isolated bacterial colony was subjected to identification and antibiotic susceptibility testing.

Identification and Antimicrobial Susceptibility Testing of Clinical Isolates

The identification and AST of the isolates were performed by an automated VITEK 2 compact system (BioMérieux, USA). All the isolates that were identified as Enterobacterales and were screened to be resistant to at least one carbapenem antibiotic (ertapenem, imipenem, or meropenem), as well as to any one of the aminoglycoside antibiotics (amikacin, gentamicin, tobramycin, and netilmicin), were included. The interpretation of AST was performed as per the Clinical and Laboratory Standards Institute (CLSI) 2024 M100 guidelines (7).

Phenotypic Detection of Carbapenem Resistance and Aminoglycoside Resistance

All the screened isolates resistant to both carbapenems and aminoglycosides were again phenotypically tested by the Kirby-Bauer disk diffusion method using imipenem 10 µg, ertapenem 10 µg, meropenem µg, amikacin 30 µg, gentamicin 10 µg, tobramycin 10 µg, and netilmicin 10 µg disks (HiMedia, India). Escherichia coli (E. coli) American Type Culture Collection (ATCC) 25922 was used as the quality control strain.

Phenotypic Detection of Carbapenemase Production

The modified carbapenem inactivation method (mCIM) tests were performed on all the isolates phenotypically resistant to carbapenem antibiotics as per the CLSI 2024 guidelines to detect carbapenemase-producing Enterobacterales (CPE) (7). For each strain tested, two tubes were prepared, each containing 2ml of Trypticase Soy Broth (TSB). One tube was EDTA-free, and the other tube was supplemented with 20µl of 0.5M EDTA. A fresh isolated colony from the test organism was inoculated into each tube using an inoculating loop.

A 10 µg meropenem disk (HiMedia, India) was dropped into each tube and incubated aerobically at 35°C for 2-4 hours. Following incubation, meropenem disks were removed from both the tubes and placed onto Mueller-Hinton Agar (MHA) plates that had been freshly inoculated with 0.5 McFarland suspension of carbapenem-sensitive Escherichia coli ATCC 25922 indicator strain. Next, the plates were incubated for 16-20 hours at 35 °C. The result of the mCIM test was interpreted as negative when the zone of inhibition was ≥19 mm, positive when it measured between 6 and 15mm, and also considered positive (intermediate) if small, pinpoint colonies appeared within the 16-18 mm zone of inhibition. E. coli ATCC 25922 was used as the quality control strain (15,16).

Molecular Detection of Antibiotic Resistance Genes

All the study isolates (n = 30), which were phenotypically confirmed to be resistant to both carbapenem and aminoglycoside antibiotics, were subsequently subjected to DNA extraction by a commercially available spin column, as per the manufacturer’s procedure (TRUPCR bacterial nucleic acid extraction kit-3B BlackBio Biotech India Ltd., Bhopal, India) (17). The purity and concentration of the extracted DNA were evaluated by measuring 1 µL of the eluted DNA using a NanoDrop-Multiskan Sky spectrophotometer to measure the optical density at 260 nm and 280 nm with an absorbance ratio of ~1.8-2.0, which is considered a purity indicator of DNA samples (18).

Genes encoding carbapenem resistance (bla_NDM, bla_OXA48, bla_KPC, bla_VIM, and bla_IMP) were detected using TaqMan hydrolysis probe-based multiplex real-time polymerase chain reaction (PCR) (QuantStudio 5, Applied Biosystems, Waltham, MA, USA) by commercially available TRUPCR UTI AST Panel kit (19). The kit has a two-tube PCR assay, in tube one, primers and probe for bla_KPC, bla_NDM, and bla_VIM, and in the second tube, primers and probe of bla_OXA-48 and bla_IMP were added. The assay was performed by preparing a final volume of 25 µL reaction, consisting of 20 µL Master Mix and 5 µL of DNA template.

The genes encoding aminoglycoside resistance genes [APH(3’)-Ia, APH(2”)-Ib, AAC(3)-IIc, AAC(6’)-Ib, and ANT(3”)-I] were detected by the conventional PCR method using previously published primers listed in supplementary Table I. The samples were amplified under different cycling conditions using a thermal cycler (Mastercycler Nexus Gradient PCR, Eppendorf, Germany) [supplementary Table II]. The thermal cycling protocol for AAC(6’)-Ib, ANT(3”)-I, and APH(3)-Ia was 95°C for 5 mins, followed by 35 cycles of 1 min at 95°C, 45 sec at 55°C, 1 min at 72°C, and final extension for 5 mins at 72°C. The PCR cycling protocol for AAC(3)-IIc and APH(2”)-Ib was 95°C for 5 mins, followed by 35 cycles of 1 min at 95°C, 45 sec at 57°C, 1 min at 72°C, and final extension for 5 mins at 72°C. After amplification, 10 µL of the amplified DNA product was separated on 2% tris-Acetate Ethylene diamine tetraacetic acid (TAE) agarose gel electrophoresis and stained with ethidium bromide (final concentration: 0.5 µg/mL). After agarose gel electrophoresis, the amplified DNA band was visualized using the gel documentation system E-Box CX5 (Vilber, France). The different amplified gel products were distinguished using the molecular weight marker in the gel. Each PCR run included Escherichia coli ATCC 25922 as a negative control to ensure the absence of non-specific amplification.

Statistical Analysis

All the data (patient demographics, clinical parameters, and outcome) were entered into an MS Excel spreadsheet version 14.0.4734.1000. Analysis of the data was performed using the GraphPad /PRISM software version 10.5.0. A p-value of <0.05 was considered statistically significant.

Ethical Clearance

Clinical samples were collected after getting approval from the Institute Ethics Committee (IEC) (Ethics approval number: KIIT/KIMS/ IEC/1857/2024), with a waiver of patient consent provided due to de-identification of collected data.

Results

During the study period, 94 clinical Enterobacterales isolates were obtained from positive blood culture samples. Of these, 30 isolates that were phenotypically confirmed to be resistant to both carbapenems and aminoglycosides were subjected to phenotypic and molecular detection of carbapenem and aminoglycoside resistance. Among these, Klebsiella pneumoniae was the most prevalent (66.7%, 20/30), followed by Escherichia coli (20%, 6/30), Proteus mirabilis (6.7%, 2/30), Enterobacter cloacae (3.3%, 1/30), and Providencia rettgeri (3.3%, 1/30). Most of the isolates were recovered from male patients (66.7%), and the most common age group was 51-60 and 61-70 years (23.3% each).

The carbapenemase production of all the 30 isolates was done by the modified carbapenem inactivation method (mCIM), and 27 (90%) tested positive, indicating that carbapenem resistance was primarily mediated by carbapenemase enzymes.

Molecular Detection of Carbapenem-Resistant Genes

Detection of carbapenem-resistant genes (bla_NDM, bla_OXA48, bla_IMP, bla_VIM, and bla_KPC) using the multiplex real-time PCR. Only bla_NDM and bla_OXA-48 genes were detected, and all other genes were undetected. The real-time PCR amplification plots of bla_NDM and bla_OXA48 are shown in Figure 1.

FIGURE 1 -. Multiplex real-time PCR amplification plot of bla_NDM and bla_OXA48.

FIGURE 2 -. Agarose gel image representing various amplifications of AME genes (a) multiplex PCR of AAC(6’)-Ib and ANT(3”)-I, lane 6-10: AAC(3)-IIc (b) APH(2”)-Ib (c) APH(3’)-I.

Molecular Detection of Aminoglycoside-Modifying Enzyme Genes

Five different aminoglycoside-modifying enzyme (AME) genes, APH(3’)-Ia, APH(2”)-Ib, AAC(3)-IIc, AAC(6’)-Ib, and ANT(3”)-I were detected in the 30 isolates using conventional PCR. Representative gel images are shown in Figures 2 (a), (b), and (c).

Detection of carbapenem resistance genes and aminoglycoside resistance genes

Among the carbapenem resistance genes, bla_NDM was detected in 12 (40%) isolates, bla_OXA-48 was found in 3 (10%) isolates, and bla_NDM + bla_OXA48 genes were detected in 13 (43.3%) isolates. In two bacterial isolates bla_NDM and bla_OXA48 genes were undetected.

Detection of AME genes revealed AAC(6’)-Ib and APH(3’)-I alone were found in 2 (6.7%) isolates each. The most common combination of aminoglycoside resistance genes was AAC(6’)-Ib+ AAC(3)-IIc+ ANT(3”)-I + APH(3’)-I detected in 4 (13.3%) isolates. No isolate harbored the ANT(3”)-I gene alone; it was present along with the other AME genes [Figure 3].

FIGURE 3 -. Distribution of the carbapenem and aminoglycoside resistance genes.

Co-occurrence of carbapenem resistance genes and aminoglycoside-resistant genes

Multiple resistance genes were found in various isolates. The most common combination of genes was bla_NDM+bla_OXA48+ AAC(6’)-Ib+ AAC(3)-IIc+ ANT(3”)-I+ APH(3’)-I (13.3%, 4/30), followed by bla_NDM+bla_OXA48+ AAC(6’)-Ib+ ANT(3”)-I (10%, 3/30) of the isolates [Table 1].

TABLE 1 -. Distribution of carbapenem resistance genes coexisting with aminoglycoside-modifying enzyme genes

Co-occurrence of genes mediating carbapenem and aminoglycoside resistance							Total Isolates (N = 30)
Carbapenem resistance genes detected		Aminoglycoside-modifying enzyme genes detected					Number of isolates	Frequency (%)
blaNDM	blaOXA48	AAC(6’)-Ib	AAC(3)-IIc	APH(2”)-Ib	ANT(3”)-I	APH(3’)-I
+	−	+	−	−	−	−	1		3.3%
+	−	−	+	−	−	−	3		10%
+	−	−	−	+	−	−	1		3.3%
+	−	−	−	−	−	+	1		3.3%
+	−	+	−	−	+	−	1		3.3%
+	−	+	+	−	−	−	2		6.7%
+	−	+	−	−	−	+	1		3.3%
+	−	+	+	−	−	+	2		6.7%
+	−	+	−	−	+	+	1		3.3%
−	+	−	−	−	−	+	1		3.3%
−	+	+	−	−	+	−	1		3.3%
−	+	+	+	−	−	+	1		3.3%
+	+	+	−	−	+	−	3		10%
+	+	+	−	−	+	+	2		6.7%
+	+	+	−	+	−	+	1		3.3%
+	+	+	+	−	−	+	2		6.7%
+	+	+	+	−	+	+	4		13.3%

TABLE 2 -. Association of bla_NDM and bla_OXA48 genes with AME genes in clinical Enterobacterales isolates

Antibiotic resistance genes	AAC(6’)-Ib	AAC(3)-IIc	APH(2”)-Ib	ANT(3’’)-I	APH(3’)-I
bla NDM positive (N = 25)	20	12	11	2	14
bla NDM negative (N = 5)	4	2	1	0	3
p -value	>0.05	0.743	0.512	0.317	0.869
bla OXA48 positive (N = 16)	15	7	10	1	12
bla OXA48 negative (N = 14)	9	7	2	1	5
p -value	0.044	0.732	0.922	0.007	0.03

Association of blaNDM and blaOXA-48 genes with aminoglycoside-modifying enzyme genes in clinical isolates

The association between the 5 AME genes and carbapenem-resistant genes was assessed. No significant association was observed between bla_NDM and any of the AME genes (p > 0.05). The bla_OXA48 gene showed an association with AAC(6’)-Ib (p = 0.044), ANT(3’’)-I (p = 0.007), and APH(3’)-I (p = 0.030) [Table 2].

In-hospital mortality in patients infected with multiple resistance genes

The rate of in-hospital mortality among patients infected with Enterobacterales isolates harboring multiple resistance genes was 43.3%. These isolates carried both carbapenem resistance genes (bla_NDM and bla_OXA48) along with one or more aminoglycoside-modifying enzymes (AMEs) genes.

Discussion

In this study, we phenotypically detected carbapenemase production in 30 clinical Enterobacterales isolates by the modified carbapenem inactivation method (mCIM). Approximately 90% of these isolates were carbapenemase producers, indicating that enzyme production plays a significant role in carbapenem resistance in Enterobacterales. This is consistent with the Verma et al. and Gallego et al. studies (16, 23). Furthermore, all mCIM-positive isolates were further tested for the EDTA carbapenem inactivation method (eCIM), and all of them were metallo-β-lactamase producers.

We found that bla_NDM was the most common carbapenem resistance gene, detected in 40% of the isolates, either individually or in combination with bla_OXA48. The bla_OXA48 gene was found in 10% of the isolates, while the co-occurrence of bla_NDM and bla_OXA48 was detected in 43.3% of the isolates. The high prevalence of these genes is consistent with reports from other parts of India and European countries, where bla_NDM and bla_OXA48 are the frequent carbapenem resistance genes found in clinical Enterobacterales isolates (24-28). Several other studies have also documented the co-carriage of bla_OXA48 and bla_NDM genes, which is in congruence with our findings (29-31). None of the isolates tested positive for the bla_VIM, bla_KPC, or bla_IMP genes in our study. Few Indian studies have similarly identified bla_NDM and bla_OXA48 to be the most common genes detected, while bla_KPC remains infrequently detected in Enterobacterales (24,25).

Among the aminoglycoside-modifying enzyme (AME) genes, both AAC(6’)-Ib and APH(3’)-I were detected individually in 6.7 % of the isolates. However, the coexistence of multiple AME genes was more frequent. The most common AME gene combination was AAC(6’)-Ib + AAC(3)-IIc + ANT(3”)-I + APH(3’)-I gene, with a positivity rate of 13.3%. The prevalence of coexisting genes has also been reported by Nie Lu et al. (32). Bacteria can acquire antimicrobial resistance genes (ARGs) through gene mutations under constant antibiotic selection pressure. ARGs are found on mobile genetic elements (MGEs), including plasmids, transposable elements, and bacteriophages (33). The coexistence of multiple resistance genes could be attributed to IncF plasmids, which are well-known carriers of extended-spectrum β-lactamases, carbapenemase, aminoglycoside-modifying enzymes, and plasmid-mediated quinolone resistance (PMQR) genes (34). A study conducted in Spain found that the plasmid conferring aminoglycoside resistance in Enterobacterales belongs to the IncF, IncFIA, or IncFIB incompatibility groups (35). Few other studies have also provided evidence suggesting that these plasmids play a crucial role in the dissemination of aminoglycoside resistance genes in drug-resistant bacteria (22).

Importantly, the study demonstrated an increased co-occurrence of carbapenem resistance genes and aminoglycoside resistance genes. The most common co-occurring resistance genes were: bla_NDM + bla_OXA48 + AAC(6’)-Ib + AAC(3)-IIc + ANT(3”)-I + APH(3’)-I, which were detected in 13.3% of the isolates, followed by bla_NDM + bla_OXA48 + AAC(6’)-Ib + ANT(3”)-I, which were found in 10% of the isolates. Wangkheimayum J et al. reported a high prevalence of carbapenem and colistin resistance in aminoglycoside-resistant Enterobacterales. However, they reported that most of the CRE isolates did not carry any carbapenemase genes, indicating an alternative resistance mechanism (22).

The present study investigated the association between all five aminoglycoside-modifying enzyme (AME) genes and carbapenem resistance genes. The bla_OXA48 gene and three specific AME genes: AAC(6’)-Ib (p = 0.044), ANT(3”)-I (p = 0.007), and APH(3’)-I (p = 0.030) were found to have a statistically significant association. This finding could be due to the co-localization of these resistance genes on conjugative plasmids, like IncF, IncL/M, or other broad-host-range plasmids, which facilitate their horizontal transmission (36,37).

The overall in-hospital mortality was 43.3% among patients harboring multiple resistance genes (bla_NDM, bla_OXA48, and at least one AME gene). This is in agreement with Baek et al., who reported a 30-day mortality rate of approximately 40.9% in CRE-infected patients harboring multiple carbapenem-resistant genes (38). A systematic review done by Falagas et al. further indicated that CRE-attributable deaths ranged from 26% to 44% (39). There are very limited studies reporting the presence of AME genes attributable to death in patients.

The presence of carbapenem resistance genes along with one or more aminoglycoside-modifying enzyme genes is a growing concern, as it limits effective treatment options. This co-occurrence of multiple resistance genes could be due to a combination of mutation accumulation or by horizontal transfer of resistance genes via plasmids, transposons, and integrons, ultimately forming clusters of resistance genes known as “antimicrobial resistance islands” (33).

The study has certain limitations; it was conducted in a single tertiary care center with a limited sample size, so the findings cannot be fully generalizable to other regions or healthcare settings. Hence, further multi-centric studies should be conducted to confirm our findings. The sequencing of the resistant genes was not done, which would have provided further confirmation and characterization of those genes. Furthermore, we documented the co-occurrence of carbapenem resistance genes and aminoglycoside-modifying enzyme genes; however, we did not analyze the clinical outcome of the patients harboring multiple resistance genes.

Conclusion

This study highlights the increasing epidemiological risk posed by multidrug-resistant organisms with multiple resistance genes. It specifies the presence of bla_NDM and bla_OXA48 in carbapenem-resistant Enterobacterales, along with circulating aminoglycoside-modifying enzyme genes in the eastern region of India. The coexistence of these genes limits the available therapeutic options, making these infections difficult to manage with an alarming mortality rate.

Acknowledgments

We would like to thank Dr. A Raj Kumar Patro, Ph.D, Consultant, Molecular Biology and Advance Diagnostics, Department of Microbiology, KIMS, for the continuous support and guidance. We would also like to acknowledge Kalinga Institute of Medical Sciences, KIIT DU, for providing financial support for carrying out this work

Other information

This article includes supplementary materials

Corresponding authors:

Nipa Singh

email: nipa.singh@kims.ac.in

Disclosures

Conflict of interest: The authors declare no conflict of interest.

Financial support: It was provided by Kalinga Institute of Medical Sciences, KIIT DU, for carrying out this work.