Carbapenemase-encoding genes in critical gram-negative bacteria isolated from ICU patients with infections and/or gastrointestinal carriage, and environmental samples in the amhara National Regional state, Ethiopia

Mizan Kindu; Feleke Moges; Ashenafi Alemu; Dawit Hailu; Rafael Joseph; Esmael Besufikad; Dareskedar Tsehay; Daniel Bashah; Zemene Tigabu; Adane Mihret; Baye Gelaw

doi:10.1371/journal.pone.0330613

Abstract

Effective infection control requires identifying and eliminating carbapenemase-producing (CP) Gram-negative bacteria (GNB) in high-risk groups like intensive care unit (ICU) patients and from contaminated environmental surfaces. This study aimed to describe the diversity of carbapenemase-encoding genes among critical GNB isolates from ICU patients with infection and/or gastrointestinal (GI) colonization, as well as from ICU environmental surfaces in the Amhara National Regional state, Ethiopia.A total of 169 carbapenem-resistant isolates were identified, including 26 from infections, 82 from GI colonization, and 61 from environmental samples. These comprised Klebsiellapneumoniae (n = 107), Escherichia coli (n = 16), Pseudomonas aeruginosa (n = 9), and Acinetobacter species (n = 37), of which 147 were analyzed.Singleplex and multiplex PCR were performed to detect predominant carbapenemase-encoding genes, including KPC (bla_KPC), MBLs (bla_IMP, bla_VIM, and bla_NDM), and OXA (bla_OXA-48, bla_OXA-23, and bla_OXA-58). PCR analysis revealed that at least one carbapenemase-encoding gene was detected in 133 (78.7%) of the 169 carbapenem-resistant isolates from patients and environmental surfaces. They were detected in 22/133(16.5%), 70/133(52.6%), and 41/133(30.8%) isolates obtained from infection, GI colonization, and environmental samples, respectively. The most prevalent carbapenemase-encoding gene, bla_NDM, was found in 101 (75.9%) of the 133 CP isolates. Other detected carbapenemase-encoding genes included bla_OXA-23 in 5 (3.8%) isolates, bla_VIM in 4 (3.0%), bla_OXA-48 in 1 (0.8%), and bla_OXA-58 in 1 (0.8%). K. pneumoniae and Acinetobacter spp. harbored all the gene types detected in this study. Co-harboring of two or more carbapenemase-encoding genes, in combination with bla_NDM, was observed in 21 of 133 (15.8%) isolates, including 7 K. pneumoniae and 14 Acinetobacter spp. isolates. However, bla_KPC and bla_IMP were not identified in any of the tested isolates. This study highlights the presence of carbapenemase-encoding genes among critical GNB across all sample source types in the study area. Moreover, the detection of isolates harboring multiple carbapenemase-encoding genes underscores the need for enhanced infection control measures in ICU settings.

Citation: Kindu M, Moges F, Alemu A, Hailu D, Joseph R, Besufikad E, et al. (2025) Carbapenemase-encoding genes in critical gram-negative bacteria isolated from ICU patients with infections and/or gastrointestinal carriage, and environmental samples in the amhara National Regional state, Ethiopia. PLoS One 20(9): e0330613. https://doi.org/10.1371/journal.pone.0330613

Editor: Dhammika Leshan Wannigama, Yamagata University Faculty of Medicine: Yamagata Daigaku Igakubu Daigakuin Igakukei Kenkyuka, JAPAN

Received: May 13, 2025; Accepted: August 5, 2025; Published: September 4, 2025

Copyright: © 2025 Kindu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data supporting the findings of this study are available within the paper and its supporting information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Carbapenems have become the last resort antibiotics for treating multidrug-resistant (MDR) Gram negative bacterial (GNB) infections, particularly in hospital settings [1]. However, the emergence of carbapenem resistant bacteria is threatening the effectiveness of these antibiotics. Carbapenemase enzymes are the most common resistance mechanism to carbapenem worldwide [2]. Initially, carbapenemases were chromosomally mediated in a few specific species, but now they are mediated by a plasmid, or both a chromosomal and a plasmid. This results in a more violent spread of resistance due to clonal expansion and horizontal gene transfer between different bacterial species and genera [3,4]. Carbapenem resistance has been labeled in Enterobacterales, mostly in Klebsiella pneumoniae compared to Escherichia coli or other Enterobacterial species, and in non-fermentativeGram-negative bacilli such as Acinetobacterbaumanniiand Pseudomonas aeruginosa. Moreover, World Health Organization (WHO) listed these carbapenem-resistant GNB isolates as pathogens of critical importance [5].

Carbapenemases can be classified into various categories according to the Ambler classification scheme as A, B, and D. Globally, the most common carbapenemases consist of K.pneumoniaecarbapenemases (KPC) are clinically the most common enzymes among the class A carbapenemases. New Delhi metallo-β-lactamases (NDM), Verona integronencodedmetallo-β-lactamases (VIM), and imipenemase (IMP) types belong to class B metallo-β-lactamases (MBLs), whereas oxacillinases, including OXA-23, OXA-24, OXA-58, and OXA-48, are part of the class D enzymes [2]. A few studies conducted in Ethiopia have provided evidence of infections caused by carbapenemase-producing (CP) GNB and have reported NDM carbapenemase being the most commonly identified enzyme [6,7].

For effective infection control, it is crucial to identify and eliminate carbapenemase-producing critical GNB among high-risk groups, such as intensive care unit (ICU) patients, and contaminated environmental sources [8]. Additionally, gastrointestinal(GI) colonized patients serve as reservoirs for CP pathogens, facilitating their dissemination [9]. Therefore, this study aimed to describe the diversity of genes encoding carbapenemase enzymes among critical GNB isolates from ICU patients with GI colonization and/or infection, as well as from ICU environmental samples in the AmharaNational Regional state, Ethiopia.

Materials and methods

Study area, patients, and specimen collection

Samples were obtained from patients and environmental surfaces in the ICUs of the University of Gondar Comprehensive Specialized Hospital (UoGCSH) and FelegeHiwot Comprehensive Specialized Hospital (FHCSH), located in the AmharaNational Regional state, North-West Ethiopia, the 1^st of June 2021 to the 30^th, December 2022.During the study period, non-duplicated clinical samples were collected from each of the 600 study participants based on clinical suspicion of infection. These included blood, urine, pus/wound swabs, and other specimens. In addition to the clinical samples, rectal swabs were collected from these study participants to identify GI colonization with CP critical GNB. Simultaneously, 384 environmental swab samples were collected from ICUs during the same period [10].

Critical GNB isolates with reduced susceptibility to carbapenem

Bacterial identification using various biochemical tests, antibiotic susceptibility testing by disk diffusion technique, and phenotypic detection of carbapenemase enzymes were performed as described in our previously published work by Mizan et al. [10]. A total of 169 carbapenem-resistant isolates were identified, including 26 from infections, 82 from gastrointestinal colonization, and 61 from environmental samples. These comprised K. pneumoniae (n = 107), E. coli (n = 16), P. aeruginosa (n = 9), and Acinetobacter spp. (n = 37) (S1 Table).

From carbapenem-resistant critical GNB isolates, rectal and environmental isolates that tested phenotypically positive for carbapenemase production using the mCIM test were subjected to carbapenemase-encoding gene detection in the case of K. pneumoniae, E. coli, and P. aeruginosa. However, for clinical samples and Acinetobacter spp., all carbapenem-resistant isolates identified by the disk diffusion test were included for carbapenemase-encoding gene detection using conventional PCR [11].Therefore, total of 147 these isolates were stored at –20 °C, and transported to the Armauer Hansen Research Institute (AHRI) for PCR analysis.

DNA extraction

DNA was extracted from carbapenem-resistant critical GNB isolates using the boiling method, as previously described [12]. Briefly, each isolate was grown overnight on nutrient agar (Oxoid, UK), and 3 to 5 colonies were suspended in 300 µL of 1 × Tris-EDTA buffer. The suspension was subjected to boiling at 94°C for 10 minutes in a water bath (Thermo Fisher Scientific, CA, USA), followed by freezing at −20°C for 10 minutes, incubation at room temperature for 1 minute, and centrifugation at 14,000 × g for 5 minutes. Finally, 150 µL of the supernatant was transferred into a nuclease-free Eppendorf tube and assessed using a Nanodrop (Thermo Scientific, USA) to measure the quality and quantity of the extracted DNA. The samples were then stored at −20°C until further analysis.

PCR-based detection of carbapenemase genes

K. pneumoniae, E. coli, P.aerugniosa and Acinetobacter spp.isolates resistant to carbapenems were analyzed for the presence of carbapenemase-encoding genes using PCR-based methods. Singleplex and multiplex PCR were performed to detect the most common carbapenemase-encoding genes, including KPC (bla_KPC), MBLs (bla_IMP, bla_VIM, and bla_NDM), and OXA (bla_OXA-48, bla_OXA-23, and bla_OXA-58), using a panel of primers as previously described [11].The sets of specific primers used for the detection of carbapenemase genes are shown in supplementary table (S2 Table).

For both singleplex and multiplex PCR, the reaction conditions were optimized by testing a range of annealing temperatures to determine the optimal annealing conditions for all primers. PCR was performed in a total volume of 25 µL, consisting of 12.5 µL (final concentration 1×) of HotStarTaq Master Mix (Qiagen, Hilden, Germany), 5 µL of genomic DNA, 0.4 µM of each primer, and 5.5 µL of nuclease-free water.

PCR amplification of carbapenemase-encoding genes was carried out under the following cycling conditions: initial denaturation at 95°C for 5 seconds, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 1 minute, and extension at 72°C for 1 minute, with a final extension at 72°C for 5 minutes. The PCR amplicons were analyzed by 1.5% agarose gel electrophoresis stained with ethidium bromide. The presence or absence of carbapenemase genes was determined using a UV transilluminator (GelDoc, Bio-Rad), and PCR product bands were compared against a 100 bp DNA ladder.

Quality control

For the optimization of the multiplex PCR, known control strains were used as positive controls. DNA samples from K. pneumoniae ATCC 1706 served as a negative control for bla_KPC, bla_NDM, bla_OXA-48, and bla_VIM. K. pneumoniae ATCC BAA-1705, K. pneumoniae ATCC BAA-2146, P. aeruginosa AR Bank #00546, and K. pneumoniae AR Bank #0039 were used as positive controls for bla_KPC, bla_NDM, bla_VIM, and bla_OXA-48, respectively, all in conjunction with carbapenemase detection. Before multiplexing the primers for carbapenemase-encoding genes, each primer was tested individually using a singleplex PCR reaction. The DNA samples for both negative and positive controls were obtained from the Ethiopian Public Health Institute

Ethical approval and consent/assent to participate

Ethical approval was obtained from the institutional review board of the University of Gondar with reference number O/VIP/RCS/05/1773/2019. Informed written consent was obtained from each study participant with the help of caregivers. Briefly, since most of the study participants were neonates, assent was taken from their parents or guardians. Moreover, consent was obtained from the caregiver for critically ill or unconscious individuals whose age was 18 years and above.

Results

A total of 169 carbapenem resistance isolates of critical GNB were recovered phenotypically. CP critical GNB isolates carrying carbapenemase-encoding genes were identified across all sample source, obtained from ICU patients with an infection and/or GI colonization, as well as from environmental surfaces (Table 1). Carbapenemase-encoding genes were detected in 133 (78.7%) of the 169 carbapenem resistance isolates. Among CP critical GNB, the majority were K. pneumoniae 87 (65.4%), followed by Acinetobacterspp. 31 (23.3%), E. coli 12 (9%) and P. aeruginosa 3 (2.3%).

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Table 1. PCR confirmed carbapenemase positive critical GNB obtained from ICU patients with GI colonization and/orinfection, as well as from ICU environmental samples in the AmharaNational Regional state, Ethiopia, 2021 to 2022.

https://doi.org/10.1371/journal.pone.0330613.t001

Characteristics of PCR confirmed carbapenemase positive ICU patients

Critical GNB carrying carbapenemase-encoding genes were detected among ICU patients with prevalence of an infection and GI colonization rate of 3.7% (22/600) and 11.3% (68/600), respectively. Five patients had both an infection and GI colonization, while two other patients had GI colonization with mixed species of CP critical GNB.Majority of the patients were from the neonatal ICU (NICU) (n = 62) (Table 2).

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Table 2. Characteristics of PCR confirmed carbapenemase positive ICU patientswith GI colonization and/or infection in the AmharaNational Regional state, Ethiopia, 2021 to 2022.

https://doi.org/10.1371/journal.pone.0330613.t002

Distribution of carbapenemase encoding genes detected among critical Gram negative bacteria

The most prevalent carbapenemase-encoding gene detected was bla_NDM, 101/133 (75.9%) followed by bla_OXA-23 gene, 5/133 (3.8%); bla_VIM 4/133 (3.0%); bla_OXA48 1/133(0.8%) and bla_OXA-58 gene 1/133(0.8%) isolates. Co-harboring of two or more types of carbapenemase-encoding genes with bla_NDM genes were observed in 21/133(15.8%) isolates. bla_{NDM +} bla_OXA-23 were detected in 9 isolates; bla_NDM + bla_VIM in 5 isolates; bla_NDM + bla_OXA-58 in 4 isolates; bla_NDM + bla_OXA-48 in 1 isolate; bla_NDM + bla_OXA-23+ bla_VIM in 1 isolate and bla_NDM + bla_OXA-23 + bla_OXA-48 in 1 isolate. However, bla_KPC, and bla_IMP genes were not detected (S1 Fig).

The bla_NDM gene was identified across all species, including 87 K. pneumoniae, 22 Acinetobacterspp., 11 E. coli, and 2 P. aeruginosa. The bla_OXA-23 gene was found in 5 Acinetobacterspp., while bla_VIM was detected in 3 Acinetobacterspp. and 1 P. aeruginosa. The bla_OXA-58 gene was identified in 1 Acinetobacterspp., and the bla_OXA-48 gene was identified in 1 E. coli isolate. Co-harboring of two or more carbapenemase-encoding genes, in combination with bla_NDM, was observed in 7 K. pneumoniae and 14 Acinetobacterspp. (Fig 1).

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Fig 1. Distribution of carbapenemase-encoding genes among critical GNB obtained from ICU patients with GI colonization and/orinfection, as well as from ICU environmental samples in the AmharaNational Regional state, Ethiopia, 2021 to 2022.

https://doi.org/10.1371/journal.pone.0330613.g001

Distribution of critical GNB carrying carbapenemase-encoding genes in different ICU environmental surfaces

Critical GNB carrying carbapenemase-encoding genes were detected on various ICU environmental surfaces, with K. pneumoniae and Acinetobacter spp. being the most frequently isolated species. K. pneumoniae carrying bla_NDM carbapenemase was primarily detected on baby incubators (n = 7), baby bed sets (n = 5), and over-bed tables (n = 3). Acinetobacter spp. exhibited a wider range of carbapenemase-encoding genes, including bla_NDM, bla_VIM, bla_OXA-23, and bla_OXA-58, with notable contamination on baby bed sets, bed rail surfaces, mattress,and over-bed tables. P. aeruginosa carrying bla_NDM was found on over-bed table (n = 1) and sink (n = 1), while E. coli carrying bla_NDM was detected on the sink (n = 1) and mattress (n = 1) (Table 3).

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Table 3. Distribution of carbapenemase-encoding genes among critical GNB in different ICU environmental surfaces in the AmharaNational Regional state, Ethiopia, 2021 to 2022.

https://doi.org/10.1371/journal.pone.0330613.t003

Distribution of carbapenemase encoding genes detected among different sample sources

Carbapenemase-encoding genes were distributed across infection, GI colonization, and environmental samples. Among K. pneumoniae isolates, bla_NDM (n = 80) was detected in infection, GI colonization, and environmental samples. Co-harbored gene, bla_NDM + bla_OXA-58 (n = 2) was found in both GI colonization and environmental sources while bla_NDM + bla_OXA-23 (n = 5) was only detected in GI colonization. In Acinetobacter spp., bla_NDM (n = 8) were present across all sample types, whereas bla_OXA-23 (n = 5) was detected from GI colonization and environmental samples. Additionally, combinations genes such as bla_NDM + bla_VIM (n = 5) and bla_NDM + bla_OXA-23 (n = 4), were identified both in infection and environmental samples. For E. coli, bla_NDM (n = 11) was detected in GI colonization and environmental, while bla_OXA-48 (n = 1) was limited to GI colonization. Among P. aeruginosa, bla_NDM (n = 2) was found in environmental samples, and bla_VIM (n = 1) was identified in an infection (Table 4).

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Table 4. Carbapenemase-encoding genes among critical GNB in different sample sources in the AmharaNational Regional state, Ethiopia, 2021 to 2022.

https://doi.org/10.1371/journal.pone.0330613.t004

Discussion

This study detected carbapenemase-encoding genes in CP critical GNB isolated from ICU environmental surface contamination, GI colonization, and infections in ICU patients, using environmental swab, rectal swab, and clinical samples, respectively. Among the CP critical GNB that carried carbapenemase-encoding genes confirmed by PCR, the most common isolates was K.pneumoniae followed by Acinetobacterspp., E. coli and P. aeruginosa. This finding is in line with reports from China that K. pneumoniae, E. coli, A.baumannii, and P. aeruginosa were the predominant isolates CP-GNB [13]. Although the frequency varied, CP critical GNB carrying carbapenemase-encoding genes, particularly K. pneumoniae and Acinetobacter spp., were detected across all sample sources, including clinical, rectal, and environmental samples. The presence of CP critical GNB carrying carbapenemase-encoding genes in all sources sample types may indicate the circulation of these isolates between the environment and patients. Further advanced studies focusing on the transmission dynamics of CP critical GNB isolates are needed to clarify these transmission pathways and inform targeted infection control strategies.

This study showed that the prevalence of carbapenemase-encoding genes among carbapenem-resistant critical GNB was 78.7%. This is comparable to a previous study conducted in Ethiopia, which reported a prevalence of 69% [14], and a study from Sudan, where the prevalence was 58.75% [15]. However, it is higher than another study conducted in Ethiopia by Seman et al., which reported a prevalence of 44.5% [6]. The variation in prevalence rates may be due to differences in the number of carbapenemase-encoding genes targeted for detection, variations in the mechanisms of carbapenem resistance, and the types of GNB included in studies.

Among carbapenemase-encoding genes, the bla_NDM gene was predominant (75.9%) in CP critical GNB isolates. bla_NDM was first reported in India, and since its initial detection, bla_NDM carrying organisms have disseminated globally [16]. In Ethiopia, the first report of bla_NDM presence was in three A. baumannii isolates [17]; however, subsequent studies have identified its occurrence in Enterobacteriaceae [7] and P. aeruginosa [14,18]. In this study, the bla_NDM gene was found across all CP critical GNB species, including K. pneumoniae, E. coli, Acinetobacter spp., and P. aeruginosa, indicating its ease of circulation within and between bacterial species. This may also explain bla_NDM genes are encoded on highly mobile, conjugative plasmids which ease horizontal inter and intra-species transfer between bacteria [19,20]. Our results are consistent with previous studies conducted in Ethiopia [7,14] and other countries, which also reported the dominance of bla_NDM genes among GNB isolates [16]. The occurrence of NDM producing GNB in Ethiopia may be explained by several factors, including frequent travel between Ethiopia and NDM-endemic countries, due to weak infection prevention and control practices in healthcare settings, clonal expansion of NDM producing isolates, and/or the efficient spread of conjugative plasmids carrying the bla_NDM gene [19,20].

Interestingly, this study identified isolates co-harboring multiple carbapenemase-encoding genes, including bla_NDM in combination with bla_OXA-23, bla_OXA-48, bla_OXA-58, and bla_VIM. Similar findings have been reported in other countries, where isolates carrying multiple carbapenemase-encoding genes have been detected [13,15,21,22]. K. pneumoniae and Acinetobacterspp. showed two or more carbapenemase-encoding genes as it was reported previously in other reports that, K. pneumoniae and Acinetobacter spp. have co-existence of multiple carbapenemase-encoding genes [14]. This co-existence of carbapenemase-encoding genes poses a significant therapeutic challenge due to limited treatment options and the potential for horizontal gene transfer, facilitating global dissemination.

The low prevalence of bla_VIM, bla_OXA-23, bla_OXA-58 and bla_OXA-48 carbapenemase-encoding genes among critical GNB was reported in this study. In contrast to global reports of a high prevalence of bla_KPC genes, we have not detected bla_KPC and bla_IMP genes among the tested isolates.This was in line with the previous studies done at Addis Ababa hospitals [6,23] and reports from Sudan [15]. Despite the first occurrence of a KPC producer dated from 1996 in the United States, nowadays, Mediterranean countries, especially Italy, Greece and Israel, are endemic for the bla_KPC gene [24]. The movement of people between Ethiopia and endemic countries, due to medical visitation, trade, employment, and unrestricted cross-border travel, may contribute to the dissemination of various carbapenemase variants within the country. Additionally, variations in the types and prevalence of carbapenemase-encoding genes among CP-GNB in different studies may be influenced by factors such as geographic region, study population, and the genetic structure of isolates [24,25].

The detection of CP K.pneumoniae, Acinetobacter spp., E. coli, and P. aeruginosa on various ICU environmental surfaces underscores the significant role of the hospital environment in harboring and potentially transmitting antimicrobial-resistant pathogens. Notably, NDM-type carbapenemases were predominantly identified, particularly in K.pneumoniae and Acinetobacterspp.. This finding aligns with previous research indicating the widespread presence of NDM variants in healthcare settings [26].The co-existence of multiple carbapenemase encoding genes, such as bla_NDM and bla_OXA-23 genes, within Acinetobacter spp. suggests a high potential for horizontal gene transfer, thereby increasing the risk of MDR outbreaks [27]. The contamination of high-contact surfaces, including baby incubators, bed rails, and sinks, further emphasizes the need for stringent infection control measures, as these areas may serve as reservoirs for CP critical GNB [26]. Moreover, these findings highlight the critical importance of routine environmental screening and the implementation of targeted disinfection strategies to mitigate the spread of resistant pathogens within ICU healthcare settings.

The frequency of CP critical GNB isolates with different carbapenemase-encoding genes across sample sources was varied. K. pneumoniae was found in both GI colonization and environmental samples, with a higher presence in GI colonization. This indicates that asymptomatic carriers and contaminated surfaces may serve as reservoirs, contributing to the spread and eventually increase in infection cases [9,28]. In contrast, Acinetobacter spp. was more frequently detected in environmental samples rather than in GI colonization. Likewise, the presence of multiple carbapenemase-encoding gene combinations, such as bla_NDM + bla_OXA-23 and bla_NDM + bla_VIM was observed in Acinetobacter spp. from both infection and environmental samples. This reinforces the possibility that patients may acquire these resistant strains from environmental surfaces during the healthcare process [29,30]. For E. coli, the majority of carbapenemase-positive isolates were identified in GI colonization samples, with fewer found in environmental samples. This may support a study suggesting that E. coli primarily remains a gut colonizer, with a lower likelihood of transmission to the hospital environment or progression to infection [28]. P. aeruginosa with carbapenemase-encoding gene, particularly bla_NDM, was detected in a sink, supporting its ability to persist in moist environments and contribute to outbreaks in critically ill patients. The presence of bla_VIM in an infection isolate further highlights the ability of P. aeruginosa to acquire and express carbapenemase genes, posing significant challenges in treatment and infection control [31,32].

One of the limitations of this study is that the environmental sampling was conducted after the COVID-19 pandemic, during a period when infection prevention and control (IPC) measures may have been intensified. This could have influenced the detection rate of critical GNB in the environment, underestimating the true level of contamination.

Conclusions

This study demonstrates the presence of carbapenemase-encoding genes among critical GNB in ICU environmental contamination, GI colonization, and infections. The detection of critical GNB carrying these genes across all sample sources suggests possible circulation of these isolates between the environment and patients, highlighting the need for further research on transmission dynamics. Moreover, the identification of isolates harboring multiple carbapenemase genes underscores the importance of strengthening infection control measures in ICU settings.

Supporting information

S1 Fig. Agarose gel electrophoresis (1.5% agarose, run for 50 min).

A. bla_NDM, bla_KPC and bla_OXA-48, Lane PC1,PC2 and PC3:positive control for bla_NDM, bla_KPC and bla_OXA-48 respectively, B. bla_OXA-58 and bla_OXA-23, Lane PC1 and PC2: positive control for bla_OXA-23 and bla_OXA-58 respectively C. bla_VIM and bla_IMP Lane PC1: positive control for bla_VIM. First line Ladder (1,00 bp), NC: negative control, Lanes 1,2,3…: represent of isolates bands.

https://doi.org/10.1371/journal.pone.0330613.s001

(TIF)

S1 Table. Row data for carbapenem resistance critical GNB obtained from ICU patients with GI colonization and/orinfection, as well as from ICU environmental samples in the Amhara National Regional state, Ethiopia, 2021 to 2022

https://doi.org/10.1371/journal.pone.0330613.s002

(XLSX)

S2 Table. Primer sequences used for the detection carbapenemase-encoding genes.

For degenerate primers: D = A, G or T; R = A or G; Y = C or T; K = G or T; W = A or T.

https://doi.org/10.1371/journal.pone.0330613.s003

(DOCX)

S1 Raw Images. The original uncropped and unadjusted images underlying all blot or gel results reported in

S1 Fig.

https://doi.org/10.1371/journal.pone.0330613.s004

(PDF)

Acknowledgments

The authors would like to thank the University of Gondar Comprehensive Specialized Hospital, FelegeHiwot Comprehensive Specialized Hospital, and the ICU healthcare workers. We also extend our gratitude to the staff members of the Microbiology and Molecular Department at the Armauer Hansen Research Institute (AHRI) for their support.

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Figures

Abstract

Introduction

Materials and methods

Study area, patients, and specimen collection

Critical GNB isolates with reduced susceptibility to carbapenem

DNA extraction

PCR-based detection of carbapenemase genes

Quality control

Ethical approval and consent/assent to participate

Results

Characteristics of PCR confirmed carbapenemase positive ICU patients

Distribution of carbapenemase encoding genes detected among critical Gram negative bacteria

Distribution of critical GNB carrying carbapenemase-encoding genes in different ICU environmental surfaces

Distribution of carbapenemase encoding genes detected among different sample sources

Discussion

Conclusions

Supporting information

S1 Fig. Agarose gel electrophoresis (1.5% agarose, run for 50 min).

S1 Table. Row data for carbapenem resistance critical GNB obtained from ICU patients with GI colonization and/orinfection, as well as from ICU environmental samples in the Amhara National Regional state, Ethiopia, 2021 to 2022

S2 Table. Primer sequences used for the detection carbapenemase-encoding genes.

S1 Raw Images. The original uncropped and unadjusted images underlying all blot or gel results reported in

Acknowledgments

References