Possible Association of Carbapenemase Production with Susceptibility Pattern and Resistance Genes Among Carbapenemase Producing Enterobacterales from Blood Isolates or Bacteremic Patients

Introduction

Bacteremia is a severe medical condition defined by the presence of bacteria in the bloodstream.^1,2 It is diagnosed with positive blood culture and is associated with systematic symptoms such as fever, chills, nausea, headache, low blood pressure, and high white blood cell counts.^3,4 The condition is associated with high morbidity and mortality rates worldwide and is known to be a commonly acquired infection in healthcare settings.^5,6

Bacteremia can be classified according to the site of infection into primary, which originates from the cardiovascular system, or secondary, which occurs due to the transmission of bacteria from another site of the body into the blood.^7,8 It can also be classified as nosocomial (hospital-acquired) or community-acquired, with differences in infectious organisms and antibiotic susceptibility.⁹ Bacteremia can indicate the presence of an actual systemic infection, such as sepsis, which often stems from sources such as the urinary tract or lungs.¹⁰

Bacteremia in the United States is reported to be shared based on data published by the National Healthcare Safety Network.¹¹ In Jordan, a study of healthcare-associated bloodstream infections (BSIs) indicates the overall incidence of health-care-associated BSIs is 8.1 per 1000 admissions, while the mortality rate caused by healthcare-associated BSIs is 5.8 per 1000 admissions.¹²

Gram-negative bacteria, including the Enterobacteriaceae, can lead to bacteremia.¹³ Members of Enterobacteriaceae are rod-shaped, Gram-negative bacteria, and some species in this family are part of the intestinal flora.¹⁴ Enterobacteriaceae encompasses common human pathogens, including Salmonella, Escherichia coli, Yersinia pestis, Klebsiella, Proteus, Enterobacter, and Shigella, which cause various infections such as cystitis, pyelonephritis, septicemia, pneumonia, peritonitis, meningitis, and device-associated infections.^15,16 Some strains of Enterobacteriaceae have become resistant to antibiotics, including penicillin, cephalosporins, and carbapenems, considered the last line of antibiotics against resistant organisms.^17,18 Mobile genes can explain this in plasmids that can spread through bacterial strains.¹⁹

Antimicrobial resistance (AMR) is one of the most pressing global health challenges, with multidrug-resistant organisms (MDROs) significantly contributing to morbidity and mortality worldwide. The World Health Organization (WHO) has identified AMR as a critical threat, with an estimated 4.95 million deaths linked to bacterial AMR in 2019.²⁰ Bloodstream infections (BSIs) caused by MDROs, particularly carbapenem-resistant Enterobacteriaceae (CRE), are associated with high mortality rates due to limited treatment options, prolonged hospital stays, and increased risk of complications such as septic shock and organ failure.^21,22

Carbapenems-resistant Enterobacteriaceae (CRE) are high-priority antibiotic-resistant pathogens identified by health reports as a major health threat.²⁰ One study demonstrated that critically ill patients who developed bloodstream infections from multidrug-resistant organisms exhibited a significantly elevated risk of mortality compared to their non-infected counterparts.²³

The prevalence of CRE poses a significant clinical challenge in Jordan and other regions with limited resources. Despite heightened awareness, critical scientific gaps remain; these include an incomplete understanding of the epidemiology and mechanisms underlying carbapenem resistance in diverse geographical settings.

This study aims to address these gaps by investigating antimicrobial susceptibility patterns, carbapenemase production, and resistance genes in Enterobacteriaceae isolated from blood cultures in Jordan. By providing insights into the prevalence and molecular characteristics of CRE, this research seeks to inform national prevention efforts and contribute to global strategies for combating AMR.

Therefore, emerging resistance of Enterobacteriaceae represents a significant challenge that requires immediate attention, and research studies are needed to inform national prevention efforts. In the current study, antimicrobial susceptibility, carbapenemase production, and resistance genes were investigated in Enterobacteriaceae isolated from blood cultures in Jordan.

Materials and Methods

Sample Collection, Identification, and Preservation

Eighty isolates of pure, non-repeated clinical Enterobacteriaceae from positive blood cultures were collected from patients in four Jordanian hospitals: King Abdullah University Hospital (KAUH), Prince Rahma Teaching Hospital, Zarqa New Governmental Hospital, and Prince Hashim bin Al-Hussein Military Hospital during the period from October 2019 to February 2020. The Research Ethics Committee (REC) of Jordan University of Science and Technology approved the study procedures. All samples were identified in the diagnostic microbiology laboratories of included hospitals using microbiological techniques/automated systems (VITEK@2, bioMérieux), and further confirmation was done in our research laboratory using the Microgen GNA-ID (Microgen Bioproducts Ltd, UK) system as the VITEK@2 identification system is not absolutely accurate.²¹ The data obtained by the Microgen GNA-ID microwell strip were designed to generate a 4-digit octal code for Enterobacteriaceae, which was used to interpret the result using the Microgen Identification System Software. Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC 19606 were used as experimental quality control. The Microgen GNA-ID system differentiates Enterobacteriaceae through simultaneous biochemical reactions, identifying microorganisms by color change after 18–24 hours at 35 ± 2°C. It uses biochemical tests based on statistical probabilities for numeric identification and compares profiles to a reference database. For sample preparation, the collected samples were sub-cultured on blood agar at 37°C for 24 hours. Fresh colonies were inoculated into nutrient broth and incubated for 18 hours. The broth was mixed with 50% sterile glycerol (1:1 ratio) and stored at −80°C for future testing.

Antimicrobial Susceptibility Testing

The Kirby-Bauer disc diffusion method on Mueller-Hinton Agar was used to examine the susceptibility of isolates to fifteen antimicrobial agents selected according to CLSI guidelines (M100, 27th ed. January 2018).¹⁵ Isolated colonies were grown for 18–24 hours on agar plates; then a 0.5 McFarland standard turbidity inoculum was then mixed with the saline. A sterile cotton swab was used to streak the inoculum across the agar surface. After drying for 5 minutes, the antibiotic discs were applied with a minimum distance of 24 mm between them. The plates were then placed under aerobic incubation at 37°C for 16–18 hours. Following the incubation period, the inhibition zone (mm) around each disc, if present, was measured in millimeters using a transparent ruler. Escherichia coli ATCC 25922 was used for quality control purposes as the control strain in susceptibility testing.

Detection of Carbapenemase Production

Carbapenemase production was assessed using three methods: chromogenic media (CHROMagar, Paris, France), the Modified Hodge Test (MHT), and the Carba NP test, to cross-validate findings.^22–24 The media were prepared according to the manufacturer’s instructions under aseptic conditions; K. pneumoniae ATCC BAA-1705 and K. pneumoniae ATCC 700603 were used for quality control according to the manufacturer’s instructions. Then, all samples were streaked onto two different chromogenic agars (CHROMagarm SuperCARBA, CHROMagar KPC) to cross-validate the results and to minimize false negatives. The plates were then incubated in aerobic conditions at 35–37°C for 18–24 hours; the results were interpreted as described by the manufacturer.

The Modified Hodge Test (MHT) was also performed to detect carbapenemase-producing Enterobacteriaceae according to CLSI guidelines (2018). A 0.5 McFarland standard suspension of E. coli ATCC^® 25922 was diluted at a 1:10 ratio and streaked on Mueller–Hinton agar, with a carbapenem susceptibility disc (Ertapenem or Meropenem) placed at the center. Colonies of the test organism were streaked from the disc’s edge to the plate’s edge, ranging between 20 and 25 mm in length, then incubated at 35°C ± 2°C for 16–20 hours. After incubation, a cloverleaf indentation at the intersection of the test organism and E. coli indicated a positive result, while no growth indicated a negative result. Positive and negative controls were K. pneumoniae ATCC^® BAA-1705 and K. pneumoniae ATCC^® BAA-1706, respectively.

In the current study, the RAPIDEC CARBA NP^® (bioMérieux, France) test was used to measure carbapenemase activity by growing isolates on Mueller–Hinton agar for 18–24 hours and as described by the manufacturer.²⁵ The results were visually assessed by comparing the colors of the test and control wells at 30 minutes and finalized within 2 hours.

Detection of Resistance Genes

Bacterial DNA was extracted from all samples using a Zymogen DNA extraction kit (Zymo Research crop. Irvine, USA) according to the protocol provided by the manufacturer. Polymerase Chain Reaction (PCR) was used to investigate the presence of resistance genes using primers targeting bla-VIM, bla-IMP, bla-KPC, bla-OXA-48, and bla-NDM. Primers were synthesized by Integrated DNA Technologies, and sequences, annealing temperature, and product size were as previously described.²⁶ A liquid stock (100 µM) of each primer was prepared from the lyophilized primers using nuclease-free water. 10 µM working stock solution was prepared for each primer for PCR use. Stock solutions were saved at −20°C until use.

PCR was performed on a BIO-RAD T100 Thermal Cycler. For amplification, 3μL of template DNA (50 ng/μL) was mixed with 2.5μL of forward and reverse primers (10μM each), 12.5μL of master mix (i-Taq DNA Polymerase), and 4.5 μL of nuclease-free water, resulting in a final volume of 25μL. The cycling conditions included an initial denaturation at 94°C for 5 minutes, followed by 40 cycles: denaturation at 94°C for 45 seconds, annealing (52°C for bla-VIM, bla-NDM, bla-OXA-48; 45°C for bla-IMP; and 62°C for bla-KPC) for 1 minute, and elongation at 72°C for 1 minute. A final extension at 72°C for 10 minutes completed the process.²⁶

After amplification, 5 µL of each PCR product was analyzed using agarose gel electrophoresis (1% agarose in Tris-Borate EDTA buffer) stained with 5 µL of Red Safe dye per 100 mL of gel (iNtron, RedSafe™ Nucleic Acid Staining Solution (20,000x)). To ensure the detection of amplicons, 100 base-pair DNA ladder (iNtron, Sizer ™ −100 DNA Marker Solution) was used for comparison. Genotyping and molecular procedures were performed at the Princess Haya Biotechnology Center.

Statistical Analysis

Statistical analysis of data was done using The Statistical Package for Social Science (SPSS) software (IBM, USA). Data were analyzed by the Pearson Chi-Square test. P value < 0.05 was considered statistically significant.

Results

Study Isolates

A total of 80 clinically pure Enterobacteriaceae isolates were collected from positive blood cultures. Most isolates were positive for Klebsiella pneumoniae (55%) followed by Escherichia coli (37.5%) and Enterobacter cloacae (5%). One isolate for each Citrobacter freundii and one isolate of Pantoea agglomerans were identified.

Antimicrobial Susceptibility Profile

Antimicrobial susceptibility testing revealed high rates of resistance among isolates to ampicillin (95%), cefazolin (81.2%), cefotaxime (70%), and ceftriaxone (67.5%). In contrast, the highest susceptibility was observed with imipenem (86.3%), meropenem (82.5%), and ertapenem (82.5%). The study also showed a high prevalence of multidrug-resistant (MDR) strains, represented by the resistant nature of 68.75% of isolates in three or more categories, conserving a consistent minimum of one drug per category. Carbapenem-resistant Enterobacteriaceae (CRE) were identified if they were resistant to ertapenem, meropenem, or imipenem with resistance rates of 13.7%, 17.5%, and 17.5% were found, respectively. Additionally, Klebsiella pneumoniae (80%) and Escherichia coli (20%) were mostly common among isolates. Table 1 summarizes the antimicrobial susceptibility profile.

Table 1 Antimicrobial Susceptibility of the Collected Isolates

Detection of Carbapenemase Production

Carbapenemase production was detected using chromogenic media (CHROMagar mSuperCARBA and CHROMagar KPC). A total of 18.75% of all isolates were Carbapenem-resistant Enterobacteriaceae (CRE), while (81.25%) were non-CRE (Table 2). Modified Hodge Test (MHT) was also performed to detect CRE (Table 2). Based on MHT results, 21.25% of all isolates were identified as CRE. Finally, RAPIDEC CARBA NP^® test (bioMérieux, France) was performed to detect Carbapenemase activity. Carba NP test detects only (10%); this might be due to less sensitivity of Carba NP test (Table 2).

Table 2 Carbapenemase Production Detection Test Results as Positive and Negative

The Association Between Carbapenemase Production and Antimicrobial Susceptibility

The association between Carbapenemase production and Antimicrobial Susceptibility results is shown in Supplementary Table 1. According to the previous tests, several significant associations (P<0.05) were observed between antibiotics and Carbapenemase production. Carbapenemase production was strongly associated (P<0.05) with resistance to all carbapenem antibiotics (Ertapenem, Imipenem, and Meropenem). Carbapenemase production was also significantly associated (P<0.05) with resistance to Cefepime, Ceftriaxone, Cefoxitin, Ciprofloxacin, and Levofloxacin.

Table 3 shows the association between multi-drug-resistant strains, Carbapenemase-resistant strains, and Carbapenemase production. Nearly all the results show a significant association (P<0.05) between the Carbapenemase production with multi-drug resistant and CRE strains.

Table 3 Association Between Multi-Drug Resistance Strains, Carbapenemase-Resistant Strains, and Carbapenemase Production

All Carbapenem Resistant Enterobacteriaceae strains were strongly associated (P<0.05) with Carbapenemase production, which means that Carbapenemase production leads to carbapenem resistance in nearly all isolates in this study. Furthermore, there was a strong concordance between multi-drug resistance strain and Carbapenemase production detected by Chromogenic media and the Modified Hodge tests (Table 3).

Detection of Resistance Genes

PCR was applied to all bacterial isolates to investigate the presence of resistance genes (bla-KPC, bla-NDM, bla-IMP, bla-VIM, and bla-OXA-48). Based on the PCR assays, 23 (28.75%) isolates were positive for one or more Carbapenemase genes. Of the 23 positive isolates for Carbapenemase genes, 6 (26%) were positive for more than one gene. Most of the carbapenemase-encoding Enterobacteriaceae isolates were Klebsiella pneumoniae (74%), followed by E. coli (26%). Overall, the NDM gene was the most predominant Carbapenemase gene detected in 12 isolates (15%), followed by KPC gene 8 (10%), OXA-48 5 (6.25%), VIM 4 (5%), and all isolates were negative to IMP-types. Table 4 represents the distribution of Carbapenemase genes among all study isolates. Figure 1 shows gels for the detection of resistance genes.

Table 4 The Distribution of Carbapenemase Genes Among All Study Isolates

Figure 1 Image of representative gel for (A) KPC gene detection. L: Ladder, PC: Positive Control, NC: Negative Control. Lanes 24, 27, 31 and 32: samples demonstrating KPC genes (498 bp), (B) VIM genes detection. L: Ladder, PC: Positive Control, NC: Negative Control. Lanes 17, 19 and 27: samples demonstrating VIM genes (390 bp), (C) OXA-48 gene detection. L: Ladder, PC: Positive Control, NC: Negative Control. Lanes 21, 35 74 and 80: samples demonstrating OXA-48 genes (238 bp), and (D) NDM gene detection. L: Ladder, PC: Positive Control, NC: Negative Control. Lanes 30, 35, 39 and 49: samples demonstrating NDM genes (521 bp).

Supplementary Table 2 shows a significant association between Carbapenemase resistance genes and Antimicrobial Susceptibility results. Bla-KPC, bla-NDM and bla-OXA-48 were significantly associated (P<0.05) with resistance to all carbapenems (Ertapenem, Imipenem and Meropenem). There was no significant association between bla-VIM and carbapenems resistance. bla-NDM showed the most significant association (P<0.05) with resistance to other antibiotic classes – other than carbapenems-.

The association between carbapenemase resistance genes and carbapenemase production is shown in Table 5. Carbapenemase production was significantly associated (P<0.05) with the presence of Carbapenemase resistant genes, except bla-VIM which showed no significant association with Carbapenemase production.

Table 5 Association Between Carbapenemase Resistance Genes and Carbapenemase Production

Discussion

Carbapenemase Resistant Enterobacteriaceae (CRE) are among the broad spectrum of Multi-Drug Resistant (MDR) Enterobacteriaceae that represents a threat to human and public health, according to the World Health Organization (WHO).²⁷ The current study explores the potential antimicrobial susceptibility, carbapenemase production, and the presence of resistance genes of Enterobacteriaceae isolated from blood cultures of bacteremic patients, which were investigated.

Among the collected pure isolates of Enterobacteriaceae-positive blood cultures, 55% of all isolates were Klebsiella pneumoniae, 37.5% were Escherichia coli, and 5% were Enterobacter cloacae. In a recent study on Carbapenem-resistant Enterobacteriaceae from patients with bacteremia in South Africa (2020), it was found that Klebsiella pneumoniae was the predominant species (78%) isolated from positive blood culture infected with Enterobacteriaceae.²⁸ Our findings are also consistent with those of the Egyptian study (2018), which found that Escherichia coli constituted 30.7% of all Enterobacteriaceae species isolated from various clinical samples, second to Klebsiella pneumoniae, which formed 38.6% of all samples.²⁹ Additionally, in a study from the United States (2017), it was shown that 90% of bloodstream infections were caused by K. pneumoniae.³⁰ Furthermore, a Jordanian study (2010) reported that Klebsiella species and Escherichia coli are the most common gram-negative microorganisms that cause bacteremia among children.³¹

Enterobacteriaceae strains become resistant to the last line antibiotics, including penicillin, cephalosporins, and carbapenems.³² In the current study, the highest rates of resistance were observed against ampicillin (90%), followed by cefazolin (76.7%), cefotaxime (70%) and ceftriaxone (65%). At the same time, the highest susceptibility rates were observed for imipenem (86.3%), followed by meropenem (82.5%) and ertapenem (81.3%). Similar trend was found in a recent study about bacteremia among Jordanian children.³¹ Much lower susceptibility rates to the carbapenem group (40%) have been observed in Enterobacteriaceae strains isolated from blood culture in South Africa.²⁸

High or intermediate Enterobacteriaceae resistance among all isolates was considered CRE. The resistance rate to imipenem was 13.7%, to meropenem was 17.5%, and to ertapenem was 17.5%. Comparable findings have been reported in Egypt.²⁹ In the disc diffusion method, 18.75% of all Enterobacteriaceae species studied were classified as CRE. Comparable findings have been reported that the resistance rate against carbapenem in nosocomial-infected patients with gram-negative bacilli was 27.17%.³³ On the contrary, two Egyptian studies found that the rates of CRE were 45% and 47%, simultaneously,^34,35 demonstrating a higher carbapenem resistance rate compared to this current study. A lower rate of carbapenem resistance was found among Enterobacteriaceae in previous studies from Jordan, Turkey, the United States, Lebanon, and Malaysia.^34,36–41

In this study, Klebsiella pneumoniae was the most common CRE species (64%), followed by Escherichia coli (20%). These findings align with a previous study from Jordan, where K. pneumoniae was also the most prevalent CRE strain (82.1%).³⁶ Similarly, a study in Egypt found K. pneumoniae (51.4%) and E. coli (28.6%) to be the dominant CRE species.²⁹

Carbapenemase production was significantly associated (P<0.05) with resistance to all carbapenem antibiotics (Ertapenem, Imipenem, and Meropenem). Carbapenemase production was also significantly associated (P<0.05) with cefepime, ceftriaxone, cefoxitin, ciprofloxacin, and levofloxacin resistance. All CRE strains were significantly associated with Carbapenemase production, which means that Carbapenemase production led to carbapenem resistance in nearly all isolates in this study. Also, there was a significant association between Multi-Drug Resistance strain (MDR) and carbapenemase production that was detected by Chromogenic media and the Modified Hodge test but not by the Carba NP test. The observed differences observed among the different detection methods could likely stem from variations in the sensitivity and specificity of each technique.

Based on the PCR assays in the current study, 28.75% of all isolates were positive for one or more of the carbapenemase genes. Of the 23 isolates positive for carbapenemase genes, 26% were positive for more than one gene. Overall, the NDM gene was the most predominant carbapenemase gene detected in 15% of isolates, followed by KPC gene (10%), OXA-48 (6.25%), VIM (5%), and all isolates were negative to IMP gene. KPC, NDM and OXA-48 were associated with resistance to all carbapenems. There was no association between VIM and carbapenem resistance. This could be due to the low prevalence of this gene in the collected sample (5%), expressivity of the gene, and utilization of alternative resistance mechanisms that do not require the expression of VIM gene. NDM showed the most significant association with resistance to other antibiotic classes – other than carbapenems-. Similar patterns, with OXA-48 and NDM as predominant genes, have been reported in nearby regions like the Arabian Gulf, Morocco, and Palestine.^42–44

In the current study, the predominant gene was NDM; this was in contrast with a study performed in the United States that showed KPC detected in more than 90% of CRE isolates from blood cultures.⁴⁵ In a study from South African, the most common Carbapenemase genes reported were OXA-48 and NDM among CRE isolated from blood culture.²⁸ In a study from Egypt, the predominant genes were KPC and VIM.²⁹ Contrary to our findings, some studies have reported KPC, IMP, and VIM genes lacking in Klebsiella pneumoniae isolates.^46,47 The variation in the prevalence of Carbapenemase genes highlights the importance of examining the spectrum of these genes in different settings/countries.

CRE remains a significant clinical challenge in Jordanian hospitals and may be spreading. Our findings provide a valuable baseline for ongoing CRE surveillance and infection control efforts across different hospital wards. In summary, this study found a relatively low prevalence of CRE compared to regional and global levels. However, the findings serve as an early warning, highlighting the need for enhanced laboratory capabilities to detect, identify, and characterize CRE more effectively.

Conclusions

The isolated Enterobacteriaceae strains demonstrated the highest resistance against Ampicillin, followed by Cefazolin, Cefotaxime, and Ceftriaxone, and the highest susceptibility rates to Imipenem, followed by Meropenem and Ertapenem. The rate of resistance strains against carbapenems was 18.75%. The prevalence of carbapenemase production according to Chromogenic Culture Media, Modified Hodge Test, and Carba NP Test was 18.75%, 21.25%, and 10%, respectively. Carba NP test showed less sensitivity in carbapenemase detection compared with other carbapenemase production detection methods. Carbapenemase production was significantly associated with resistance to all of carbapenems antibiotics. Carbapenemase production was also significantly associated with resistance to Cefepime, Ceftriaxone, Cefoxitin, Ciprofloxacin, and Levofloxacin. All Carbapenem-Resistant Enterobacteriaceae strains were significantly associated with carbapenemase production. There was a significant association between multi-drug resistance strain and carbapenemase production detected by Chromogenic media and the Modified Hodge test. The prevalence of carbapenemase resistance genes bla-KPC, bla-NDM, bla-IMP, bla-VIM, and bla-OXA-48 was 10%, 15%, 0%, 5% and 6.25%, respectively. Bla-KPC, bla-NDM and bla-OXA-48 were significantly associated with resistance to all carbapenems. bla-NDM showed the most significant association with resistance to other antibiotic classes other than carbapenems. Carbapenemase production was significantly associated with the presence of carbapenemase-resistant genes. Bla-VIM showed no significant association with carbapenem resistance or carbapenemase production.

One of the limitations of this study lies in its relatively small sample size of 80 isolates, which may not comprehensively represent the broader population of bacteremic patients in Jordan. Additionally, the study was conducted in only four hospitals, potentially limiting the generalizability of the findings to other regions or healthcare settings. Furthermore, the reliance on specific diagnostic methods, such as the Carba NP test, showed lower sensitivity compared to other techniques like the Modified Hodge Test and chromogenic media. These limitations should be considered when interpreting the results and designing future studies.