Molecular epidemiology of Carbapenemase-encoding genes and comparative evaluation of carbapenem MIC with genotypic carbapenem resistance in Klebsiella isolates from neonatal sepsis cases

INTRODUCTION

The past two decades have witnessed an increase in the incidence and spread of multidrug-resistant organisms like Carbapenem-resistant Enterobacteriales (CRE). CRE poses a considerable threat to mankind as they are resistant to almost all the available beta-lactam antimicrobials,including one or more carbapenem drugs along with fluoroquinolones, and aminoglycosides. The treatment options are either very few or non-existent for this group of multidrug-resistant bacteria.^[1]

Klebsiella pneumoniae (K. pneumoniae) is the most notorious member of CRE group that is frequently associated with life-threatening infections. This pathogen can be resistant to the repertoire of available antibiotics. The isolation of K. pneumoniae in increased frequencies from hospital environments,including intensive care units especially in developing countries is alarming.^[2-4] K. pneumoniae is very frequently associated with oxacillinases (OXA)-48 like and New Delhi metallo-b-lactamase (NDM) carbapenemase-encoding genes globally. Acquisition and transmission of potent transferable plasmid-encoded carbapenemases such as Klebsiella pneumoniae Carbapenemase (KPC), NDM, Imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase-48 like enzymes is common in multi-drug resistant and hypervirulent strains of Klebsiella spp.^[3]

India is endemic to CRE harboring carbapenemase-encoding genes of OXA-48,such as OXA-181 and OXA-232 and NDM-1 variants, which were first described in K. pneumonia.^[5,6] The sporadic outbreaks of various members of CRE possessing other carbapenemase-encoding genes such as IMP, KPC, and VIM have also been reported over recent years.^[7,8] Noticeably, the circulating carbapenemase strains of pathogenic bacteria may vary geographically or even at different locations within the same healthcare setting. Phenotypic determination of potential carbapenem resistance due to the presence of carbapenemase-encoding gene and its impact on the susceptibility to other antimicrobial agents varies widely according to the individual carbapenemase-encoding gene present.^[1] OXA-48 like carbapenemases is known to possess weak carbapenem hydrolytic activity and, thus, is likely to be missed by phenotypic detection methods.^[9,10] Therefore, the molecular identification of resistance mechanisms in circulating strains of CRE pathogens is crucial for healthcare settings to effectively manage life-threatening infections without unwarranted delay. Furthermore, the availability of information regarding the prevalent resistant strains can play an important role in containment of these infections.

Neonatal sepsis is the third leading cause of neonatal mortality. As per United Nations Children’s Fund, the neonatal mortality rate in India was 21.7/1000 live births in the year 2022.^[11] Carbapenem-resistant K. pneumoniae is often known for frequently causing outbreaks in neonatal intensive care units (NICUs), thus warrants its molecular characterization pertaining to the presence of carbapenemase-encoding genes.^[12] Often the inadequacy of molecular diagnosis of CRE and their underlying resistance mechanisms in resource-limited settings contribute to the inadvertent spread of these resistant isolates. The growing complexities surrounding the treatment of life-threatening infections caused by CRE demands an understanding of its local epidemiology for providing adequate empirical coverage and antimicrobial resistance control initiatives.

Numerous studies have addressed the dissemination of CRE, but the data on neonatal sepsis are limited. Earlier studies reported the presence of a single carbapenemase gene in K. pneumoniae, but recent years have shown the emergence of coproduction of multiple carbapenemases.^[13,14] Furthermore, NDM producing K. pneumoniae was more prevalent previously in neonatal infections. Besides, recent years have noted a significant emergence of highly transferable OXA-48 class D carbapenemase in Enterobacteriales.^[15,16] Therefore, the present study attempted to determine the molecular epidemiology of Carbapenemase-encoding genes and comparative evaluation of carbapenem minimum inhibitory concentration (MIC) with genotypic carbapenem resistance in Klebsiella isolates from neonatal sepsis cases.

MATERIALS AND METHODS

A prospective study was conducted by the molecular laboratory of the Department of Microbiology in collaboration with NICU and the Department of Pediatrics. Post obtaining the approval from the Institutional Ethics Committee-Human Research, the study was extended for over a period of one year, starting July 2019 to June 2020. The first 100 neonates admitted to NICU with clinical signs and symptoms suggestive of neonatal sepsis who had blood cultures positive for Klebsiella spp. were included in the study. Sepsis screening was performed in the clinically suspected cases. The criteria included total leukocyte count (<500/mm³), absolute neutrophil count (<1800/mm³), immature to a total neutrophil ratio (>20%), C-reactive protein (>1 mg/dL), and micro erythrocyte sedimentation rate (15 mm or more in the 1^st h). Sepsis screening was considered positive if any two of these parameters were positive.^[17]

All these non-duplicate Klebsiella spp. isolates from blood samples of neonatal sepsis patients were identified and processed further using the standard laboratory guidelines. Antimicrobial susceptibility testing (AST) was performed by modified Kirby Bauer disc diffusion method using the amikacin (30 μg), aztreonam (30 μg), azithromycin (15 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefixime (5 μg), ciprofloxacin (5 μg), cotrimoxazole (25 μg), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), and piperacillin-tazobactam (100/10 μg) antibiotic discs (HiMedia laboratories, India).

Minimum Inhibitory Concentration (MIC for imipenem and meropenem was determined by Epsilometer-test using Imipenem Ezy MIC™ Strip (IPM) (0.002–32 μg/mL) and Meropenem Ezy MIC™ Strip (MRP) (0.002–32 μg/mL) (HiMedia Laboratories, India). K. pneumoniae ATCC BAA-1705 and K. pneumoniae ATCC BAA-1706 were used as positive and negative controls. For both the antibiotics, imipenem and meropenem, isolates were considered susceptible (MIC ≤1 μg/mL, intermediate (MIC of 2 μg/mL), and resistant (MIC ≥4 μg/mL) after incubation of 18 h at 35–37°C. The AST and interpretation of results were performed as per Clinical and Laboratory Standards Institute guidelines.^[18]

The deoxyribonucleic acid (DNA) was extracted (HIPURATM bacterial genomic DNA purification kit from HiMedia laboratories, India) by spin-column procedure. All the isolates of Klebsiella spp. were tested for the presence of beta-lactamase Klebsiella pneumoniae carbapenemase (blaKPC), beta-lactamase New Delhi metallo-β- lactamases-1(blaNDM-1), beta-lactamase imipenemase (blaIMP), beta-lactamase Verona imipenemase (blaVIM) genes by multiplex polymerase chain reaction (PCR) and uniplex PCR for beta-lactamase oxacillinase-48 (blaOXA-48) using the primer sequences for target genes mentioned in Table 1.^[19,20]

Cycling conditions for blaKPC, blaNDM-1, blaIMP, and blaVIM genes Multiplex PCR: ^[19] For amplification, 30 cycles were done at 94°C for 1 min, 54°C for 1 min, and 72°C for one and a half minutes followed by a final extension step of 5 min at 72°C.

Cycling conditions for blaOXA-48 gene Uniplex PCR:^[20] For amplification, initial denaturation at 94°C for 10 min; 30 cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 1 min followed by a final elongation step at 72°C for 7 min. The annealing temperature was optimal at 57°C.

The amplified products were then run on 1.5% Agarose gel (HiMedia laboratories India) at 100 Volts for 45 min to 1 h and viewed under UV light (G: BOX, SynGene). The gel was then photographed using GeneSnap from SynGene [Figure 1].

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Figure 1:

Gel electrophoresis showing bands of DNA and 100bp DNA ladder in GeneSnap software. 1: Positive control for blaNDM-1 gene; 2,9: Negative control (Distilled water); 5, 12: DNA ladder; 3,4,6,7,8: Test (positive for blaNDM-1 gene); 10: Positive control for blaOXA-48 gene; 11,13: Test (positive for blaOXA-48 gene). blaNDM-1: beta-lactamase New Delhi metallo-β-lactamases-1, blaOXA-48: Oxacillinases.

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RESULTS

Distribution of carbapenemase-encoding genes among Klebsiellas spp. isolates

The overall prevalence of carbapenemase-encoding genes in Klebsiella spp. isolates from blood samples of neonatal sepsis cases admitted in NICU was 16% (16/100). The distribution of various carbapenemase-encoding genes is shown in Table 2 and Figure 2. The most predominant carbapenemase-encoding gene was blaOXA-48 gene (12%), followed by blaNDM-1 gene (6%). None of the isolates harbored carbapenemase-encoding genes of blaIMP, blaVIM, and blaKPC. The coexpression of both blaOXA-48 and blaNDM-1 was observed in 2% of isolates.

Table 2: Distribution of carbapenemase-encoding genes in Klebsiella spp. isolates from blood samples of neonatal sepsis cases admitted in NICU (n=100).

Carbapenemase-encoding gene/s	Present (%)	Absent (%)
KPC	0	100
NDM-1	6	94
IMP	0	100
VIM	0	100
OXA-48	12	88
Coexpression of blaOXA-48 and blaNDM-1	2	98
Total	16	82

KPC: Klebsiella pneumoniaeCarbapenemase, NDM: New Delhi metallo-β-lactamase, IMP: Imipenemase, VIM: Verona integron-encoded metallo-beta-lactamase, OXA-48-like: Oxacillinases, NICU: Neonatal intensive care unit. n: Total number of tested isolates.

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Figure 2:

Prevalence of carbapenemase-encoding genes in Klebsiella spp. isolates from blood samples of neonatal sepsis cases admitted in neonatal intensive care unit (n = 100). n: Specify total number of tested isolates, KPC: Klebsiella pneumoniae Carbapenemase, NDM-1: New Delhi metallo-β-lactamase-1, IMP: Imipenemase, VIM: Verona integron-encoded metallo-beta-lactamase, OXA-48-like: Oxacillinases.

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DISCUSSION

Neonatal sepsis remains the third leading cause of neonatal mortality. The bacteriological profile responsible for neonatal sepsis varies geographically. The most frequent offenders of early-onset sepsis and late-onset sepsis in developing nations like India are K. pneumoniae and Staphylococcus aureus.^[21] The production of carbapenemases by Enterobacterales isolates especially Klebsiella spp., associated with invasive infections, gut colonization, or hospital-acquired infections, has been detected across the globe.^[22,23] These superbugs causing infections in vulnerable newborns are present everywhere, including the birth canal, incubators, hands of healthcare workers, and even the medical equipment like nasogastric tubes. Premature or low-birth-weight infants needing extended hospital stay or dependency on life support systems have significantly elevated risk of infection by these pathogens. The carbapenemase producers play a significant role in triggering outbreaks by transferring plasmid-encoded carbapenemase genes, thus posing an exceptionally difficult challenge in the containment of these infections.^[24] Many Indian studies have documented the high prevalence of bloodstream infections caused by multidrug resistant Klebsiella spp. isolates in high-risk settings of NICUs.^[25,26]

The predominant mechanism of resistance in the present study was the presence of blaOXA-48 (12%) followed by blaNDM-1 (6%). The coexpression of both blaOXA-48 and blaNDM-1 was observed in 2% of isolates. However, another study from India has reported blaOXA48-like expression in 13%, blaNDM in 19%, and coexpression of blaNDM and blaOXA48-like in 28% isolates bloodstream isolates of K. pneumonia.^[27] In line with our findings, this research has also reported the predominant carbapenem-encoding genes as blaNDM and blaOXA48. Laolerd et al.,^[28] have reported the presence of at least one of the carbapenemase genes in 77.7% of CRE isolates (predominantly K. pneumoniae). The same study also documented that the prevalence of blaNDM (71.75%) and blaOXA48-like (50.22%) genes among CRE isolates and coexpression of both blaNDM and blaOXA48-like genes was 32.73% among K. pneumoniae isolates.^[28] The differences in the distribution of blaNDM , blaOXA48, and their coexpression can be attributed to the geographical variations.

The present study documented blaOXA-48 carbapenem-encoding gene to be most prevalent in a tertiary care high-volume center of north India. The OXA-48-like enzymes are hidden threats as they possess a low level of carbapenem hydrolytic activity. This mechanism of resistance remains underreported by standard laboratory procedures and subsequent treatment failures.^[10] In the present study, intermediate and susceptible MIC values were also observed along with resistant MIC values for imipenem in blaOXA-48 harboring isolates. This could be probably due to the absence of coresistance mechanisms in OXA-48 like producers. In the absence of other resistance mechanisms, OXA-48, like producers,can show lower or intermediate MIC values for carbapenems. Thus, their true prevalence rates are hard to estimate.^[29,30] In our study, all the carbapenemase gene-harboring isolates had resistant MIC values for meropenem. Thus, meropenem is a better predictor of carbapenemase production in comparison to imipenem.

The frequent use of carbapenems and other beta-lactam antibiotics has led to increased resistance in the bacteria,and the rapid fluctuation in their susceptibility pattern challenges the treatment options for serious infections. Current guidelines from the World Health Organization recommend the use of ampicillin, extended-spectrum cephalosporins (cefotaxime), and aminoglycosides (gentamicin and amikacin) for the treatment of neonatal sepsis. This protocol is widely followed in various healthcare settings.^[31] However, the production of AmpC beta-lactamases and extended-spectrum beta-lactamases has led to increasing dependency on carbapenems as next line of treatment. Among carbapenems, meropenem is the preferred carbapenem in neonates as the safety profiles of other carbapenems are not well documented in this patient group.^[32]

The OXA-48 producers hydrolyze penicillins with greater efficiency and carbapenems at a lower extent, sparing expanded-spectrum cephalosporins, whereas MBLs (NDM-1) are more potent, they can hydrolyze almost all available beta-lactam antibiotics, including carbapenems along with extended-spectrum cephalosporins, aminoglycosides, aztreonam, and fluoroquinolones.^[33] Optimizing treatment strategies for these infections become a challenge due to the lack of knowledge about the circulating carbapenemases. We compared the distribution of susceptibility to routine antimicrobial agents in isolates with and without genotypic carbapenem resistance. Despite the inability of OXA-48 producers to hydrolyze expanded-spectrum cephalosporins, high resistance rates (94%) to these cephalosporins were observed in our study. This additional resistance to expanded-spectrum cephalosporins among OXA-48 producers can be due to the presence of other mechanisms of resistance including coexpression of both OXA-48 and NDM-1. Imipenem was the most effective antimicrobial agent in the absence of any carbapenemase-encoding gene and amikacin was most effective in the presence of carbapenemase-encoding genes followed by imipenem. Similar results showing carbapenem drugs being most effective against Klebsiella were also reported in study by Mohsen et al.^[34]

Considering the widespread prevalence of multidrug-resistant Klebsiella spp. as a causative agent of life-threatening infections, it is crucial to understand the mechanisms of resistance behind the last resort drugs of carbapenems. Furthermore, their therapeutic utility can be largely restricted by the presence of certain carbapenemase-encoding genes.^[35] Thus, the determination of carbapenem resistance by phenotypic methods should be supplemented with genotypic assessment. The knowledge of prevailing strains of carbapenemases can aid in the implementation of antibiotic cycling and formulation of effective hospital antibiotic policies. This has the potential to significantly impact patient care in terms of reduced morbidity, mortality, and duration of hospital stay. However, further elaborative studies with larger sample sizes and diverse clinical conditions are warranted.

Carbapenems are broad-spectrum higher-level antibiotics and are often considered as last resort antibiotics. The emergence of carbapenemases is a major healthcare problem. The susceptibility of carbapenem-producing CRE to carbapenem drugs may vary depending on the molecular class of carbapenemase produced. The distribution of circulating strains is not uniform and varies geographically. Therefore, the determination of prevalence of carbapenemase-encoding genes may be of paramount importance in the development of effective antibiotic policies at various levels.

CONCLUSIONS

Early detection and knowledge of the prevalence of carbapenemase encoding genes significantly benefit patient care by lowering morbidity, mortality, and hospitalization duration. This also impacts the formulation of combination antibiotic therapies, and hospital antibiotic policies and forms the basis of antibiotic cycling as an important component of infection control in NICU.