European Journal of Cardiovascular Medicine

Bloodstream infections (BSIs) pose a significant global health challenge, contributing to substantial morbidity and mortality, particularly in healthcare settings. The causative agents, antimicrobial susceptibility patterns, and clinical outcomes associated with BSIs vary across different regions. In hospitals, these infections lead to prolonged hospital stays, increased healthcare costs, and high mortality rates, with approximately 200,000 cases and a mortality rate of 20-50% reported worldwide [1, 2].

The United States experiences a considerable burden of BSIs, ranking them as the 10th leading cause of death, with an incidence of 76-100 cases per 100,000 individuals [3, 4]. Pediatric intensive care units (PICUs) face a particularly high prevalence of nosocomial infections, with BSIs accounting for 28% of these cases. Risk factors for pediatric BSIs include invasive procedures, underlying medical conditions, and antibiotic use [5, 6]. Neonates are also vulnerable to BSIs, with rates ranging from 4 to 24%, influenced by factors such as prematurity, catheterization, and antibiotic therapy [5-7].

BSIs can progress to severe sepsis and septic shock, characterized by systemic inflammation, organ dysfunction, and hypotension. These conditions carry high mortality rates, especially in neonates and pediatric patients [8, 9]. Risk factors for severe sepsis include age, underlying diseases, and immunosuppression [10, 11].

Gram-negative bacteria are the predominant pathogens causing BSIs, with common culprits including Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., Haemophilus influenzae, and various streptococci and staphylococci species [12, 13]. Early and accurate diagnosis through methods like automated blood culture systems, such as BacT/Alert 3D/60, is crucial for effective management and improved patient outcomes [14]. While these systems offer advantages in diagnostic efficiency, their high costs can limit their accessibility in certain settings [15].

The escalating challenge of antimicrobial resistance underscores the need for continuous surveillance of pathogen susceptibility patterns to guide appropriate antibiotic therapy and prevent treatment failures [16].

The present study was designed to assess the drug resistance pattern among the gram negative isolates and to evaluate the distribution of genetic markers of drug resistance among the gram negative isolates in a tertiary care hospital in Cuttack, Odisha.

Study Design and Setting

This prospective study was conducted between November 2023 and July 2024 in the Department of Microbiology at SCBMCH, Cuttack, Odisha.

Study Population

A total of 558 pediatric patients, including neonates, with suspected bloodstream infections admitted to the PICUs were enrolled.

Inclusion and Exclusion Criteria

All patients with suspected bloodstream infections admitted to the PICU were included. Patients who had received antibiotics within seven days prior to clinical presentation were excluded.

Data Collection

Detailed clinical information, including age, sex, and clinical presentation, was recorded for each patient. Two blood cultures were collected from each patient, with a 15-30 minute interval between samples. Blood cultures were processed using the BacT/ALERT 3D system (bioMérieux, Marcy-l’Étoile, France). Positive cultures were subcultured on blood agar and MacConkey agar.

Microbiological Analysis

Isolates were identified using standard microbiological methods. Antimicrobial susceptibility testing was performed using the broth microdilution method to determine minimum inhibitory concentrations (MICs). Advanced Expert System (AES) analysis was conducted to characterize resistance patterns. DNA was extracted from isolates, and its quality and quantity were assessed using a UV-VIS spectrophotometer.

A total of 558 clinically suspected cases of sepsis were evaluated in this study. Out of these, 192 cases (34.4%) were culture positive for various microorganisms (Table 1).

Table 1: Culture Positive Cases Among Clinically Suspected Cases of Sepsis

Among the 192 culture positive cases, the distribution of microorganisms was as follows (Table 2): 

Table 2: Distribution of Microorganism Among Culture Positive Cases

Of the 97 Gram-negative isolates, a significant proportion were found to be multi-drug resistant (MDR). Specifically, 75 isolates (77.3%) were classified as MDR (Table 3).

Table 3: Distribution of Multi-Drug Resistance (MDR) Among the Gram Negative Isolates

Genotypic analysis for extended-spectrum beta-lactamase (ESBL) genes revealed that all isolates carried the TEM gene, while 18 (24%) contained both SHV and CTX genes (Table 4).

Table 4: Detection of ESBL by Genotypic Method Targeting 3 Different Genes

When carbapenemase production was assessed, none of the isolates were identified as producers via phenotypic methods (Modified Hodge Test); however, 29 isolates (38.6%) were found to produce carbapenemase according to genotypic methods targeting the NDM-1 gene (Table 5).

Table 5: Comparison of Carbapenemase by Phenotypic and Genotypic Method

Additionally, a breakdown of the isolates showed that 12 (50%) of Acinetobacter baumannii, 10 (58.8%) of Klebsiella pneumoniae, and 5 (41.6%) of Burkholderia cepacia complex were identified as carbapenemase producers using genotypic methods (Table 6).

Table 6: Detection of Carbapenemase by Genotypic Method Targeting 2 Different Genes

In the assessment of AmpC production, the results indicated that 4 isolates (5.33%) were identified as AmpC producers through phenotypic methods, while 9 isolates (12%) were detected via genotypic methods (Table 7).

Table 7: Comparison of AmpC by Phenotypic and Genotypic Method

The effectiveness of the various detection methods for resistant isolates was compared (Table 8). Using phenotypic methods, ESBL was detected in 27 isolates (36%), while no carbapenemase producers were identified, and AmpC producers were found in 4 isolates (5.3%). In contrast, genotypic methods identified 100% of isolates as ESBL producers, 38.6% as carbapenemase producers, and 12% as AmpC producers.

Table 8: Comparison of Methods Used for Detection of Resistance of Isolates

Bloodstream infections (BSI) are a significant cause of morbidity and mortality among pediatric patients. Effective management of BSI relies on prompt clinical suspicion, early diagnostic measures, timely initiation of rational antimicrobial therapy, and comprehensive supportive care [17]. Blood cultures are essential for diagnosing and managing BSI.

In our study, blood culture positivity was found in 192 (34.4%) of cases, which is consistent with findings by Surase et al. (32%) and Parihar et al. (28.9%) [204, 205]. Blood culture positivity rates vary across studies from different regions, such as Goel et al. (9.2%), Nasa et al. (10.6%), Mathur et al. (10.6%), Lunagaria et al. (16.9%), Arora et al. (20.02%), Sharma et al. (33.9%), and Remirez Barba et al. (39%) [18].

In our study, Gram-negative bacteria, Gram-positive bacteria, and yeasts were isolated in 50.5%, 43.3%, and 6.2% of cases, respectively, in automated blood culture systems. These findings are similar to Lunagaria et al., who isolated Gram-negative bacteria, Gram-positive bacteria, and yeasts in 55.3%, 40%, and 4.7% of cases, respectively [19]. Most studies report a higher prevalence of Gram-negative bacteria compared to Gram-positive bacteria [20].

Our study observed that 75 (77.3%) of the Gram-negative bacteria isolated were multidrug-resistant (MDR), while Gupta et al. reported a 72.1% MDR rate among Gram-negative bacteria isolates [21]. Acinetobacter baumannii (32%) was the predominant isolate, followed by Klebsiella pneumoniae (22.6%) in clinically suspected bloodstream infections. In contrast, Livadiotti et al. found Klebsiella pneumoniae (27%) as the most common isolate [22], indicating possible geographical variation in the spectrum of microorganisms.

The antibiotic resistance pattern among Gram-negative isolates in this study showed that most isolates were resistant to Piperacillin/Tazobactam (96%) and cefepime (90%), similar to findings by Vanitha et al. [23]. Acinetobacter baumannii was highly resistant to Piperacillin/Tazobactam (100%) and Cefuroxime (100%), followed by ceftriaxone (95.8%). Similarly, Klebsiella pneumoniae showed high resistance to ampicillin (100%), followed by Cefoperazone/Sulbactam (94.1%). Colistin (97.3%) and tigecycline (97.3%) were the most effective antibiotics for all Gram-negative bacterial isolates, including non-fermenters, consistent with Lunagaria et al., who found colistin (80.9%) and tigecycline (66%) as the most sensitive antibiotics [24].

In this study, 36% of isolates were detected as Extended Spectrum β-Lactamase (ESBL) producers by phenotypic methods (Combined double disc test), while 100% of isolates were detected as ESBL producers by genotypic methods (targeting the TEM gene). Bajpai et al. reported 51.2% as ESBL producers by phenotypic methods and 48.7% by genotypic methods [25]. In our hospital settings, the TEM gene (100%) predominated over SHV (24%) and CTX (24%) genes responsible for ESBL production. This result aligns with Yazdi et al. (87.1% TEM, 70.6% SHV, 30.8% CTX) but differs from studies by Eftekhar et al., where SHV (43.1%) exceeded TEM (35.2%); Shahid et al., where CTX (28.8%) exceeded SHV (13.7%); and Ahmed et al., where CTX (71.4%) exceeded TEM (55.1%) [26]. Several other studies worldwide have shown variable results. In the majority of Indian and Chinese studies, the TEM gene predominated over SHV and CTX. ESBL producers in our study were found to be 100% by PCR (targeting the TEM gene), higher than the findings of Bajpai et al. (48.7%), Kavitha et al. (32%), and Arora et al. (34.4%) [27]. Among 75 ESBL producers, co-existence of all three genes (TEM, CTX, SHV) was observed in 9.3% of isolates, higher than Kaur et al.'s study, which observed co-existence in only 6.45% of isolates [28]. Co-existence of two genes was highly observed with the TEM and CTX genes in 17.3% of isolates, followed by TEM and SHV genes in 10.6% of isolates.

The higher incidence of ESBL production could be due to the injudicious use of antibiotics in hospitalized patients and geographic variation. Carbapenems are known as the last resort for treating infectious diseases, playing a key role in managing severe hospital-acquired infections. The recent emergence of carbapenemase-producing Gram-negative isolates mediating carbapenem resistance is a concerning trend [29]. None of the isolates were detected as carbapenemase producers by phenotypic methods (Modified Hodge Test), but 29 (38.6%) isolates were detected as carbapenemase producers by genotypic methods (targeting the NDM-1 gene). In our study, 29 (38.6%) isolates had the NDM-1 gene, and 11 (14.6%) had the KPC gene. In comparison, Somily et al. reported 35.7% with the NDM-1 gene and none with the KPC gene [26]. The NDM-1 (New Delhi metallo-β-lactamase) gene, first described in a Swedish patient of Indian origin in 2009, is currently one of the predominant resistant genes. NDM strains are usually resistant to nearly all antibiotics except colistin and tigecycline [30].

This study observed that 38.6% of MDR Gram-negative bacteria had carbapenemase resistance, with Klebsiella pneumoniae (58.8%) and Acinetobacter baumannii (50%) being the majority of carbapenemase producers. Various studies have reported that most carbapenemase producers are Acinetobacter spp. and Pseudomonas spp. [31]. The prevalence of carbapenemase-producing isolates was 38.6% in our study (genotypic method), consistent with Somily et al.'s findings, which reported a prevalence of 35.7% [32].

In this study, 5.3% of isolates were detected as AmpC β-lactamase (AmpC) producers by phenotypic methods (Disc potentiation test), and 12% by genotypic methods (targeting the AmpC gene). Rania et al. reported 4.1% and 28.5% detection of AmpC producers by phenotypic and genotypic methods, respectively [33]. The prevalence of AmpC β-lactamase-producing isolates was 12% (genotypic method) in our study, lower than other Indian studies [32]. AmpC β-lactamase production was mostly seen in Klebsiella pneumoniae (17.6%), followed by Acinetobacter baumannii (8.3%), differing from Singhal et al.'s study, where AmpC production was higher in Acinetobacter spp. (28.57%), followed by E. coli (6.97%) and Klebsiella spp. (6.18%) [34].

By phenotypic methods, we detected 27 (36%) isolates as ESBL producers, 4 (5.3%) as AmpC producers, and none as carbapenemase producers. However, genotypic methods detected 75 (100%) isolates as ESBL producers, 9 (12%) as AmpC producers, and 29 (38.6%) as carbapenemase producers. The higher incidence of MDR isolates with resistant genes may be due to the injudicious use of antibiotics and geographical variation.

In this study, we investigated the genotypic and phenotypic drug resistance patterns of Gram-negative bacteria isolated from bloodstream infections among pediatric patients in a tertiary care hospital in Odisha. The findings highlight a significant prevalence of multidrug-resistant (MDR) organisms, underscoring the critical need for continuous monitoring and stringent antibiotic stewardship. Our study revealed a high prevalence of MDR organisms, with a substantial proportion of Gram-negative isolates exhibiting resistance to multiple commonly used antibiotics, including third-generation cephalosporins and carbapenems. This resistance poses a serious challenge to the effective management of bloodstream infections in pediatric patients, leading to limited therapeutic options and necessitating the use of more potent and often more toxic antibiotics.

Overall, our research underscores the critical importance of continuous surveillance of antimicrobial resistance patterns, the implementation of effective infection control practices, and the promotion of judicious antibiotic use to combat the growing threat of multidrug-resistant Gram-negative bacteria in pediatric patients. These measures are essential to improving clinical outcomes and ensuring the sustainable use of existing antibiotics in the fight against resistant infections.

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