Five-year trends in distribution and antimicrobial resistance of gram-negative

Authors

Abstract

AimHealthcare-associated urinary tract infections are common in intensive care units (ICUs), and increasing antimicrobial resistance among Gram-negative (GN) bacteria complicates treatment. This study aimed to evaluate the distribution and antibiotic resistance rates of GN microorganisms isolated from adult ICU patients.
MethodsUrine culture samples obtained from ICU patients between January 2021 and June 2025 were evaluated in the Medical Microbiology Laboratory of Haydarpaşa Numune Training and Research Hospital. Only the first positive isolate from each patient was included. Cultures with bacterial growth of ≥10⁵ CFU/mL were analyzed. Identification was performed using MALDI-TOF MS, and antimicrobial susceptibility testing was conducted using the VITEK 2 system. Results were interpreted according to EUCAST criteria. Statistical analyses were performed using SPSS version 22.0, and p<0.05 was considered significant.
ResultsThe most frequently isolated pathogens were E.coli (35.3%), K.pneumoniae (27.0%), P.aeruginosa (14.4%), and A.baumannii (7.5%). A significant difference in microorganism distribution was observed over the years (p=0.031). E.coli isolates showed highest resistance to ampicillin (67.7%) and ciprofloxacin (54.8%). K.pneumoniae isolates demonstrated high multidrug resistance, particularly against ceftazidime, ceftriaxone, cefepime, and ciprofloxacin, with a significant increase in meropenem resistance over time (p=0.030). P.aeruginosa isolates showed highest resistance to ceftazidime and cefepime, with increasing cefepime resistance (p=0.003). A.baumannii isolates exhibited high resistance to nearly all tested antibiotics.
ConclusionE.coli and K.pneumoniae were the predominant GN pathogens isolated from ICU urine cultures. High multidrug resistance rates, especially among K.pneumoniae, P.aeruginosa, and A.baumannii, emphasize the importance of continuous local surveillance, rational antimicrobial use, and strict infection control measures in ICUs.

Keywords

Introduction

Urinary tract infections (UTIs) are among the most common healthcare-associated infections observed in patients hospitalized in intensive care units (ICUs) and are associated with substantial increases in morbidity, mortality, and healthcare costs.¹ Despite advances in modern medicine, factors such as prolonged urinary catheterization, invasive procedures, immunosuppression, and underlying comorbidities significantly increase the risk of infection in ICU patients.² In this high-risk patient population, the development of UTIs not only prolongs hospital stay but also contributes to the selection and dissemination of resistant microorganisms due to increased antibiotic exposure.³
Gram-negative (GN) bacteria are the most frequently isolated pathogens in healthcare-associated UTIs. In addition to members of the Enterobacterales family such as Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae), non-fermentative GN bacilli including Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) represent major causative agents.⁴ In recent years, the increasing prevalence of extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing strains has markedly limited the effectiveness of antimicrobial therapy and created significant challenges in the selection of empirical treatment regimens.⁵ Consequently, infections caused by multidrug-resistant (MDR) GN bacteria have become increasingly difficult to manage and are associated with higher morbidity and mortality rates.⁶ The rapid dissemination of resistant strains, particularly among ICU patients, reduces the success of empirical therapy and highlights the urgent need for updated treatment strategies and antimicrobial stewardship interventions.⁷ Therefore, continuous surveillance of the species distribution and antimicrobial resistance profiles of GN bacteria isolated in ICUs is of critical importance for guiding infection control policies and optimizing empirical treatment approaches.³
The present study aimed to evaluate the distribution of GN microorganisms isolated from urine cultures of adult ICU patients in our hospital and to determine the antimicrobial resistance profiles of these isolates.

Materials and Methods

Urine culture samples obtained from patients hospitalized in the intensive care units (ICUs) between January 2021 and June 2025 were included in the study at the Medical Microbiology Laboratory of Haydarpaşa Numune Training and Research Hospital. In cases where multiple positive cultures were detected from the same patient, only the isolate obtained from the first positive urine culture was included in the analysis, while repetitive isolates were excluded. Urine samples were quantitatively inoculated onto 5% sheep blood agar and Eosin Methylene Blue agar plates and incubated at 37°C for 18–24 hours. Cultures demonstrating bacterial growth of ≥10⁵ CFU/mL and considered clinically significant were included in the analysis.
Microorganism identification was performed using conventional microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (VITEK MS, bioMérieux, France). Antimicrobial susceptibility testing was carried out using the automated VITEK 2 system (bioMérieux, France) and, when necessary, results were confirmed by disk diffusion and/or gradient diffusion testing. Antimicrobial susceptibility results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria valid for the corresponding year. The distribution of isolated Gram-negative bacteria and their antimicrobial resistance rates were evaluated.
Ethical ApprovalThe study was approved by the Haydarpaşa Numune Training and Research Hospital Non-Interventional Clinical Research Ethics Committee (Date: 22.07.2025; Decision No: HNEAH-GOAEK/KK/2025/107).
Statistical AnalysisStatistical analyses were performed using SPSS version 21.0. Categorical variables were expressed as number and percentage. Comparisons were performed using the chi-square test and chi-square test for trend. A two-sided p value <0.05 was considered statistically significant.
Reporting GuidelinesThis study was reported in accordance with the STROBE guideline.

Results

During the study period, a total of 570 Gram-negative bacilli were isolated from urine cultures obtained from patients hospitalized in the ICU. Analysis of the isolate distribution revealed that the most frequently isolated pathogen was Escherichia coli (E. coli) at 35.3% (n=201), followed by Klebsiella pneumoniae (K. pneumoniae) at 27.0% (n=154), Pseudomonas aeruginosa (P. aeruginosa) at 14.4% (n=82), and Acinetobacter baumannii (A. baumannii) at 7.5% (n=43). Other pathogens were isolated at lower frequencies (Figure 1).
The annual distribution of Gram-negative (GN) bacteria isolated from urine culture samples is presented in Table 1. Analysis of yearly distribution demonstrated a statistically significant difference in microorganism distribution over the study period (p=0.031). The proportion of Escherichia coli (E. coli) was 33.5% in 2021 and reached its highest level in 2023 at 41.2%. The highest isolation rate of Klebsiella pneumoniae (K. pneumoniae) was observed in 2025 (38.6%), whereas Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) showed their highest isolation rates in 2021, at 21.3% and 11.0%, respectively (Table 1).
In Escherichia coli (E. coli) isolates, the highest resistance rates were observed for ampicillin (67.7%), ciprofloxacin (54.8%), and ceftazidime (44.3%). Antibiotic resistance rates remained generally similar throughout the study period, and no statistically significant differences were detected between years (p>0.05) (Table 2).
In Klebsiella pneumoniae (K. pneumoniae) isolates, the highest resistance rates were observed for ceftazidime (66.6%), ceftriaxone (77.2%), cefepime (80.5%), and ciprofloxacin (75.2%) (Table 3). During the study period, a statistically significant difference in antibiotic resistance rates was detected only for meropenem resistance in K. pneumoniae isolates (p=0.030, Table 3). Resistance rates for the other antibiotics remained statistically similar between years (p>0.05) (Table 3).
In Pseudomonas aeruginosa (P. aeruginosa) isolates, the highest resistance rates were observed for cefepime (37.0%) and ceftazidime (40.6%), whereas the lowest resistance rate was detected for amikacin (20.6%). Comparison of antibiotic resistance rates across the study years demonstrated a statistically significant increase only in cefepime resistance (p=0.003). No statistically significant differences were found in resistance rates for the other antibiotics between 2021 and 2025 (p>0.05) (Table 4).
High resistance rates against all tested antibiotics were detected in Acinetobacter baumannii (A. baumannii) isolates. The highest resistance rates were observed for ciprofloxacin (88.8%), gentamicin (71.8%), and amikacin (64.2%). No statistically significant differences were identified in antibiotic resistance rates between 2021 and 2025 (p>0.05) (Table 5).

Discussion

In this study, the distribution and antibiotic resistance profiles of Gram-negative bacteria isolated from urine cultures of adult intensive care unit patients between 2021 and 2025 were evaluated. Among the isolates, Escherichia coli (E. coli) (35.3%) and Klebsiella pneumoniae (K. pneumoniae) (27.0%) were the most frequently identified pathogens, followed by Pseudomonas aeruginosa (P. aeruginosa) (14.4%) and Acinetobacter baumannii (A. baumannii) (7.5%). The significant differences observed in microorganism distribution over the years indicate that the epidemiology of urinary tract infection pathogens in the ICU is dynamic. In addition, the significant increase in meropenem resistance among K. pneumoniae isolates and cefepime resistance among P. aeruginosa isolates highlights the critical importance of regular surveillance of local resistance data for guiding empirical treatment decisions.
In our study, the highest resistance rates among Escherichia coli (E. coli) isolates were observed against ampicillin, ciprofloxacin, and third-/fourth-generation cephalosporins. The high resistance rates to ceftriaxone and ciprofloxacin particularly suggest that extensive use of broad-spectrum antibiotics, fluoroquinolone exposure, and extended-spectrum beta-lactamase (ESBL) production may play important roles in the development of resistance among ICU patients. In the large-scale multicenter study conducted by Xu et al., involving 28 centers, high resistance rates were similarly reported among E. coli isolates against several antibiotics, particularly ampicillin, trimethoprim/sulfamethoxazole, and ciprofloxacin..⁸ Similarly, Khalid et al. demonstrated that ESBL-positive Escherichia coli (E. coli) isolates exhibited significantly higher resistance profiles compared with ESBL-negative strains, with notably increased resistance rates particularly against beta-lactam antibiotics and fluoroquinolones (p<0.001).⁹ A large-scale urinary tract infection study published from Türkiye in 2026 similarly reported high resistance rates to ciprofloxacin, ceftriaxone, and ceftazidime among Gram-negative uropathogens isolated from hospitalized patients, whereas resistance rates to amikacin and carbapenems remained relatively low.¹⁰ These findings indicate that the resistance patterns observed in Escherichia coli (E. coli) isolates in our study are largely consistent with the current literature. In this respect, the E. coli resistance data obtained in our study are in agreement with previously published reports. Nevertheless, the absence of meropenem resistance and the lack of a significant increase in ertapenem resistance over the years are clinically encouraging findings. These results suggest that carbapenems, when used with appropriate indications, remain effective therapeutic options for urinary tract infections caused by E. coli. However, preserving this effectiveness requires the prudent and restricted use of carbapenems in accordance with antimicrobial stewardship principles.
Klebsiella pneumoniae (K. pneumoniae) isolates emerged as the most resistant member of the Enterobacterales family in our study. The high resistance rates observed for ceftriaxone, cefepime, piperacillin/tazobactam, and ciprofloxacin indicate that this pathogen represents a major challenge for empirical therapy in the ICU setting. The most remarkable finding was the significant increase in meropenem resistance over the years. The increasing prevalence of carbapenem-resistant K. pneumoniae in Türkiye and worldwide has been largely associated with the dissemination of carbapenemases, particularly OXA-48-like, NDM, and KPC enzymes. Current Infectious Diseases Society of America (IDSA) guidelines also emphasize that treatment strategies for carbapenem-resistant Enterobacterales (CRE) infections should be guided by the underlying resistance mechanism, highlighting ceftazidime-avibactam for OXA-48-like carbapenemase producers and ceftazidime-avibactam plus aztreonam or cefiderocol for metallo-beta-lactamase-producing isolates.¹¹
In our study, the increasing trend of resistance to ceftazidime-avibactam and colistin among Klebsiella pneumoniae (K. pneumoniae) isolates is also noteworthy. Recent studies from various countries have reported progressively increasing extensive drug resistance patterns, particularly among carbapenem-resistant and ICU-associated K. pneumoniae urinary isolates. Moreover, the emergence of resistance to novel beta-lactam/beta-lactamase inhibitor combinations such as ceftazidime-avibactam has become an important clinical concern.^12,13,14,15 In a study conducted in Türkiye involving multidrug-resistant ICU isolates, susceptibility to ceftazidime-avibactam was reported to be high for Klebsiella pneumoniae (K. pneumoniae); however, the authors emphasized that the emergence of resistance to this agent should be closely monitored.¹⁶ Furthermore, recent meta-analysis data from Türkiye demonstrating an increasing prevalence of colistin-resistant Klebsiella pneumoniae (K. pneumoniae) isolates over time support the rising trend of colistin resistance observed in our study.¹⁷ Therefore, in ICU-associated urinary tract infections caused by Klebsiella pneumoniae (K. pneumoniae), treatment selection should not rely solely on conventional antibiogram results but should also be guided, whenever possible, by the carbapenemase type and local susceptibility data for ceftazidime-avibactam and colistin.
The highest resistance rates among Pseudomonas aeruginosa (P. aeruginosa) isolates were detected against ceftazidime and cefepime, with a particularly notable increase in cefepime resistance over the study period. P. aeruginosa is known to rapidly develop beta-lactam resistance through intrinsic resistance mechanisms, including porin loss, efflux pump activation, and AmpC beta-lactamase production. In the multicenter analysis conducted by Xu et al., resistance rates in P. aeruginosa isolates were generally lower than those observed in other Gram-negative pathogens; however, the authors emphasized that moderate resistance rates to certain agents, such as ticarcillin/clavulanate, warrant caution in empirical treatment selection.⁸ Studies conducted in Türkiye evaluating Gram-negative bacteria isolated from intensive care unit urine cultures have similarly demonstrated that susceptibility to aminoglycosides and colistin in Pseudomonas aeruginosa (P. aeruginosa) remains relatively preserved, whereas resistance to beta-lactams and carbapenems continues to represent a clinically significant problem.¹⁸ In our study, the lower resistance rate to amikacin compared with other antimicrobial agents suggests that amikacin may still represent an important therapeutic option for multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) isolates. Nevertheless, the increasing trend in carbapenem and colistin resistance observed over the years indicates that single-agent empirical therapy may become progressively more risky, particularly in ICU-associated urinary tract infections.
The markedly high antibiotic resistance rates observed in Acinetobacter baumannii (A. baumannii) isolates indicate that this pathogen remains one of the most problematic nosocomial agents in intensive care units. In our study, the high resistance rates detected against ciprofloxacin, gentamicin, amikacin, and carbapenems suggest that the majority of isolates exhibited a multidrug-resistant phenotype. Similarly, studies published in recent years have reported very high resistance rates among A. baumannii isolates against aminoglycosides, fluoroquinolones, cephalosporins, and carbapenems, whereas colistin and tigecycline have largely preserved their activity in many centers.¹⁹ The 2024 Infectious Diseases Society of America (IDSA) guidelines also emphasize that the treatment of carbapenem-resistant Acinetobacter baumannii (A. baumannii) infections is highly challenging and should be guided by antimicrobial susceptibility results in consultation with infectious diseases specialists.¹¹ In contrast, the absence of colistin resistance observed in our study represents a noteworthy and encouraging finding, suggesting that colistin has largely preserved its in vitro activity against Acinetobacter baumannii (A. baumannii) isolates in our center. However, considering the nephrotoxicity risk and the potential for the development of resistance, the use of this agent should be carefully managed in accordance with antimicrobial stewardship principles.²⁰

Limitations

This study has several limitations. First, due to its retrospective and single-center design, the generalizability of the findings to all intensive care units may be limited. In addition, only microbiological data were evaluated; therefore, the absence of clinical parameters such as patient characteristics, comorbidities, history of antibiotic use, invasive procedures, and mortality data may have restricted the clinical interpretation of the results. Furthermore, molecular analyses for resistance mechanisms were not performed, preventing the evaluation of the distribution of ESBLs, carbapenemases, and other resistance genes. Nevertheless, the inclusion of long-term surveillance data and the presentation of current resistance patterns of urinary Gram-negative pathogens in the intensive care unit represent important strengths of our study.

Conclusion

In conclusion, our study demonstrated a high burden of antimicrobial resistance among Gram-negative bacteria isolated from urine cultures in the intensive care unit, with particularly notable increases in resistance to certain critical antibiotics among Klebsiella pneumoniae (K. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa) isolates. Although the preservation of carbapenem susceptibility in Escherichia coli (E. coli) isolates is an encouraging finding, the high resistance rates to ceftriaxone and ciprofloxacin indicate that empirical treatment strategies should be selected cautiously. The significant increase in meropenem resistance in K. pneumoniae isolates, together with the increasing trend of resistance to ceftazidime-avibactam and colistin, suggests that available treatment options are becoming progressively limited. Therefore, empirical treatment protocols in intensive care units should be regularly updated according to local surveillance data, and antimicrobial stewardship practices, infection control measures, and strategies aimed at reducing catheter-associated urinary tract infections should be implemented in an integrated manner.

Declarations

Abbreviations

A.baumannii: Acinetobacter baumannii
CFU: Colony-forming unit
ESBL: Extended-spectrum beta-lactamase
EUCAST: European committee on antimicrobial susceptibility testing
GN: Gram-negative
ICU: Intensive care unit
MALDI-TOF MS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
MDR: Multidrug-resistant
P.aeruginosa: Pseudomonas aeruginosa
SPSS: Statistical package for the social sciences
UTI: Urinary tract infection
VITEK: Automated microbial identification and susceptibility testing system

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About This Article

Received:

May 11, 2026

Accepted:

June 17, 2026

Published Online:

June 19, 2026