Phenotypic detection of extended-spectrum beta-lactamase produced by Klebsiella pneumoniae isolated from different clinical samples from intensive care unit, Khamis Military Hospital, Abha, Kingdom of Saudi Arabia

INTRODUCTION

K. pneumoniae, a gram-negative bacillus, is a significant contributor to healthcare-associated infections within hospital intensive care units (ICUs). These infections encompass a spectrum including bloodstream infections, urinary tract infections, ventilator-associated pneumonia, and surgical site infections.¹ The pathogenicity of K. pneumoniae is attributed to its lipopolysaccharides, cell wall proteins, and envelope receptors, which facilitate its binding to host cells while evading the human immune system.² The growing concern arises from the increasing resistance demonstrated by these bacteria, particularly against carbapenem antibiotics, a class previously reserved as the drug of last resort for severe gram-negative bacterial infections.^3,4

Carbapenem resistance in Enterobacteriaceae, including K. pneumoniae, is governed by two primary mechanisms. The first entails the production of enzymes such as ESBLs and AmpC beta-lactamases that hydrolyze cephalosporins. The second mechanism involves the production of carbapenemases, a lactamase capable of hydrolyzing a wide range of antibiotics including carbapenems. These carbapenemases include K. pneumoniae carbapenemases (KPC), metallo-beta-lactamases such as New Delhi metallo-beta-lactamase-1 (NDM-1), metalloproteinases active on imipenem (abbreviated as IMP), Verona Integron metallo-beta-lactamase (VIM), and oxacillinase-48-like carbapenemases (abbreviated as OXA-48).⁵ Notably, KPC demonstrates clonal expansion and genetic exchange capabilities, leading to enhanced resistance development and resilience in human reservoirs through biofilm formation.⁶

The resistance mechanism presented by NDM-1 has a particularly concerning therapeutic implication. First reported in 2009, NDM-1 confers resistance to a vast majority of available antibiotics, leaving only limited options like colistin and tigecycline effective. Moreover, the genes encoding NDM-1 exhibit a propensity for horizontal transfer to other species within the Enterobacteriaceae family, exacerbating the challenge of antibiotic resistance dissemination.⁷

Among the ESBLs, three main types are recognized and were named based on their genes: TEM, SHV, and CTX-M. These plasmid-mediated enzymes are associated with genes such as blaTEM, responsible for hydrolyzing penicillins and first-generation cephalosporins, and blaCTX-M, which exhibits a preference for cefotaxime.^8,9 Another ESBL gene, SHV, underpins plasmid-mediated resistance to ampicillin and is frequently identified in enterobacteria like K. pneumoniae and Escherichia coli.¹⁰

Given this complex landscape of antimicrobial resistance, this study aimed to isolate and identify ESBLs production in K. pneumoniae strains originating from diverse clinical samples within the ICU of Khamis Military Hospital in Abha, Kingdom of Saudi Arabia. Few studies in Saudi Arabia described the pattern of ESPLs production by K. pneumoniae in hospitals settings, which highlight the urgent need for conducting this research. This study holds significance in contributing to the understanding of antibiotic resistance patterns in a clinical context, aiding in the formulation of effective therapeutic strategies and measures to counter the escalating challenge of multidrug-resistant bacterial infections.

METHODS

This research was a retrospective observational study conducted at Khamis Military Hospital, Abha, Saudi Arabia. The study aimed to isolate and identify ESBLs produced by K. pneumoniae in various clinical samples obtained from patients admitted to the ICU during the period from December 2021 to April 2022. A total of 114 isolates of K. pneumoniae were obtained from clinical samples collected within the ICU. These isolates were derived from different types of infections. Clinical samples were collected and stored as per standard procedures to maintain their integrity and prevent contamination.

To include the clinical samples in the study, all samples underwent microbiological identification. During the initial Identification phase, the collected clinical isolates were subjected to initial identification through conventional methods which involved preliminary testing to identify potential K. pneumoniae isolates based on their morphological and biochemical characteristics. During the confirmation of isolates phase, the identified isolates were further confirmed using advanced microbiological techniques. This confirmation step aimed to ensure the accuracy of the initial identification and confirm the isolates as K. pneumoniae bacteria. Antimicrobial susceptibility testing was then carried out on the confirmed K. pneumoniae isolates to assess their response to various antibiotics. This testing was conducted using the Vitek 2 identification system, a product of BioMerieux.¹¹ The testing was performed in strict adherence to the manufacturer's instructions to ensure consistent and reliable results.

Additionally, data about the clinical samples were collected from patients records, microbiological test results, and antimicrobial susceptibility profiles. Demographic information of the patients from whom the samples were obtained, and sample details were recorded. The collected data were subjected to statistical analysis using appropriate tools. Descriptive statistics were used to summarize the demographic and clinical characteristics of the patient population from whom the samples were obtained. The percentages of ESBL-producing K. pneumoniae and carbapenemases were calculated and presented in table. The authors obtained approval from the Scientific Research and Conference Committee of Najran University, Saudi Arabia. Permission for data collection was obtained from Khamis Military Hospital Ethics Internal Review Board Committee, and the data collected was used for research purposes only.

Following the microbiological identification phase, a total of 114 isolates of K. pneumoniae were meticulously collected from diverse clinical samples originating from patients within ICU. Among this collection, the distribution of isolates across different sources was as follows: tracheal secretions contributed to 29 (25.43%)

RESULTS

isolates, urine samples contributed to 31 isolates (27.19%), blood specimens contributed to 31 isolates (27.19%), wound swabs contributed to 12 isolates (10.52%), and sputum samples contributed to 11 (9.6%) isolates. In addition, 55 (48.24%) isolates originated from male patients, and 59 (51.75%) originated from female patients (Table 1).

Among the isolated K. pneumoniae bacteria, the percentage of ESBL production constituted 28.94% (33 isolates). Among these ESBL-producing isolates, 5 (15.15%) were extracted from tracheal secretions, 19 (57.57%) from urine samples, 5 (15.15%) from blood samples, and 4 (12.12%) from wound swabs.

Regarding the distribution of carbapenemases production, among the isolated K. pneumoniae, 43 isolates (37.7%) had the KPC and NDM carbapenemases, while 34 isolates (29.8%) had the OXA-48 carbapenemases. The co-occurrence of ESBL mechanism of resistance with the presence of KPC and NDM carbapenemases was found in 2 isolates (1.7%) only, while the co-occurrence of ESBL mechanism of resistance with the presence of OXA-48 carbapenemase was also found in 2 isolates (1.7%) only (Table 1).

DISCUSSION

The emergence of ESBL producing K. pneumoniae strains within high-risk settings such as ICUs and their subsequent spread from hospital environments to the community poses a grave challenge to public health. The virulence factors associated with ESBL-producing K. pneumoniae, particularly those linked to its capsule, play a pivotal role in its pathogenicity, thereby exacerbating the intricacies involved in infection management. Previous studies have also emphasized the importance of ESBL-producing K. pneumoniae’s virulence factors in the complexity of infections management and their potential impact on public health.^12,13 In concurrence with previous findings, this study sheded light on K. pneumoniae's significance as a prominent ICU pathogen, capable of rapidly adapting and expanding its resistance spectrum against multiple antimicrobial agents.¹⁴

The insights gleaned from this investigation stood to heighten physician awareness regarding early diagnosis and targeted antimicrobial interventions to prevent potential complications. The presented data will serve to enhance clinical decision-making, enabling healthcare providers to initiate appropriate treatment strategies promptly.¹⁴ Early detection and responsive therapeutic strategies are critical to mitigate the challenges posed by ESBL-producing K. pneumoniae strains.

This study findings underscored the distribution patterns of multi-drug resistant K. pneumoniae isolates, revealing a substantial prevalence among various clinical samples. Notably, 27.19% of resistant K. pneumoniae were sourced from both urine and blood, with a comparable prevalence of 25.43% in tracheal aspirates, 10.52% in wound swabs, and 9.6% in sputum cultures. This aligned with earlier research that had identified K. pneumoniae as a key culprit in urinary tract infections, bloodstream infections, and surgical wound-related infections.^15,16

Within the confines of Khamis Military Hospital, this study scrutinized 114 isolates of K. pneumoniae for their carbapenem resistance profiles. The analysis revealed a spectrum of resistance mechanisms, with ESBL producers constituting 28.94%, OXA-48 carbapenemase production at 29.82%, combined KPC and NDM carbapenemases production at 37.71%, ESBL and OXA-48 carbapenemase co-occurrence at 1.7%, and a similar prevalence of 1.7% for combined ESBL, KPC and NDM resistance. The outcomes of this study were in tandem with reports from North Africa and West Africa, which had also indicated notably elevated phenotypic resistance rates.^3,17

A precedent for high percentages of ESBL-producing strains and a remarkable incidence of multidrug resistance has been documented previously in Saudi Arabia, specifically within the Armed Forces Hospital in Al-Kharaj, during the period spanning November 2004 to October 2007.¹⁸

In the current landscape, the escalation of K. pneumoniae infections was amplified by the increasing incidence of severe cases, coupled with a dearth of effective therapeutic options. This challenging scenario is compounded by the emergence of K. pneumoniae strains that have acquired enhanced virulence or antibiotic resistance traits. The intricacies of these newly evolved factors, their implications in distinct patient cohorts, and the potential targets for intervention remain areas of ongoing research.¹⁹

CONCLUSION

This study's culmination highlights the prominence of K. pneumoniae as a formidable ICU-associated pathogen. Predominantly identified in urine, blood, and respiratory sources, the pervasive presence of multi-drug resistant K. pneumoniae necessitates vigilant attention from infection control authorities, particularly within ICU settings. Recommendations stemming from this study advocate for routine in-vitro assays as a mean to expedite tailored treatments, thereby mitigating the potential complications that vulnerable ICU patients face. The study's implications resonate far beyond its immediate scope, emphasizing the urgency for a comprehensive approach to address the evolving challenges of infectious diseases in healthcare settings.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Scientific Research and Conference Committee of Najran University, Saudi Arabia, under No: 4/3/2021, dated October 16, 2021, and was conducted in accordance with the Declaration of Helsinki.

REFERENCES

1. 1. 1. Calfee DP. Recent advances in the understanding and management of Klebsiella pneumoniae. F1000 Res. 2017;6:1760.
2. 2. Czekaj T, Ciszewski M. Klebsiella pneumoniae NDM-new emerging superbacteria. Med Rodz. 2015;1:12-27.
3. 3. Kotb S, Lyman M, Ismail G, Fattah M, Girgis SA, Etman A, et al. Epidemiology of carbapenem-resistant Enterobacteriaceae in Egyptian intensive care units using national healthcare-associated infections surveillance data, 2011–2017. Antimicrob Resist Infect Control. 2020;9:2.
4. 4. Xu L, Sun X, Ma X. Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae. Ann Clin Microbiol Antimicrob. 2017;16(1):18.
5. 5. Reyes J, Aguilar AC, Caicedo A. Carbapenem-resistant Klebsiella pneumoniae: microbiology key points for clinical practice. Int J Gen Med. 2019;12:437-46.
6. 6. Molton JS, Tambyah PA, Ang BSP, Ling ML, Fisher DA. The global spread of healthcare-associated multidrug-resistant bacteria: a perspective from Asia. Clin Infect Dis. 2013;56(9):1310-8.
7. 7. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010;10(9):597-602.
8. 8. Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001;14(4):933-51.
9. 9. Rawat D, Nair D. Extended-spectrum ß-lactamases in gram negative bacteria. J Glob Infect Dis. 2010;2(3):263.
10. 10. Sharma M. Prevalence and antibiogram of extended spectrum β-lactamase (ESBL) producing Gram negative bacilli and further molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. J Clin Diagnostic Res. 2013;7(10):2173-7.
11. 11. Bobenchik AM, Hindler JA, Giltner CL, Saeki S, Humphries RM. Performance of Vitek 2 for antimicrobial susceptibility testing of Staphylococcus spp. and Enterococcus spp. Carroll KC, ed. J Clin Microbiol. 2014;52(2):392-7.
12. 12. Paterson DL, Bonomo RA. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev. 2005;18(4):657-86.
13. 13. Cheng HY, Chen YS, Wu CY, Chang HY, Lai YC, Peng HL. RmpA Regulation of capsular polysaccharide biosynthesis in Klebsiella pneumoniae CG43. J Bacteriol. 2010;192(12):3144-58.
14. 14. Falagas ME, Tansarli GS, Karageorgopoulos DE, Vardakas KZ. Deaths attributable to carbapenem-resistant Enterobacteriaceae infections. Emerg Infect Dis. 2014;20(7):1170-5.
15. 15. Cienfuegos-Gallet AV, Ríos AM, Viana P, Brinez F, Castro C, Villamil G, et al. Risk factors and survival of patients infected with carbapenem-resistant Klebsiella pneumoniae in a KPC endemic setting: a case-control and cohort study. BMC Infect Dis. 2019;19(1):830.
16. 16. Vading M, Nauclér P, Kalin M, Giske CG. Invasive infection caused by Klebsiella pneumoniae is a disease affecting patients with high comorbidity and associated with high long-term mortality. PLoS One. 2018;13(4):0195258.
17. 17. ElMahallawy HA, Zafer MM, Amin MA, Ragab MM, Al-Agamy MH. Spread of carbapenem resistant Enterobacteriaceae at tertiary care cancer hospital in Egypt. Infect Dis (Auckl). 2018;50(7):560-4.
18. 18. Ahmad S, Al-Juaid NF, Alenzi FQ, Mattar EH, Bakheet OES. Prevalence, antibiotic susceptibility pattern and production of extended-spectrum beta-lactamases amongst clinical isolates of Klebsiella pneumoniae at Armed Forces Hospital in Saudi Arabia. J Coll Physicians Surg Pak. 2009;19(4):264-5.
19. Salabi A, Walsh TR, Chouchani C. Extended spectrum β-lactamases, carbapenemases and mobile genetic elements responsible for antibiotics resistance in Gram-negative bacteria. Crit Rev Microbiol. 2013;39(2):113-22.