Antimicrobial Susceptibility of Klebsiella pneumoniae Isolated from Intensive and Non-intensive Care Units Patients: A One-year Retrospective Study in a Tertiary Healthcare Hospital, Saudi Arabia

Antimicrobial-resistant Klebsiella pneumoniae(K. pneumoniae) constitutes a major global health warning and is significantly implicated in severe infections associated with increased morbidity and mortality. As hospitalized patients in the ICU are more vulnerable to severe infections with increased cost of treatment and prolonged hospital stays, we aimed to compare antimicrobial susceptibility of K. pneumoniae obtained from intensive care unit (ICU) and non-intensive care unit (non-ICU) patients as well as to investigate potential impact of antimicrobial resistance on patient outcome. A retrospective, cross-sectional study conducted on ICU and non-ICU patients having K. pneumoniae infection during 2021 at Prince Mohammed bin Abdulaziz Hospital (PMAH) in Riyadh. Data regarding K. pneumoniae and their antimicrobial susceptibility, were retrieved and analyzed through R Software. 229 K. pneumoniae were isolated, 33.2% from ICU patients, and 66.8% from other departments. Most of the patients were males (66.8%) belonged to the older age group ( 62.9%). The isolates were obtained from endotracheal aspirate, sputum, blood, urine and wound samples. The ICU patients developed higher resistance to all examined antibiotics than non-ICU (p<0.001). More than 60% of ICU Klebsiella isolates were extended-spectrum b -lactamases (ESBL) and multidrug resistant (MDR) compared to non-ICU isolates (p<0.001). The most effective drugs were amikacin, imipenem, and meropenem, but their effectiveness substantially decreased against MDR strains. There was a statistically significant difference between the MDR, ESBL, and sensitive groups regarding hospital stay and mortality (P< 0.001). ICUs have exhibited a remarkable increase in MDR K. pneumoniae , which has a negative impact on patient outcomes.


INTRODUCTION
Since intensive care unit (ICU) patients are critically ill and frequently subjected to invasive procedures, they are more prone to severe infections caused by aggressive organisms.Furthermore, antimicrobial resistance is one of the most significant threats to ICU patients as it complicates their treatment, along with prolonged hospital stays, high costs, and severe adverse outcomes. 1According to the World Health Organization (WHO), "AMR is one of the top 10 global public health threats facing humanity" .2The uncontrolled use of antimicrobials, the overworked healthcare workforce, and the prolonged exposure to invasive equipments are important causes of antimicrobial resistance especially during pandemics. 3 n t h e re c e nt ye a rs , K l e bs i e l l a pneumoniae (K.pneumoniae), one of the major Enterobacterales, has emerged as a clinically significant pathogen because of increased antibiotic resistance and its propensity to cause serious outcomes. 4It has been ranked the second among Gram-negative bacteria in causing hospital-acquired infections. 5K. pneumoniae is recognized as the primary cause of serious nosocomial infections such as respiratory infections, urinary tract infections, soft tissue infections, bacteremia, and sepsis. 6They are also responsible for device-associated infections in ICU patients. 7The pathogenicity of K. pneumoniae is influenced by the presence of capsule, lipopolysaccharide (LPS), and other cell wall proteins that allow the organism to bind to the host cells and protect against phagocytosis. 8urthermore, the ability of K. pneumonie to form a thick layer of biofilm is considered as an important virulence factor.Biofilms permit bacteria to attach to biotic or abiotic surfaces, protect microorganisms from opsonization by antibodies, phagocytosis, and removal via the ciliary action of epithelial cells. 9In addition, resistance to antibacterial and disinfectant agents is noticeably more among bacterial populations in biofilms than free-living planktonic cells. 10ultidrug resistance (MDR) is defined as "acquiring non-sensitivity to one or more agents in at least three groups of antimicrobials". 11ltidrug"resistant gram-negative bacteria (MDR-GNB) are the leading cause of limited therapeutic options or even antimicrobial treatment failure among critically ill patients. 12It has been revealed that indiscriminate use of antimicrobials is the main drive for these multidrug-resistant organisms' (MDROs) appearance and rapid spread. 13Recently, K. pneumoniae producing extended-spectrum b-lactamases and carbapenemases have significantly spread and exhibited a high degree of multidrug resistance. 14They become capable of hydrolyzing most antibiotics, including cephalosporins and carbapenems, which are the primary treatment options for the severe infections caused by Gram-negative bacilli. 15ccording to the WHO, MDR K. pneumoniae pose a great public health threat and considered highpriority pathogens for research and development of novel antibiotic therapies. 16he lack of effective treatment for multidrug-resistant pathogens and the resulting increase in mortality has led to an urgent need for finding a radical solution to reduce the nightmare of spread of MDR bacteria all over the world. 17ne of these important solutions is an increased focus on the preventive strategies against the transmission of these pathogens.Several organizations have established prevention plans to implement an infection prevention and control criteria in order to break the chain of transmission and halt the spread of MDR-GNB. 18The WHO urges clinicians to implement antimicrobial stewardship as a potential solution to this substantial increase in antimicrobial resistance. 19ntimicrobial stewardship programs have been linked to great reduction in antibiotics use, hospital stay, and medical costs with significant decrease in mortality. 20Nevertheless, before the application of any stewardship program, surveillance of antimicrobial resistance is required to provide data on the prevalent MDROs and their antimicrobial resistance pattern. 21Several studies have shown the importance of routine microbiological surveillance in guiding the empirical antimicrobial treatment which is essential for tackling nosocomial infections.Providing routine information on the isolates' susceptibility could offer timely evidence regarding their resistance trend rather than the traditional methods of surveillance networks. 22So, we aimed at this work to do surveillance study to determine antimicrobial resistance pattern of K. pneumoniae obtained from ICU and non-ICU patients in Prince Mohammed bin Abdulaziz Hospital in Saudi Arabia, as well as to investigate potential impact of antimicrobial resistance on patients outcomes.

Study setting and design
A one-year retrospective, crosssectional study was conducted from January 2021 to December 2021 at Prince Mohammed bin Abdulaziz Hospital (PMAH), one of the Ministry of Health hospitals and a major referral hospital in Riyadh with 500 beds.The hospital serves patients over the age of 12 years with diagnostic and therapeutic means of the second and third levels of internal medicine specialties and surgical acute and chronic ones.

Data collection
The demographic and clinical data regarding patients having K. pneumoniae infections were retrieved from the patients' medical records and microbiology laboratory information system at PMAH.For each patient, the following information was collected: age, sex, the type of sample positive with K. pneumoniae, the results of antimicrobial susceptibility of isolates, and the clinical outcome, including length of hospital stay and mortality.
K. pneumoniae were isolated from various microbiological samples, including (endotracheal aspirate, sputum, blood, urine, and wound) from ICU and non-ICU patients (1 per patient).In this study, we aimed to compare the antimicrobial susceptibility results of K. pneumoniae isolated from ICU patients to those from non-ICU departments and investigate the potential effects of antimicrobial resistance on patients' outcome.

Processing of microbiological specimens and identification of isolated organisms
The samples were processed, and the isolates were identified in accordance with the microbiology laboratory standard procedures. 23All the media used in the isolation and identification of the pathogens were from (Oxoid, UK).All the collected specimens were inoculated onto the conventional culture media and allowed to grow for 18-24 h at 37°C.If no growth appears, incubation of the agar plates will continue for 48 hours.Based on microscopic examination and staining reactions, colonial morphology, and the VITEK 2 compact system (bioMerieux, Craponne, France), K. pneumoniae isolates were identified.

Antimicrobial susceptibility
Kirby-Bauer disk diffusion test was performed to determine bacterial isolates' susceptibility against antibiotics, and the results were interpreted based on the Clinical Laboratory Standards Institute (CLSI) guidelines. 24he antibiotic discs used were obtained from Oxoid.Controls such as P.aeruginosa (ATCC 27853) and E. coli (ATCC 25922) were utilized for antimicrobial susceptibility tests.
• ESBL: A difference of 5 mm in the zone size between ceftazidime and ceftazidime+ clavulanic acid discs confirms ESBL production. 25 MDR: The Centers for Disease Control and Prevention (CDC) defines MDR isolates as acquired non-sensitivity to one or more agents in at least three groups of antimicrobials. 26ased on the antimicrobial susceptibility, patients were categorized into three groups: ESBL-K.pneumoniae group (patients with ESBL-K.pneumoniae infections), MDR-K.pneumoniae group (patients with MDR-K.pneumoniae infections), non-ESBL, non-MDR K. pneumoniae group (patients with susceptible K. pneumoniae Infections).

Statistical analysis Data management & descriptive statistics
After discussing the protocol and the study objectives as well as data collection and verification, all data was fed into R Software version 3.5.2(2018-12-20) -"Eggshell Igloo." for statistical analysis.Quantitative data were represented as mean & Standard deviation, while qualitative categorical variables were represented by numbers and percentages.

Analytical statistics
• All the comparative analysis was done using the Chi-square test or Fisher exact test.
Statistically significant results are considered if the p-value ≤ 0.05.

RESULTS
The current study aims to provide background information on the pattern of local antibiotic resistance, which is necessary for prescribing empirical antibiotic treatment against certain pathogens.This data will also be significant as a guide for implementing antimicrobial stewardship.Thus, the study will present the antimicrobial susceptibility pattern of one of the most prevalent organisms, K. pneumoniae, retrieved from different sites of infection among both ICU and non-ICU patients as well as the clinical sequelae during the 1-year retrospective study.

Distribution of K. pneumoniae isolates, among ICU and non-ICU patients, according to age, gender, and sample types
The study was conducted on 229 K. pneumoniae isolates collected from ICU patients (33.2%) and non-ICU patients (66.8%).More than 50% of patients belonged to the older age group (63% e"60 years), and the mean age of ICU and non-ICU patients was 55 and 65 years, respectively.The ratio of males to females was 2.04 /1.The ratio of males to females in the ICU and non-ICU departments was 2.5:1 and 1.9:1, respectively.

Antimicrobial susceptibility of K. pneumoniae isolates
Isolates obtained from ICU patients were more resistant to all antibiotics than those isolated from non-ICU patients.The rate of ampicillin resistance was the highest, with 100% resistance among ICU Klebsiella isolates and 98.0% among non-ICU isolates without any statistically significant difference (p=0.553).Then, ceftriaxone showed significantly higher resistance, 82.9% among ICU isolates vs. 51.0%among  2).

Clinical outcome of patients
Regarding the clinical outcome, the length of hospital stay was significantly higher (IQR:16.0days) among the patients with MDR -K.pneumoniae infection compared to patients with ESBL-K.pneumoniae infection (IQR: 11.0 days) and patients with susceptible strains (IQR:8.5 days ) (P<0.001) (Figure 3).The correlation of mortality with antimicrobial resistance showed that,out of the 20 deaths (8.7%), 13 occurred among MDR patients, five among the ESBL group, and two among patients with susceptible strains with clinically significant difference (p= 0.004) (Table 5) (Figure 4).

DISCUSSION
K. pneumoniae is one of the main pathogens in the hospitals that causes various nosocomial infections with MDR pattern.ESBL producing strains are of major concern globally.In the United States, Carbapenem-resistant klebsiella have increased from 1.6 to 10.4% during the period from 2001 till 2011, and have provoked  the worldwide attention due to challenging treatment. 27n the current study, most K. pneumoniae isolates were retrieved from elderly male patients.This finding could be due to the predominantly male population in this hospital.Infections generally occur in males more than females because of the variations in the levels of sex steroids and differences in sex chromosomelinked genes. 28Increased occurrence of infection in the elderly could be referred to defective immune system and defense mechanisms, ICU admission,invasive mechanical ventilation, and frequent exposure to antimicrobials.Comorbidities as diabetes mellitus,cardiovascular diseases and malignancy are also considered risk factors. 29In several studies, similar to our results, K. pneumoniae and other gram-negative bacteria predominated in elderly male patients. 30,31oreover, most isolates, especially those from ICU, were isolated from respiratory and blood samples, while those from non-ICU were retrieved from urine and wound.This could be due to the prevalence of invasive devices as ventilators and central venous catheters and their associated infections in ICU rather than non-ICU.Similarly, other studies revealed the predominance of respiratory tract infections in the ICU. 32The study by Rao et al., also showed the highest isolation rate of K. pneumoniae from sputum (51.85%), followed by blood samples (11.11%). 33Contrary to our study, K. pneumoniae were primarily isolated from blood, urine, 11 and ear discharge in other studies. 34In the present study, we investigated the antimicrobial susceptibility of K. pneumoniae isolated from ICU and non-ICU patients.As previously reported in different studies, 35 antimicrobial resistance was higher among isolates obtained from ICU compared to non-ICU patients.This finding can be attributed to increased usage of these agents and lack of adherence to infection control measures in the ICU department. 36Klebsiella spp.isolated from hospitals have different resistance mechanisms, such as ESBLs, AmpC, and carbapenemases, which render the isolates resistant to certain penicillins, cephalosporins. 37and carbapenems. 38n our study, in ICU, higher resistance rates were detected toward ampicillin (100%),3 rd generation cephalosporin (82.9%,80.3%),and trimethoprimsulphamethoxazole (77.6%).In comparison, lower rates were observed toward amoxicillinclavulanate (69.7%), 2 nd, and 4 th -generation cephalosporins (67.1%,64.5%),quinolones (65.8%), aminoglycosides (65.8%), piperacillin-tazobactam (61.8%) and carbapenems with resistance rates (60.5%).Higher resistance to the commonly used antibiotics is challenging and could adversely affect outcomes.Failure of treatment and the need to save an effective alternative safer drug continues to be an urgent need.Our results align with Wani et al., who reported the highest resistance among ICU patients toward ampicillin (%95) followed by third and fourth generations cephalosporins. 39 previous study in KSA also reported higher resistance of K. pneumoniae isolates obtained from ICU toward cephalosporins and trimethoprim/ sulfamethoxazole (60%).31 These studies also reported lower resistance toward quinolones, aminoglycosides, piperacillin-tazobactam and carbapenems similar to our results.In addition, a study done by Sader et al., similarly observed higher resistance among ICU patients toward third generation cephalosporins but it differs from our results that it shows also higher resistance towards carbapenems.40 On the contrary, the study done by Ghenea et al. showed the highest resistance of Klebsiella strains in the ICU toward amoxicillin/clavulanate (85.31%), and 1 st generation cephalosporins, cefazoline (78.90%) but in consistence with our results, the resistance to quinolones, ciprofloxacin (30.57%), and meropenem (27.78%) was lower.34 Another study revealed distinct susceptibility profiles of K. pneumoniae isolated from ICU patients with higher resistance toward aztreonam, ticarcillin-clavulanic acid, ciprofloxacin, and levofloxacin while relatively lower resistance was detected toward colistin and tigecycline.41 The susceptibility profile may differ according to the geographic area, antimicrobial agents used, and adherence to infection control standards and antimicrobial stewardship.
Our study revealed that the most effective antibiotic toward Klebsiella isolates was amikacin, which has a susceptibility rate of 42.1% and 79.1% among ICU and non-ICU patients, respectively.Imipenem and meropenem were also effective, with a susceptibility rate of 39.5% among ICU patients and 77.8, and 77.1 among non-ICU patients, respectively.In consistence with our results, Kumari et al. also informed that amikacin antibiotic is the most effective, with a sensitivity rate of 69.5%. 42Leelarasamee et al. also considered amikacin as an effective drug and reported 43% resistance rate. 43In addition to amikacin, other agents, including meropenemvaborbactam, ceftazidime-avibactam, and colistin, were observed by Sader et al., to be also effective against K. pneumoniae isolated from ICU and non-ICU patients. 40n increased number of MDR bacteria in the ICU is referred to several contributing factors including excessive use of antibiotics, prolonged ICU stay, use of invasive medical devices, comorbidities and lack of hygienic measures and isolation precautions.This causes a problem in patients' treatment, necessitating adherence to rigorous infection control procedures in healthcare settings and warranting the proper usage of antimicrobials. 44In the current study, it was observed that MDR K. pneumoniae isolates were higher among ICU patients (60.5%) compared to non-ICU (21.6%).While higher prevalence of ESBL was noticed in non-ICU departments (30.1% in non-ICU vs. 15.8% in ICU) .The increased use of cephalosporins, which promote the emergence of ESBL-producing isolates, may account for the higher prevalence of ESBL in non-ICU departments. 45Analogous to us, Sader et al., reported the isolation of resistant phenotypes, such as MDR K. pneumoniae, which have very few treatment choices, more common from ICU rather than non-ICU patients. 46However, Altaf Bandy and Bilal Tantry reported the isolation of 39.3%,13% MDR, and ESBL K. pneumoniae, respectively, with considerably higher rates from clinical specimens of intensive care units (p < 0.01) in comparison to the samples obtained from non-ICU departments. 47The prevalence of MDR or ESBL depends on different factors, such as the regional guidelines for antimicrobial prescription and use, self-administration of antibiotics, and infection control guidelines. 48Carbapenems are the lastresort antibiotics for the treatment of Gramnegative ESBLs and the main defense against MDR Gram-negative infections.However, our study results proved that carbapenem resistance among MDR isolates is very high and alarming.Imipenem and meropenem reported effectiveness against ESBL strains in the current study in both ICU and non-ICU (100%,100%, 97.8%, and 95.8%, respectively), while their efficacy toward MDR strains decreased significantly in ICU and totally lost among non-ICU (2.2%, 2.2%, 0.0%, and 0.0%, respectively).This higher resistance toward carbapenems among MDR strains suggest carbapenemase production by the bacteria. 47Hawkey et al., declared that there is an increasing fear of the rise in carbapenem resistance among MDR-GNB. 49Furthermore, Indrajith et al. reported that 58% of MDR K. pneumoniae were carbapenem-resistant. 50 The global rise in Carbapenem-resistant strains is a public health threat that aroused worldwide attention, because of the restricted therapy.It is linked to increased rate of mortality and poor outcomes regardless of the chosen antibiotic therapy. 51Routine surveillance system in hospitals is very important for judicious detection of carbapenem susceptibility and the empirical therapy to control these infections.In addition, the use of new antimicrobials, such as ceftazidimeavibactam, meropenem-vaborbactam, tigecycline, and colistin, should be approached against MDR strains. 52,53The current study revealed a significant association between MDR, ESBL production by K. pneumoniae and poor patient outcome including increased mortality, and hospital stay (p<0.001).This finding can be explained by the delayed or inappropriate use of the proper antibiotics.Comorbid conditions ( like pneumonia, diabetes mellitus, renal disease), immunocompromised state and use of invasive devices could also affect patient outcome. 54In consistence with our study, Siwakoti et al. also reported a significant association between MDR GNB infection and hospital stay and mortality. 55A study done by Ben-David D et al. 56 also reported higher mortality among patients with carbapenem-resistant K. pneumoniae than those with carbapenem susceptible klebsiella infection (48% vs.17%).Cosgrove et al. 57 also found a significant association between the resistance of Enterobacter spp.toward third generation cephalosporins and patient mortality (Relative risk, 5.02).Contrary to our findings, other studies 58,59 reported a non-significant association between antimicrobial resistance and patient outcome.

CONCLUSION
There is a significant increase in ESBL and MDR producing K. pneumoniae in hospital settings, particularly ICUs.The prevalence of resistance in ICUs more than any other hospital department, can be linked to increased burden of co-morbidity in ICU patients, the use of immunosuppressive medications, the uncontrolled use of antibiotics like prolonged use of broadspectrum ones, lack of strict adherence to infection control measures as poor compliance to hand hygiene and isolation precautions especially during pandemics.Carbapenems are the main line of treatment against ESBL strains, but more effective agents for carbapenem resistant strains such as colistin, tigecycline, and Ceftazidime/avibactam should also be considered to save patients life.Alternative strategies that could control MDR K. pneumoniae including phytochemicals and bacterial metabolites such as biosurfactants are also enabling further research.Hospital-acquired infections caused by resistant pathogens leads to increased patient mortality, prolonged hospitalization, and extra costs for medical care with serious economic burden.The situation is alarming and the health authorities should provide unlimited attention to decrease the risk of nosocomial infections.Hospitals should implement the proposals of CDC and WHO to decrease nosocomial infections.

Strengths and limitations
The current study is the first work done in the region to compare the antibiogram of one of the most common bacteria in healthcare settings (K.pneumoniae ) between ICU and non-ICU.
However, being a single-center study that does not reflect the whole region is the main limitation.The lack of information about the history of improper use of empirical antibiotics could also be considered.

Recommendations
The inclusion of other Gram-negative and Gram-positive bacteria is compulsory to provide a comprehensive picture of antibiotic resistance.A study of the underlying molecular basis of antimicrobial resistance of MDR-K.pneumoniae should also be done.

Figure 2 .Figure 3 .
Figure 2. Pattern of resistance among ICU and non-ICU Klebsiella isolates

Table 1 .
Comparative analysis for baseline patients' characteristics & sample types of ICU vs. non-ICU patients *Data are represented as count (%)

Table 2 .
Comparative analysis for different antimicrobial susceptibility of ICU vs. non-ICU klebsiella isolates

MDR, and sensitive groups among ICU Klebsiella isolates vs. non-ICU
MDR K. pneumoniae were found in 60.5% of ICU isolates but only in 21.6% of non-ICU isolates (P<0.001).ICU klebsiella isolates had a significantly lower prevalence of ESBL (15.8%) than non-ICU isolates (30.1%) (P<0.001).About half of the K. pneumoniae isolates (48.4%) obtained from non-ICU patients were significantly susceptible and did not show either ESBL or MDR resistance, while only 23.7% of ICU ones were susceptible (p<0.001)(Table3)(Figure

Table 4 .
Comparative analysis for different antibiotics susceptibility of ESBL & MDR klebsiella isolates from ICU vs. non-ICU